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Abcam isl1
Genome-wide downstream targets of <t>ISL1</t> during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
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1) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

2) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

3) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

4) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

5) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

6) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

7) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

8) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

9) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

10) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

11) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

12) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

13) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

14) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

15) Product Images from "Mouse DRG Cell Line with Properties of Nociceptors"

Article Title: Mouse DRG Cell Line with Properties of Nociceptors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0128670

MED17.11 express the early neuronal markers FOX3 (NeuN), Isl1 and Tuj1, and can be transfected with GFP. Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.
Figure Legend Snippet: MED17.11 express the early neuronal markers FOX3 (NeuN), Isl1 and Tuj1, and can be transfected with GFP. Immunolabelling of MED17.11 cells cultured in permissive conditions for large T antigen expression. GFP transgene expression in MED17.11. Scale bar is 100 μm.

Techniques Used: Transfection, Cell Culture, Expressing

16) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

17) Product Images from "New insights insights into human female reproductive tract development"

Article Title: New insights insights into human female reproductive tract development

Journal: Differentiation; research in biological diversity

doi: 10.1016/j.diff.2017.08.002

Cervical development. (A–D) are sagittal sections of the female reproductive tract of a 16 week fetus (A=H E stain, B=ISL1 immunostain, C–D=keratin 19 immunostain). Boundaries between the vagina, cervix and uterus are nebulous up to 18 weeks, when vaginal fornices become apparent (F). (B) ISL1 immunostaining is strong in vaginal stroma, absent in uterine stroma, with a sharp fall off in staining intensity at the mid-point of the uterovaginal canal (red arrow in B). Keratin 19 immunostaining (C–D) may also be indicative of vaginal-exocervical-endocervical boundaries. Cervical glands are prominent at 18 weeks of gestation (E).
Figure Legend Snippet: Cervical development. (A–D) are sagittal sections of the female reproductive tract of a 16 week fetus (A=H E stain, B=ISL1 immunostain, C–D=keratin 19 immunostain). Boundaries between the vagina, cervix and uterus are nebulous up to 18 weeks, when vaginal fornices become apparent (F). (B) ISL1 immunostaining is strong in vaginal stroma, absent in uterine stroma, with a sharp fall off in staining intensity at the mid-point of the uterovaginal canal (red arrow in B). Keratin 19 immunostaining (C–D) may also be indicative of vaginal-exocervical-endocervical boundaries. Cervical glands are prominent at 18 weeks of gestation (E).

Techniques Used: Staining, Immunostaining

18) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

19) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

20) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

21) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

22) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

23) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

24) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

25) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

26) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

27) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

28) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

29) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

30) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

31) Product Images from "A Regulatory Pathway Involving Notch1/?-Catenin/Isl1 Determines Cardiac Progenitor Cell Fate"

Article Title: A Regulatory Pathway Involving Notch1/?-Catenin/Isl1 Determines Cardiac Progenitor Cell Fate

Journal: Nature cell biology

doi: 10.1038/ncb1906

Identification of genes affected by stabilized β-Catenin in cardiac progenitors. a, Lateral view of Rosa YFP ; Isl1 Cre ; β-catenin(ex3) loxP embryo at E9.0 showing YFP + cells in precardiac mesoderm (pm). b, Histograms of YFP + cell populations from control ( Isl1 Cre , left) and stabilized β-cat ( Isl1 Cre ; β-catenin(ex3) loxP , right) embryos. c, A heatmap of expression arrays showing significantly downregulated cardiac genes (green) in stabilized β-catenin pm cells. Color bar indicates fold change in log 2 scale. d, qPCR data of downregulated genes in FACS-purified cardiac progenitors with stabilized β-Catenin (Top). These genes were similarly affected in pm of Notch1 loss-of-function embryos (Bottom). Data are mean ± s. d.; n =3; * P
Figure Legend Snippet: Identification of genes affected by stabilized β-Catenin in cardiac progenitors. a, Lateral view of Rosa YFP ; Isl1 Cre ; β-catenin(ex3) loxP embryo at E9.0 showing YFP + cells in precardiac mesoderm (pm). b, Histograms of YFP + cell populations from control ( Isl1 Cre , left) and stabilized β-cat ( Isl1 Cre ; β-catenin(ex3) loxP , right) embryos. c, A heatmap of expression arrays showing significantly downregulated cardiac genes (green) in stabilized β-catenin pm cells. Color bar indicates fold change in log 2 scale. d, qPCR data of downregulated genes in FACS-purified cardiac progenitors with stabilized β-Catenin (Top). These genes were similarly affected in pm of Notch1 loss-of-function embryos (Bottom). Data are mean ± s. d.; n =3; * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, FACS, Purification

