Structured Review

Millipore fa j3ncz
JMJ703 is a histone H3K4 demethylase. (A) Schematic presentation of JMJ703 protein structure. Fragment spanning amino acids 139–498 was obtained for crystal growth and structural determination. The region between amino acids 113–700 was used for enzymatic assays. (B) In vivo histone H3K4 demethylase activity of JMJ703. Constructs containing the JMJ703 fragment tagged by FLAG-HA <t>(FA-J3NCZ)</t> were transfected into tobacco leaf cells. Nuclei expressing FA-J3NCZ (stained with anti-HA) were examined for H3K4 methylation by anti-H3K4me1/2/3 and anti-H3K27me3 (indicated by arrows). At least 30 nuclei expressing JMJ703 per transfection were observed and imaged. Bar = 25 µm. (C) In vitro demethylase activity of JMJ703. Bulk histones were incubated with three quantities of FA-J3NCZ and analyzed by Western blots using antibodies of H3K4me1/2/3. The same blots were analyzed by anti-H3 to control loadings. FA-J3NCZ levels were revealed by anti-HA. (D) Characterization of c-JMJ703 binding to H3K4me0/1/2/3, H3K9me0 and H3K36me0 peptides using surface plasmon resonance. Curves for different concentration of peptide are differentially colored and labeled.
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1) Product Images from "Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice"

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1003239

JMJ703 is a histone H3K4 demethylase. (A) Schematic presentation of JMJ703 protein structure. Fragment spanning amino acids 139–498 was obtained for crystal growth and structural determination. The region between amino acids 113–700 was used for enzymatic assays. (B) In vivo histone H3K4 demethylase activity of JMJ703. Constructs containing the JMJ703 fragment tagged by FLAG-HA (FA-J3NCZ) were transfected into tobacco leaf cells. Nuclei expressing FA-J3NCZ (stained with anti-HA) were examined for H3K4 methylation by anti-H3K4me1/2/3 and anti-H3K27me3 (indicated by arrows). At least 30 nuclei expressing JMJ703 per transfection were observed and imaged. Bar = 25 µm. (C) In vitro demethylase activity of JMJ703. Bulk histones were incubated with three quantities of FA-J3NCZ and analyzed by Western blots using antibodies of H3K4me1/2/3. The same blots were analyzed by anti-H3 to control loadings. FA-J3NCZ levels were revealed by anti-HA. (D) Characterization of c-JMJ703 binding to H3K4me0/1/2/3, H3K9me0 and H3K36me0 peptides using surface plasmon resonance. Curves for different concentration of peptide are differentially colored and labeled.
Figure Legend Snippet: JMJ703 is a histone H3K4 demethylase. (A) Schematic presentation of JMJ703 protein structure. Fragment spanning amino acids 139–498 was obtained for crystal growth and structural determination. The region between amino acids 113–700 was used for enzymatic assays. (B) In vivo histone H3K4 demethylase activity of JMJ703. Constructs containing the JMJ703 fragment tagged by FLAG-HA (FA-J3NCZ) were transfected into tobacco leaf cells. Nuclei expressing FA-J3NCZ (stained with anti-HA) were examined for H3K4 methylation by anti-H3K4me1/2/3 and anti-H3K27me3 (indicated by arrows). At least 30 nuclei expressing JMJ703 per transfection were observed and imaged. Bar = 25 µm. (C) In vitro demethylase activity of JMJ703. Bulk histones were incubated with three quantities of FA-J3NCZ and analyzed by Western blots using antibodies of H3K4me1/2/3. The same blots were analyzed by anti-H3 to control loadings. FA-J3NCZ levels were revealed by anti-HA. (D) Characterization of c-JMJ703 binding to H3K4me0/1/2/3, H3K9me0 and H3K36me0 peptides using surface plasmon resonance. Curves for different concentration of peptide are differentially colored and labeled.