Isl1 targets Myocd and β-Catenin regulates Bhlhb2 to repress Smyd1 . a, Relative expression levels of Myocd and Smyd1 in FACS-purified control and Isl1 knockdown (KD) CPCs, determined by qPCR (mean ± s. d.; n =4; * P
Figure Legend Snippet: Isl1 targets Myocd and β-Catenin regulates Bhlhb2 to repress Smyd1 . a, Relative expression levels of Myocd and Smyd1 in FACS-purified control and Isl1 knockdown (KD) CPCs, determined by qPCR (mean ± s. d.; n =4; * P

Techniques Used: Expressing, FACS, Purification, Real-time Polymerase Chain Reaction

Notch1 loss-of-function causes CPC expansion and increases free β-Catenin levels. a–f, Control embryos. g–l, Isl1 Cre , Notch1 flox/flox embryos (N1-KO). a , g , Lateral views of ED10.5 embryos. b, c, h, i, Lateral ( b , h ) or frontal ( c , i ) view of embryos focused on cardiac regions showing absence of right ventricle (rv) in mutants. d , e , j , k , Transverse sections (H E) of embryos ( d, j ) with enlargement of boxed areas ( e, k ) showing hyperplasia of precardiac progenitors (asterisk). f , l, Phosphohistone3 (Ph3, red) and Isl1 (green) immunostaining of transverse sections through the precardiac region. To compensate for the severe downregulation of Isl1 in Notch1 mutant embryos, Isl1 signals were amplified with the TSA system. DAPI (blue) was used to counterstain the nuclei. m , Percentage of ph3-positive cells in precardiac mesoderm region shown in e and k (mean ± s. d.; n =4; * P
Figure Legend Snippet: Notch1 loss-of-function causes CPC expansion and increases free β-Catenin levels. a–f, Control embryos. g–l, Isl1 Cre , Notch1 flox/flox embryos (N1-KO). a , g , Lateral views of ED10.5 embryos. b, c, h, i, Lateral ( b , h ) or frontal ( c , i ) view of embryos focused on cardiac regions showing absence of right ventricle (rv) in mutants. d , e , j , k , Transverse sections (H E) of embryos ( d, j ) with enlargement of boxed areas ( e, k ) showing hyperplasia of precardiac progenitors (asterisk). f , l, Phosphohistone3 (Ph3, red) and Isl1 (green) immunostaining of transverse sections through the precardiac region. To compensate for the severe downregulation of Isl1 in Notch1 mutant embryos, Isl1 signals were amplified with the TSA system. DAPI (blue) was used to counterstain the nuclei. m , Percentage of ph3-positive cells in precardiac mesoderm region shown in e and k (mean ± s. d.; n =4; * P

Techniques Used: Immunostaining, Mutagenesis, Amplification

Isl1 loss-of-function results in expansion of CPCs and suppression of their myocardial and smooth muscle lineages. a, YFP expression in control ( Rosa YFP , Isl1 Cre/+ , left) and Isl1-null ( Rosa YFP , Isl1 Cre/Cre , right) embryos at the 5-somite stage. Arrows indicate YFP + CPCs. Scale bars, 50 µm. b, Quantification of YFP + cells in indicated embryos at somite 5 (mean ± s. d.; n =3; * P
Figure Legend Snippet: Isl1 loss-of-function results in expansion of CPCs and suppression of their myocardial and smooth muscle lineages. a, YFP expression in control ( Rosa YFP , Isl1 Cre/+ , left) and Isl1-null ( Rosa YFP , Isl1 Cre/Cre , right) embryos at the 5-somite stage. Arrows indicate YFP + CPCs. Scale bars, 50 µm. b, Quantification of YFP + cells in indicated embryos at somite 5 (mean ± s. d.; n =3; * P