Techniques Used: In Vivo, Activity Assay, Construct, Transfection, Expressing, Staining, Methylation, In Vitro, Incubation, Western Blot, Binding Assay, SPR Assay, Concentration Assay, Labeling

2) Product Images from "REST-dependent epigenetic remodeling promotes the in vivo developmental switch in NMDA receptors"

Article Title: REST-dependent epigenetic remodeling promotes the in vivo developmental switch in NMDA receptors

Journal: Nature neuroscience

doi: 10.1038/nn.3214

REST increases transiently, is recruited to and coincides with epigenetic marks of repression at the grin2b promoter during rat hippocampal postnatal development a , Representative Western blots of whole hippocampal lysates showing that REST increases, GluN2B declines, and GluN2A increases during postnatal development (see full-length blot in Supplementary Fig. 1a ). b , Time course showing that REST protein increases transiently at P14–15 (n = 5). c , GluN2B mRNA exhibits a long-term decline during postnatal development, assessed by RT-qPCR. The decline was highly significant from P15 through P60 ( vs . P3; n = 5). d,e , Time course showing that whereas GluN2B protein declines after P21 (n = 6), GluN2A protein increases markedly from P8 to P16 and remains high up to P60 (n = 3). Data were normalized to corresponding values at P3. f , GluN1 mRNA is not altered during postnatal development (n = 6). g , Representative Western blot ( left panel ) and summary data ( right panel ) show REST protein expression in the nuclear fraction of the hippocampal cell body layer, which is enriched for neurons. Note that REST abundance in the neuronal nuclear fraction increases strikingly by P14–15 (n = 4). Data were normalized to corresponding data at P9. See full-length blot in Supplementary Fig. 12 ). h , Map of the rat grin2b gene indicating location of RE1 motifs contained within the proximal (PR1; gray box ) and distal (PR2; white box ) regions of the grin2b promoter probed by chromatin immunoprecipitation (ChIP). i,j , REST occupancy is markedly enriched at the grin2b proximal (PR1, grey bars ) (n = 6) and distal PR2 ( white bars ) (n = 3) promoters at P15 but declines by P60. k,l , REST is not enriched at RE1 sites within the grin2a (n = 9), nor grin1 (n = 6) promoters. Inset, same data depicted with expanded y-axis. m,n , CoREST (n = 3) and G9a (n = 3) are enriched at PR1 by P15. o,p , Increase in H3K9me3 (n = 3) and H3K27me3 (n = 6) (marks of repression) at P15. q , Decrease in trimethylation of H3K4 (n = 3) (mark of active transcription), at PR1. r , MeCP2 occupancy is enriched at grin2b PR1 by P15 with a sharp increase by P60 (n = 3). All samples were normalized to input and to corresponding values at P3. Summary data represent the mean ± s.e.m. *p
Figure Legend Snippet: REST increases transiently, is recruited to and coincides with epigenetic marks of repression at the grin2b promoter during rat hippocampal postnatal development a , Representative Western blots of whole hippocampal lysates showing that REST increases, GluN2B declines, and GluN2A increases during postnatal development (see full-length blot in Supplementary Fig. 1a ). b , Time course showing that REST protein increases transiently at P14–15 (n = 5). c , GluN2B mRNA exhibits a long-term decline during postnatal development, assessed by RT-qPCR. The decline was highly significant from P15 through P60 ( vs . P3; n = 5). d,e , Time course showing that whereas GluN2B protein declines after P21 (n = 6), GluN2A protein increases markedly from P8 to P16 and remains high up to P60 (n = 3). Data were normalized to corresponding values at P3. f , GluN1 mRNA is not altered during postnatal development (n = 6). g , Representative Western blot ( left panel ) and summary data ( right panel ) show REST protein expression in the nuclear fraction of the hippocampal cell body layer, which is enriched for neurons. Note that REST abundance in the neuronal nuclear fraction increases strikingly by P14–15 (n = 4). Data were normalized to corresponding data at P9. See full-length blot in Supplementary Fig. 12 ). h , Map of the rat grin2b gene indicating location of RE1 motifs contained within the proximal (PR1; gray box ) and distal (PR2; white box ) regions of the grin2b promoter probed by chromatin immunoprecipitation (ChIP). i,j , REST occupancy is markedly enriched at the grin2b proximal (PR1, grey bars ) (n = 6) and distal PR2 ( white bars ) (n = 3) promoters at P15 but declines by P60. k,l , REST is not enriched at RE1 sites within the grin2a (n = 9), nor grin1 (n = 6) promoters. Inset, same data depicted with expanded y-axis. m,n , CoREST (n = 3) and G9a (n = 3) are enriched at PR1 by P15. o,p , Increase in H3K9me3 (n = 3) and H3K27me3 (n = 6) (marks of repression) at P15. q , Decrease in trimethylation of H3K4 (n = 3) (mark of active transcription), at PR1. r , MeCP2 occupancy is enriched at grin2b PR1 by P15 with a sharp increase by P60 (n = 3). All samples were normalized to input and to corresponding values at P3. Summary data represent the mean ± s.e.m. *p

Techniques Used: Western Blot, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation

3) Product Images from "Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters"

Article Title: Barrier-to-Autointegration Factor 1 (BAF/BANF1) Promotes Association of the SETD1A Histone Methyltransferase with Herpes Simplex Virus Immediate-Early Gene Promoters

Journal: mBio

doi: 10.1128/mBio.00345-15

Effect of BAF depletion on viral chromatin. ChIP assays were performed to determine the levels of histone H3, the H3K9me3 heterochromatin mark, and the H3K4me3 euchromatin mark on the ICP27 IE gene promoter. (A to C) HeLa cells transfected with NT control or BAF-specific siRNA were infected with HSV-1 at an MOI of 1 and fixed at the postinfection times indicated (top panels) or 2 hpi (bottom panels). Chromatin was prepared and immunoprecipitated with antibodies specific for H3 (A), H3K9me3 (B), and H3K4me3 (C), and the amounts of ICP27 promoter sequences were determined by quantitative PCR. The top panels present two independent experiments, and the bottom panels present three or more independent experiments. Lines connect values of individual experiments. (D and E) ChIP assays were performed to determine the levels of BAF (D) and SETD1A (E) on viral gene promoters. HA-BAF was expressed transiently in HeLa cells that were then infected with HSV-1 at an MOI of 5. FLAG-SETD1A was expressed transiently in BAF-depleted HeLa (siBAF) or control (NT) cells that were then infected with HSV-1 at an MOI of 5. At 2 hpi, the cells were fixed and ChIP was performed with antibodies specific for HA or FLAG. The levels of individual HA-BAF- or FLAG-SETD1A-associated promoter sequences were determined by quantitative PCR. The histogram shows the mean values and standard deviations from three independent experiments. *, P
Figure Legend Snippet: Effect of BAF depletion on viral chromatin. ChIP assays were performed to determine the levels of histone H3, the H3K9me3 heterochromatin mark, and the H3K4me3 euchromatin mark on the ICP27 IE gene promoter. (A to C) HeLa cells transfected with NT control or BAF-specific siRNA were infected with HSV-1 at an MOI of 1 and fixed at the postinfection times indicated (top panels) or 2 hpi (bottom panels). Chromatin was prepared and immunoprecipitated with antibodies specific for H3 (A), H3K9me3 (B), and H3K4me3 (C), and the amounts of ICP27 promoter sequences were determined by quantitative PCR. The top panels present two independent experiments, and the bottom panels present three or more independent experiments. Lines connect values of individual experiments. (D and E) ChIP assays were performed to determine the levels of BAF (D) and SETD1A (E) on viral gene promoters. HA-BAF was expressed transiently in HeLa cells that were then infected with HSV-1 at an MOI of 5. FLAG-SETD1A was expressed transiently in BAF-depleted HeLa (siBAF) or control (NT) cells that were then infected with HSV-1 at an MOI of 5. At 2 hpi, the cells were fixed and ChIP was performed with antibodies specific for HA or FLAG. The levels of individual HA-BAF- or FLAG-SETD1A-associated promoter sequences were determined by quantitative PCR. The histogram shows the mean values and standard deviations from three independent experiments. *, P

Techniques Used: Chromatin Immunoprecipitation, Transfection, Infection, Immunoprecipitation, Real-time Polymerase Chain Reaction