Techniques Used: Expressing

Increased levels of Isl1 promote myocardial differentiation. a, Schematic diagram of differentiation of Myh7-GFP ES cells with Isl1 overexpression. b, c, Relative expression levels of Isl1 on ED6 EBs ( b ), and endothelial ( Flk1 ), cardiac sarcomeric ( Actc1, Mlc2v, Myh7 ) and smooth muscle ( Sma ) genes on day 8 EBs ( c ), determined by qPCR. d, FACS analyses on ED 9 EBs to identify % of cells entering myocardial-lineage. Data are mean ± s. e. m.; n =3; * P
Figure Legend Snippet: Increased levels of Isl1 promote myocardial differentiation. a, Schematic diagram of differentiation of Myh7-GFP ES cells with Isl1 overexpression. b, c, Relative expression levels of Isl1 on ED6 EBs ( b ), and endothelial ( Flk1 ), cardiac sarcomeric ( Actc1, Mlc2v, Myh7 ) and smooth muscle ( Sma ) genes on day 8 EBs ( c ), determined by qPCR. d, FACS analyses on ED 9 EBs to identify % of cells entering myocardial-lineage. Data are mean ± s. e. m.; n =3; * P

Techniques Used: Over Expression, Expressing, Real-time Polymerase Chain Reaction, FACS

32) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

33) Product Images from "Direct and indirect requirements of Shh/Gli signaling in early pituitary development"

Article Title: Direct and indirect requirements of Shh/Gli signaling in early pituitary development

Journal: Developmental biology

doi: 10.1016/j.ydbio.2010.09.024

Gli2 mutant pituitaries have normal patterning but show defects in proliferation. (A-F) RNA in situ hybridization of wild type (WT) and Gli2 mutants using probes of Lhx3 , Prop1 and Gata2 . (G-J) Immunostaining of Pax6 and Isl1. (K-P) Analysis of proliferation
Figure Legend Snippet: Gli2 mutant pituitaries have normal patterning but show defects in proliferation. (A-F) RNA in situ hybridization of wild type (WT) and Gli2 mutants using probes of Lhx3 , Prop1 and Gata2 . (G-J) Immunostaining of Pax6 and Isl1. (K-P) Analysis of proliferation

Techniques Used: Mutagenesis, RNA In Situ Hybridization, Immunostaining

34) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

35) Product Images from "Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin DevelopmentA Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel cell Development"

Article Title: Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin DevelopmentA Cascade of Wnt, Eda, and Shh Signaling Is Essential for Touch Dome Merkel cell Development