4) Product Images from "An essential role for UTX in resolution and activation of bivalent promoters"

Article Title: An essential role for UTX in resolution and activation of bivalent promoters

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1516

UTX loss blocks the RA-induced bivalency resolution and activation of most bivalent Hoxa and Hoxb genes in mouse ESCs. ( A and B ) ChIP-Seq density profiles for H3K27me3 (red) and H3K4me3 (blue) at the mouse Hoxa / Hoxb (A) and Hoxc / Hoxd (B) cluster genes. H3K4me3 and H3K27me3 levels at the Hoxa – d promoters in WT, RA-induced WT, Utx -null and RA-induced Utx -null V6.5 mouse ESCs were analyzed using ChIP-Seq. The vertical axis shows ChIP-Seq signals at a maximal value of 100. ( C and D ) Effect of UTX knockdown on RA-induced changes in expression levels of Hoxa/Hoxb (C) and Hoxc / Hoxd (D) cluster genes. mRNA levels of Hoxa – d cluster genes in WT, RA-induced WT, Utx -null and RA-induced Utx -null V6.5 mouse ESCs were analyzed using quantitative RT-PCR. Cells were treated with 0.2 μM RA. Data are presented as the mean ± SEM (error bars) of at least three independent experiments. * P
Figure Legend Snippet: UTX loss blocks the RA-induced bivalency resolution and activation of most bivalent Hoxa and Hoxb genes in mouse ESCs. ( A and B ) ChIP-Seq density profiles for H3K27me3 (red) and H3K4me3 (blue) at the mouse Hoxa / Hoxb (A) and Hoxc / Hoxd (B) cluster genes. H3K4me3 and H3K27me3 levels at the Hoxa – d promoters in WT, RA-induced WT, Utx -null and RA-induced Utx -null V6.5 mouse ESCs were analyzed using ChIP-Seq. The vertical axis shows ChIP-Seq signals at a maximal value of 100. ( C and D ) Effect of UTX knockdown on RA-induced changes in expression levels of Hoxa/Hoxb (C) and Hoxc / Hoxd (D) cluster genes. mRNA levels of Hoxa – d cluster genes in WT, RA-induced WT, Utx -null and RA-induced Utx -null V6.5 mouse ESCs were analyzed using quantitative RT-PCR. Cells were treated with 0.2 μM RA. Data are presented as the mean ± SEM (error bars) of at least three independent experiments. * P

Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR

UTX loss interferes with the RA-induced bivalency resolution in the RA-inducible bivalent genes Hoxa3, Hoxb2, Hoxc4 and Hoxd4 in mouse ESCs. Quantitative ChIP analysis was performed to analyze the effect of UTX loss on the RA-induced changes of H3K4me3, H3K27me3 and H3 levels at the Hoxa3 ( A ), Hoxb2 ( B ), Hoxc4 ( C ), Hoxd4 ( D ), Hoxa10 ( E ), Hoxb13 ( F ), Hoxc10 ( G ) and Hoxd10 ( H ) in RA-untreated and RA-treated WT V6.5 mouse ESCs as well as RA-untreated and RA-treated Utx -null V6.5 mouse ESCs. Cells were treated with 0.2 μM RA for 0 or 5 days.
Figure Legend Snippet: UTX loss interferes with the RA-induced bivalency resolution in the RA-inducible bivalent genes Hoxa3, Hoxb2, Hoxc4 and Hoxd4 in mouse ESCs. Quantitative ChIP analysis was performed to analyze the effect of UTX loss on the RA-induced changes of H3K4me3, H3K27me3 and H3 levels at the Hoxa3 ( A ), Hoxb2 ( B ), Hoxc4 ( C ), Hoxd4 ( D ), Hoxa10 ( E ), Hoxb13 ( F ), Hoxc10 ( G ) and Hoxd10 ( H ) in RA-untreated and RA-treated WT V6.5 mouse ESCs as well as RA-untreated and RA-treated Utx -null V6.5 mouse ESCs. Cells were treated with 0.2 μM RA for 0 or 5 days.