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1006151

Loss of EED does not affect the hair follicle microenvironment, but leads to upregulation of Merkel cell differentiation genes. (A-E) Shh and Wnt pathways do not appear to be majorly altered in PRC-null developing skin. (A) RT-qPCR analysis of Shh pathway genes shows no significant difference in their expression in P0 EED cKO (K14-Cre; EED flox/flox ) compared to control epidermis, while Shh expression is slightly reduced in EED-null compared to control epidermis (Gli1, p = 0.2000; Gli2, p = 0.1143; Gli3, p = 0.3429; Ptch1, p = 0.1143; Shh, p = 0.0286). RT-qPCR analysis of Wnt pathway genes shows no significant difference in expression of most genes in P0 EED cKO compared to control epidermis (Wnt3, p = 0.4857; Wnt4, p = 0.1143; Wnt7a, p = 0.3429; Wnt7b, p = 0.4857; Wnt10a, p = 0.2000; Wnt10b, p = 0.0286; Tcf3, p = 0.6857; Tcf4, p = 0.4857; Dkkl1, p = 0.6857; Axin2, p = 0.2000; Sp5, p = 0.0286). (B,C) Immunohistochemistry staining for β-catenin does not show major differences in expression or nuclear staining in first wave (B) or second wave (C) hair follicles in EED cKO skin compared to control at E16. Note that, as has been previously described, the stratum corneum is prematurely acquired in the PRC2-null E16 embryo [ 51 ]. (D,E) In situ hybridization for Gli1 RNA (D) and Shh RNA (E) shows no major changes in expression in Ezh1/2 2KO (K14-Cre; Ezh1 del/del ;Ezh2 flox/flox ) skin compared to control at E16. (F) FACS scheme for Merkel cell (MC) sorting. After gating on singlets and live cells, EpCAM-APC staining was used to gate on all epidermal cells and Atoh1-GFP labels Merkel cells. EpCAM-APC(+) Atoh1-GFP(-) cells were sorted as epidermal controls. (G) ChIP-qPCR showing significantly lower levels of H3K27me3 at Merkel genes, Isl1 , Sox2 , and Atoh1 , in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells. (Neuro, p = 0.0411; Olig1, p = 0.0200; Isl1, p = 0.0022; Sox2, p = 0.0194; Atoh1, p = 0.0050). (H) RT-qPCR showing specific expression of MC signature genes Isl1 , Sox2 , and Atoh1 in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells ( Isl1 , p = 0.0004; Sox2 , p = 0.0004; Atoh1 , p = 0.0004) (I) IF staining showing that Krt8(+) (K8) MCs have the H3K27me3 mark in P0 control Krt14(+) (K14) epidermis. Krt14(+) cells serve as a positive control for H3K27me3 staining. Quantification of H3K27me3 staining intensity (below) (Kruskal-Wallis test p
Figure Legend Snippet: Loss of EED does not affect the hair follicle microenvironment, but leads to upregulation of Merkel cell differentiation genes. (A-E) Shh and Wnt pathways do not appear to be majorly altered in PRC-null developing skin. (A) RT-qPCR analysis of Shh pathway genes shows no significant difference in their expression in P0 EED cKO (K14-Cre; EED flox/flox ) compared to control epidermis, while Shh expression is slightly reduced in EED-null compared to control epidermis (Gli1, p = 0.2000; Gli2, p = 0.1143; Gli3, p = 0.3429; Ptch1, p = 0.1143; Shh, p = 0.0286). RT-qPCR analysis of Wnt pathway genes shows no significant difference in expression of most genes in P0 EED cKO compared to control epidermis (Wnt3, p = 0.4857; Wnt4, p = 0.1143; Wnt7a, p = 0.3429; Wnt7b, p = 0.4857; Wnt10a, p = 0.2000; Wnt10b, p = 0.0286; Tcf3, p = 0.6857; Tcf4, p = 0.4857; Dkkl1, p = 0.6857; Axin2, p = 0.2000; Sp5, p = 0.0286). (B,C) Immunohistochemistry staining for β-catenin does not show major differences in expression or nuclear staining in first wave (B) or second wave (C) hair follicles in EED cKO skin compared to control at E16. Note that, as has been previously described, the stratum corneum is prematurely acquired in the PRC2-null E16 embryo [ 51 ]. (D,E) In situ hybridization for Gli1 RNA (D) and Shh RNA (E) shows no major changes in expression in Ezh1/2 2KO (K14-Cre; Ezh1 del/del ;Ezh2 flox/flox ) skin compared to control at E16. (F) FACS scheme for Merkel cell (MC) sorting. After gating on singlets and live cells, EpCAM-APC staining was used to gate on all epidermal cells and Atoh1-GFP labels Merkel cells. EpCAM-APC(+) Atoh1-GFP(-) cells were sorted as epidermal controls. (G) ChIP-qPCR showing significantly lower levels of H3K27me3 at Merkel genes, Isl1 , Sox2 , and Atoh1 , in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells. (Neuro, p = 0.0411; Olig1, p = 0.0200; Isl1, p = 0.0022; Sox2, p = 0.0194; Atoh1, p = 0.0050). (H) RT-qPCR showing specific expression of MC signature genes Isl1 , Sox2 , and Atoh1 in FACS-sorted Merkel cells compared to FACS-sorted epidermal cells ( Isl1 , p = 0.0004; Sox2 , p = 0.0004; Atoh1 , p = 0.0004) (I) IF staining showing that Krt8(+) (K8) MCs have the H3K27me3 mark in P0 control Krt14(+) (K14) epidermis. Krt14(+) cells serve as a positive control for H3K27me3 staining. Quantification of H3K27me3 staining intensity (below) (Kruskal-Wallis test p

Techniques Used: Cell Differentiation, Quantitative RT-PCR, Expressing, Immunohistochemistry, Staining, In Situ Hybridization, FACS, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control

Shh signaling activity is required for Merkel cell formation. (A-C) IF stainings for Merkel cell (MC) markers Krt20 (K20) (A,C), Isl1 (B), Krt8 (K8) (B), and Sox2 (C) show a complete absence of Merkel cells in E18 Shh KO (Shh EGFPcre/EGFPcre ) mice compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Shh KO E18 skin (right panel of B) (Krt8 p = 0.0005 and Krt20 p = 0.0001). (D) TUNEL staining shows no increase in apoptosis in the skin of E18 Shh KO mice. (E) IF stainings for Merkel cell markers Krt18 (K18) and Sox2 show a complete absence of Merkel cells in E16 Shh KO mice when compared to control. Sox2 also labels the dermal condensate (dc). (F) TUNEL staining shows no increase in apoptosis in the Krt14(+) basal layer of E16 Shh KO mice when compared to control. Note that cells undergoing cornification in the suprabasal layers are TUNEL(+), as previously reported [ 33 ]. (G,I,J) IF stainings for Merkel cell (MC) markers Krt20 (G,I), Krt8 (J), Isl1 (J), and Sox2 (I) show a complete absence of Merkel cells in P0 Shh epidermis-conditional knockout (Shh cKO) (K14-Cre; Shh flox/flox ) mice compared to control. Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Shh cKO P0 skin (right panel of J) (both p
Figure Legend Snippet: Shh signaling activity is required for Merkel cell formation. (A-C) IF stainings for Merkel cell (MC) markers Krt20 (K20) (A,C), Isl1 (B), Krt8 (K8) (B), and Sox2 (C) show a complete absence of Merkel cells in E18 Shh KO (Shh EGFPcre/EGFPcre ) mice compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Shh KO E18 skin (right panel of B) (Krt8 p = 0.0005 and Krt20 p = 0.0001). (D) TUNEL staining shows no increase in apoptosis in the skin of E18 Shh KO mice. (E) IF stainings for Merkel cell markers Krt18 (K18) and Sox2 show a complete absence of Merkel cells in E16 Shh KO mice when compared to control. Sox2 also labels the dermal condensate (dc). (F) TUNEL staining shows no increase in apoptosis in the Krt14(+) basal layer of E16 Shh KO mice when compared to control. Note that cells undergoing cornification in the suprabasal layers are TUNEL(+), as previously reported [ 33 ]. (G,I,J) IF stainings for Merkel cell (MC) markers Krt20 (G,I), Krt8 (J), Isl1 (J), and Sox2 (I) show a complete absence of Merkel cells in P0 Shh epidermis-conditional knockout (Shh cKO) (K14-Cre; Shh flox/flox ) mice compared to control. Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Shh cKO P0 skin (right panel of J) (both p

Techniques Used: Activity Assay, Mouse Assay, TUNEL Assay, Staining, Knock-Out

Shh signaling activity in the epidermis is required for Merkel cell formation. (A-C) IF stainings for Merkel cell markers Krt8 (K8) (A,C), Krt20 (K20) (B), Sox2 (B), and Isl1 (C) show a highly significant reduction in the number of Merkel cells in P0 Smoothened epidermis-conditional knockout (Smo cKO) (K14-Cre; Smo flox/flox ) mice when compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Smo cKO P0 skin (right panel of B) (both p
Figure Legend Snippet: Shh signaling activity in the epidermis is required for Merkel cell formation. (A-C) IF stainings for Merkel cell markers Krt8 (K8) (A,C), Krt20 (K20) (B), Sox2 (B), and Isl1 (C) show a highly significant reduction in the number of Merkel cells in P0 Smoothened epidermis-conditional knockout (Smo cKO) (K14-Cre; Smo flox/flox ) mice when compared to control (ctrl). Quantification of Krt8(+) and Krt20(+) Merkel cells in control and Smo cKO P0 skin (right panel of B) (both p