Techniques Used: Chromatin Immunoprecipitation

UTX resolves the bivalency of many RA-inducible bivalent genes during RA-driven differentiation of mouse ESCs. ( A and C ) Comparison of average ChIP-Seq reads densities for H3K4me3 and H3K27me3 between RA-treated and untreated WT cells (A) and between RA-treated and untreated Utx -null cells (C). The average values of three biological replicates of the 637 RA-inducible bivalent genes (top), the rest of bivalent genes (middle) and non-bivalent genes (bottom) were plotted from -5K to 5K around the transcription start site (TSS). ( B and D ) ChIP-Seq enrichment profiles for H3K4me3 (first column) and H3K27me3 (second column) levels and for RA-induced changes in H3K4me3 (third column) and H3K27me3 (fourth column) levels in WT (B) and Utx -null (D) mouse ESCs. ( E ) Comparison of ChIP-Seq reads densities for H3K4me3 and H3K27me3 between RA-treated and untreated WT cells and between RA-treated and untreated Utx -null cells. The 316 RA-inducible bivalent genes downregulated by UTX loss were used.
Figure Legend Snippet: UTX resolves the bivalency of many RA-inducible bivalent genes during RA-driven differentiation of mouse ESCs. ( A and C ) Comparison of average ChIP-Seq reads densities for H3K4me3 and H3K27me3 between RA-treated and untreated WT cells (A) and between RA-treated and untreated Utx -null cells (C). The average values of three biological replicates of the 637 RA-inducible bivalent genes (top), the rest of bivalent genes (middle) and non-bivalent genes (bottom) were plotted from -5K to 5K around the transcription start site (TSS). ( B and D ) ChIP-Seq enrichment profiles for H3K4me3 (first column) and H3K27me3 (second column) levels and for RA-induced changes in H3K4me3 (third column) and H3K27me3 (fourth column) levels in WT (B) and Utx -null (D) mouse ESCs. ( E ) Comparison of ChIP-Seq reads densities for H3K4me3 and H3K27me3 between RA-treated and untreated WT cells and between RA-treated and untreated Utx -null cells. The 316 RA-inducible bivalent genes downregulated by UTX loss were used.

Techniques Used: Chromatin Immunoprecipitation

During RA-driven differentiation of human NT2/D1 cells, UTX is responsible for the bivalency resolution and activation of most RA-inducible bivalent HOXA and HOXB genes. ( A ) ChIP-Seq density profiles of H3K27me3 (red) and H3K4me3 (blue) at the human HOXA and HOXB cluster genes in NT2/D1 cells. The vertical axis shows the coverage for histone methylation set at a maximal value of 100. ( B ) Effect of UTX knockdown on the RA-induced changes in H3K27me3, H3K4me3 and H3 levels at the HOXA and HOXB cluster promoters in NT2/D1 cells. shLuc- or shUTX-6-treated cells were cultured in media containing 10 μM RA for 0 or 4 days. Chromatin levels for histone marks were analyzed using a quantitative ChIP assay. PCR values for each time point were normalized to input. The relative occupancy represents the fold change in chromatin levels from day 0 to day 4 (4d/0d). ( C ) Effect of UTX knockdown on the RA-induced changes in the expression levels of HOXA and HOXB cluster genes. shLuc- or shUTX-6-treated NT2/D1 cells were incubated with 10 μM RA for 0 or 6 days. mRNA levels were analyzed using quantitative RT-PCR. Data are presented as the mean ± SEM (error bars) of least three independent experiments. * P
Figure Legend Snippet: During RA-driven differentiation of human NT2/D1 cells, UTX is responsible for the bivalency resolution and activation of most RA-inducible bivalent HOXA and HOXB genes. ( A ) ChIP-Seq density profiles of H3K27me3 (red) and H3K4me3 (blue) at the human HOXA and HOXB cluster genes in NT2/D1 cells. The vertical axis shows the coverage for histone methylation set at a maximal value of 100. ( B ) Effect of UTX knockdown on the RA-induced changes in H3K27me3, H3K4me3 and H3 levels at the HOXA and HOXB cluster promoters in NT2/D1 cells. shLuc- or shUTX-6-treated cells were cultured in media containing 10 μM RA for 0 or 4 days. Chromatin levels for histone marks were analyzed using a quantitative ChIP assay. PCR values for each time point were normalized to input. The relative occupancy represents the fold change in chromatin levels from day 0 to day 4 (4d/0d). ( C ) Effect of UTX knockdown on the RA-induced changes in the expression levels of HOXA and HOXB cluster genes. shLuc- or shUTX-6-treated NT2/D1 cells were incubated with 10 μM RA for 0 or 6 days. mRNA levels were analyzed using quantitative RT-PCR. Data are presented as the mean ± SEM (error bars) of least three independent experiments. * P

Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Methylation, Cell Culture, Polymerase Chain Reaction, Expressing, Incubation, Quantitative RT-PCR

5) Product Images from "H2A.Z.1 crosstalk with H3K56-acetylation controls gliogenesis through the transcription of folate receptor"

Article Title: H2A.Z.1 crosstalk with H3K56-acetylation controls gliogenesis through the transcription of folate receptor

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky585

H2A.Z.1 directly and collaboratively interacts with ASF1a to regulate the acetylation of H3K56. ( A ) E15.5 NPCs were infected with a lentivirus encoding control-shRNA or H2A.Z.1-shRNA and cultured for 3 days. Western blot analyses of H3K56ac, H3K9ac, H3K27ac, H3K4me3 and H3K36me3 expressions in the cultured NPCs. ( B ) Quantification of H3K56ac expression in control and H2A.Z.1 knockdown progenitor cultures. Data are represented as means ± S.E.M. ( n = 3; Student's t-test; * P
Figure Legend Snippet: H2A.Z.1 directly and collaboratively interacts with ASF1a to regulate the acetylation of H3K56. ( A ) E15.5 NPCs were infected with a lentivirus encoding control-shRNA or H2A.Z.1-shRNA and cultured for 3 days. Western blot analyses of H3K56ac, H3K9ac, H3K27ac, H3K4me3 and H3K36me3 expressions in the cultured NPCs. ( B ) Quantification of H3K56ac expression in control and H2A.Z.1 knockdown progenitor cultures. Data are represented as means ± S.E.M. ( n = 3; Student's t-test; * P

Techniques Used: Infection, shRNA, Cell Culture, Western Blot, Expressing

6) Product Images from "The ATP-dependent chromatin remodeler Chd1 is recruited by transcription elongation factors and maintains H3K4me3/H3K36me3 domains at actively transcribed and spliced genes"

Article Title: The ATP-dependent chromatin remodeler Chd1 is recruited by transcription elongation factors and maintains H3K4me3/H3K36me3 domains at actively transcribed and spliced genes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx321