Techniques Used: Activity Assay, Knock-Out, Mouse Assay

36) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

37) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

38) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

39) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

40) Product Images from "ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells"

Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw301

Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
Figure Legend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

Techniques Used: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P
Figure Legend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

Techniques Used: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P
Figure Legend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.
Figure Legend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

Techniques Used: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P
Figure Legend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P
Figure Legend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

Techniques Used: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software

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Immunohistochemistry:

Article Title: Intravitreal homocysteine-thiolactone injection leads to the degeneration of multiple retinal cells, including photoreceptors
Article Snippet: .. Immunohistochemical analysis and quantification of labeled cells For immunohistochemical analysis, the retinal tissue sections were boiled in a citrate buffer (pH 6.0) for 20 min for antigen retrieval and were then incubated, respectively, with one of the following antibodies: rabbit anti-homocysteine (1/50, cat. ab15154; Abcam); mouse anti-rhodopsin (1/400, cat. Ab81702; Abcam ) ; mouse anti-calbindin (1/50, cat. sc-74462; Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-Pax-6 antibody (1/50, cat. sc-32766; Santa Cruz Biotechnology); rabbit anti-Islet-1 (1/150, cat. Ab20670; Abcam); sheep anti-Chx-10 (1/150, cat. ab16141; Abcam); or rabbit anti-GFAP (1/250, cat. 2301–1; Epitomics, Burlingame, CA). .. The preparations were then incubated with a horseradish peroxidase-conjugated secondary antibody (1/200), either antimouse, antirabbit, or antisheep IgG (Jackson ImmunoResearch Laboratories, Inc.).

Labeling:

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    Genome-wide downstream targets of <t>ISL1</t> during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).
    Isl1, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

    Journal: Nucleic Acids Research

    Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

    doi: 10.1093/nar/gkw301

    Figure Lengend Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation. ( A ) Relative mRNA expression of pluripotency ( Oct4, Sox2 and Nanog ), mesoderm ( Mesp1 ), cardiac progenitor ( Isl1, Fgf10, Mef2c and Myocd ) and cardiomyocyte ( α-MHC, β-MHC and Troponin T ) markers in cardiac differentiation of ESCs was measured by qRT-PCR at indicated time points. 18S rRNA was used as an internal control. Data are mean ± SD, n = 4. ( B ) ChIP-seq ISL1-binding regions were mapped relative to their nearest downstream genes. Annotation includes whether a peak is in the first intron, other introns, 5′ UTR, last intron, exons (coding), 3′ UTR, intergenic. ( C ) GO functional clustering of genes associated with ISL1 ChIP-seq peaks (top 10 categories are shown). ( D ) The binding of ISL1 in EBs at day 7 of cardiac differentiation, H3K4me1 and H3K27ac in embryonic day 14.5 of heart tissue on representative ISL1 target genes, Myocd, Fgf10, Tbx3, Gata6, Smarcd3 and Tbx20 . (E) Overlay of RNA-seq and ChIP-seq results revealed 472 genes as potential direct targets of ISL1 in day 7 EBs of differentiation. (F and G) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1 (top 10 categories are shown).

    Article Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation ISL1 is expressed in cardiac progenitors and is required for their proliferation, survival, and differentiation.

    Techniques: Genome Wide, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Functional Assay, RNA Sequencing Assay

    JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

    Journal: Nucleic Acids Research

    Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

    doi: 10.1093/nar/gkw301

    Figure Lengend Snippet: JMJD3 regulates cardiac differentiation. ( A ) Schematic diagram of the knockout Jmjd3 using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in B are shown by arrows. Donor plasmid utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), mPGK, neomycin resistance (NeoR), T2A, EGFP, polyA sequence, and right homolog arm (RHR). ( B ) PCR genotyping of WT and Jmjd3 +/− ESCs. The size of PCR product of wild type ESCs is ∼2 kb, and is ∼1 kb of the Jmjd3 -KO ESCs. ( C ) Relative mRNA expression of Jmjd3 in wild type and Jmjd3 +/− ESCs using primers specific target to exon 9–10. ( D ) Relative mRNA expression of pluripotency genes ( Oct4, Sox2 and Nanog ) in wild type and Jmjd3 +/− ESCs. ( E ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( F ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from wild type and Jmjd3 +/− ESCs. ( G ) ChIP of nuclear extracts of day 7 EBs differentiated from wild type and Jmjd3 +/− ESCs using anti- H3K27me3. qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild type EBs. ( H ) Relative mRNA expression of Jmjd3, Isl1 and ISL1's downstream genes ( Myocd, Mef2c, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 ) in day 7 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( I ) Relative mRNA expression of cardiomyocyte genes ( α-MHC and β-MHC ) in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. Doxycycline was supplemented to the culture medium since day 5. ( J ) Percentage of beating EBs in day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. ( K ) Western blot analysis of total protein extracts of day 12 EBs differentiated from ish Jmjd3 in the presence or absence of Dox. β-Tubulin served as a loading control. Data in C–J are mean ± SD, n = 3. * P

    Article Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation ISL1 is expressed in cardiac progenitors and is required for their proliferation, survival, and differentiation.

    Techniques: Knock-Out, CRISPR, Plasmid Preparation, Sequencing, Polymerase Chain Reaction, Expressing, Chromatin Immunoprecipitation, Binding Assay, In Situ Hybridization, Western Blot

    ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

    Journal: Nucleic Acids Research

    Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

    doi: 10.1093/nar/gkw301

    Figure Lengend Snippet: ISL1 and JMJD3 form a transcriptional regulatory complex. ( A ) ChIP-seq density heatmaps of ISL1 and JMJD3 on ISL1 binding sites, Input served as a negative control. ( B ) Overlay of JMJD3 and ISL1 ChIP-seq results revealed 602 genes as potential direct targets of ISL1/JMJD3 in EBs at day 7 of differentiation. ( C ) GO functional clustering of genes allowed for identification of cellular functions directly regulated by ISL1/JMJD3 (top 10 categories are shown). (D and E) ChIP of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-ISL1 ( D ) and anti-HA ( E ) or IgG as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. (F and G) re-ChIP of material from anti-ISL1 ChIP elutions using anti-HA ( F ), or from anti-HA ChIP elutions using anti-ISL1 ( G ). IgG served as control. qRT-PCRs were performed using primers targeting ISL1 binding sites on Mef2c and Myocd enhancers. ChIP enrichments are normalized to Input and are represented as fold change relative to IgG. ( H and I ) ChIP of nuclear extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox using anti-ISL1 (H) or anti-HA (I). qRT-PCRs were performed using primers targeting ISL1 binding sites in the enhancers of Mef2c, Myocd, Bmp2, Fgf10, Gata6, Smarcd3, Tbx3 and Tbx20 . ChIP enrichments are normalized to Input and are represented as fold change relative to wild Dox- EBs. Data in D–I are mean ± SD, n = 3. * P

    Article Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation ISL1 is expressed in cardiac progenitors and is required for their proliferation, survival, and differentiation.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Negative Control, Functional Assay, In Situ Hybridization

    JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

    Journal: Nucleic Acids Research

    Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

    doi: 10.1093/nar/gkw301

    Figure Lengend Snippet: JMJD3 physically interacts with ISL1 at cardiac progenitor stage. ( A ) Co-immunoprecipitation of nuclear extracts from HEK293T cells transfected with a Flag construct or Flag tagged of ISL1 expression plasmid using anti-Flag were analysis by anti-JMJD3 and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. ( B ) Schematic diagram of the endogenous tagging N-ternimal of JMJD3 with Flag-HA epitope (JMJD3-NFH) using Crispr/Cas9. CRISPR-Cas9 cleavage site (exon4, 5.5 kb downstream of the TSS) is shown by scissor. Positions of the genotype primers used in C are shown by arrows. Donor ultramer utilized for targeting Jmjd3 locus is shown: left homolog arm (LHR), Flag-HA sequence, and right homolog arm (RHR). ( C ) PCR genotyping of wild type (WT) and Jmjd3-NFH ESCs. The size of PCR product of wild type ESCs is 299 bp, and the Jmjd3-NFH ESCs is 368 bp. ( D ) Western blot analysis of total protein extracts of WT and JMJD3-NFH ESCs. β-Tubulin served as a loading control. ( E ) ISL1 (red) and HA (green) immunostaining on day 7 EBs differentiated from JMJD3-NFH ESCs. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 20 μm. ( F ) Co-immunoprecipitation of nuclear extracts of day 7 EBs differentiated from JMJD3-NFH ESCs using anti-FLAG (F) or anti-ISL1 ( G ) or IgG as a control were analysis by anti-HA and anti-ISL1 immunoblotting. Five percent of total lysates were loaded as input. E1 and E2 represented two times elution with glycine. ( H and I ) Schematic representation of ISL1 (H) or JMJD3 (I) and its domains and GST pull-down assays. GST pull-down assays with purified GST-fused proteins and in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) were analyzed with anti-FLAG (H) or ISL1 (I) immunoblotting. Five percent of total in vitro transcribed/translated JMJD3-Flag (H) or ISL1 (I) was loaded as input. Coomassie brilliant blue staining (CBB staining) was applied to detect the purified GST-fused protein expression, * indicate the predicted proteins.

    Article Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation ISL1 is expressed in cardiac progenitors and is required for their proliferation, survival, and differentiation.

    Techniques: Immunoprecipitation, Transfection, Construct, Expressing, Plasmid Preparation, CRISPR, Sequencing, Polymerase Chain Reaction, Western Blot, Immunostaining, Staining, Purification, In Vitro

    ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

    Journal: Nucleic Acids Research

    Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

    doi: 10.1093/nar/gkw301

    Figure Lengend Snippet: ISL1 perturbation impairs demethylation of H3K27me3 at the enhancers of its downstream cardiac-specific target genes. ( A and B ) ChIP of nuclear extracts from day 3, 5 and 7 EBs differentiated from ESCs using anti-ISL1, anti-H3K4me1, anti-H3K4me3, anti-H3K27me3 and anti-H3K27ac or IgG as the control. qRT-PCRs were performed using primers flanking conserved ISL1 binding sites in the Mef2c enhancer (A) and the Myocd enhancer (B). Data are mean ± SD, n = 3. P

    Article Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation ISL1 is expressed in cardiac progenitors and is required for their proliferation, survival, and differentiation.

    Techniques: Chromatin Immunoprecipitation, Binding Assay

    ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

    Journal: Nucleic Acids Research

    Article Title: ISL1 and JMJD3 synergistically control cardiac differentiation of embryonic stem cells

    doi: 10.1093/nar/gkw301

    Figure Lengend Snippet: ISL1 modulates the demethylase activity of JMJD3. ( A ) Relative JMJD3/UTX demethylase activity of nuclear extracts from day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. ( B ) Western blot analysis of total cell lysates of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. β-Tubulin served as a loading control. ( C ) Relative mRNA expression of Isl1, Jmjd3 and Utx of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. (D) Western blot analysis of histone extracts of day 7 EBs differentiated from ish Isl1 ESCs in the presence or absence of Dox. Histone H3 served as a loading control. (E) Relative JMJD3/UTX demethylase activity of nuclear extracts from NIH-3T3 cells transient transfected with control or ISL1 expression construct. (F) Western blot analysis of total cell lysates of NIH-3T3 cells transient transfected with control or ISL1 expression construct. β-Tubulin served as a loading control. (G) Western blot analysis of histone extracts of NIH-3T3 cells transient transfected with control or ISL1 expression construct. Histone H3 served as a loading control. Optical densities of protein bands were quantified by Image J software and relative expression levels of H3K27me3 to Histone H3 were shown in D and G. Data in A, C and E are mean ± SD, n = 3. *** P

    Article Snippet: Genome-wide downstream targets of ISL1 during cardiac progenitor differentiation ISL1 is expressed in cardiac progenitors and is required for their proliferation, survival, and differentiation.

    Techniques: Activity Assay, In Situ Hybridization, Western Blot, Expressing, Transfection, Construct, Software