Chd1 occupancy in the wild-type (WT) strain and deletion mutants of candidate recruitment factors. ( A ) Top: the heat map shows Chd1 occupancy in the indicated deletion mutants at the 100 genes where Chd1 occupancy was highest in WT cells. Each row indicates a gene. Chd1 occupancy was measured by counting Chd1 Chromatin immunoprecipitation sequencing (ChIP-seq) reads from the transcription start site (TSS) to transcription termination site (TTS) and normalizing by transcript length and sequencing depth. The level of occupancy of Chd1 is depicted in a green-to-red color scheme (green—low; red—high occupancy of Chd1) after being standardized into a z-score per row. Bottom: boxplots summarize the distribution of Chd1 occupancy values shown above. ( B ) Metagene average occupancy profiles of Chd1 in candidate recruitment factor deletions. The y-axis in the plots shows normalized Chd1 ChIP-seq read density (reads per million, RPM) obtained by averaging the read counts for the most actively transcribed genes, identified based on the occupancy of RNAPII Ser-5P in the WT strain under normal growth conditions (536 genes, see ‘Materials and Methods’ section). The x-axis shows the distance from 1 kb upstream to 2 kb downstream of the TSS. Each plot includes a positive control (Chd1 occupancy measured in the WT strain, blue) and a negative control (a mock ChIP, green) to compare Chd1 occupancy in the selected deletion mutants (red). The dark line is the mean and the shaded envelope indicates the 95% confidence interval for the mean Chd1 ChIP-seq read density. PAF1, CTR1 and SPT4 mutants show a highly significant difference in Chd1 occupancy. The arrows indicate the genomic regions showing the strongest and most significant differences in occupancy. RTF1, SET1 and SET2 mutants show a marginal difference in Chd1 occupancy.
Figure Legend Snippet: Chd1 occupancy in the wild-type (WT) strain and deletion mutants of candidate recruitment factors. ( A ) Top: the heat map shows Chd1 occupancy in the indicated deletion mutants at the 100 genes where Chd1 occupancy was highest in WT cells. Each row indicates a gene. Chd1 occupancy was measured by counting Chd1 Chromatin immunoprecipitation sequencing (ChIP-seq) reads from the transcription start site (TSS) to transcription termination site (TTS) and normalizing by transcript length and sequencing depth. The level of occupancy of Chd1 is depicted in a green-to-red color scheme (green—low; red—high occupancy of Chd1) after being standardized into a z-score per row. Bottom: boxplots summarize the distribution of Chd1 occupancy values shown above. ( B ) Metagene average occupancy profiles of Chd1 in candidate recruitment factor deletions. The y-axis in the plots shows normalized Chd1 ChIP-seq read density (reads per million, RPM) obtained by averaging the read counts for the most actively transcribed genes, identified based on the occupancy of RNAPII Ser-5P in the WT strain under normal growth conditions (536 genes, see ‘Materials and Methods’ section). The x-axis shows the distance from 1 kb upstream to 2 kb downstream of the TSS. Each plot includes a positive control (Chd1 occupancy measured in the WT strain, blue) and a negative control (a mock ChIP, green) to compare Chd1 occupancy in the selected deletion mutants (red). The dark line is the mean and the shaded envelope indicates the 95% confidence interval for the mean Chd1 ChIP-seq read density. PAF1, CTR1 and SPT4 mutants show a highly significant difference in Chd1 occupancy. The arrows indicate the genomic regions showing the strongest and most significant differences in occupancy. RTF1, SET1 and SET2 mutants show a marginal difference in Chd1 occupancy.

Techniques Used: ChIP-sequencing, Chromatin Immunoprecipitation, Sequencing, Positive Control, Negative Control

7) Product Images from "Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells"

Article Title: Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsv013

Heterochromatin clustering in the Zscan4 + cells. (A) Co-immunostaining of ES cells with an HP1α antibody (green) and a Zscan4 antibody (not shown). Red, DAPI. Scale bars, 5 µm. More examples are shown in Supplementary Fig. S2A . (B) Size distribution of nuclear foci stained with an HP1α antibody in the Zscan4 + cells (red bars) and in the Zscan4 − cells (blue bars). Average areas of each focus was 3.6 and 1.5 µm 2 in the Zscan4 + cells and in the Zscan4 − cells, respectively. n = 40. (C) Number distribution of nuclear foci stained with an HP1α antibody in the Zscan4 + cells (red bars) and in the Zscan4 − cells (blue bars). Average numbers of foci in each nucleus were 2.4 and 4.7 in the Zscan4 + cells and in the Zscan4 − cells, respectively. n = 60. See also Supplementary Fig. S6 .
Figure Legend Snippet: Heterochromatin clustering in the Zscan4 + cells. (A) Co-immunostaining of ES cells with an HP1α antibody (green) and a Zscan4 antibody (not shown). Red, DAPI. Scale bars, 5 µm. More examples are shown in Supplementary Fig. S2A . (B) Size distribution of nuclear foci stained with an HP1α antibody in the Zscan4 + cells (red bars) and in the Zscan4 − cells (blue bars). Average areas of each focus was 3.6 and 1.5 µm 2 in the Zscan4 + cells and in the Zscan4 − cells, respectively. n = 40. (C) Number distribution of nuclear foci stained with an HP1α antibody in the Zscan4 + cells (red bars) and in the Zscan4 − cells (blue bars). Average numbers of foci in each nucleus were 2.4 and 4.7 in the Zscan4 + cells and in the Zscan4 − cells, respectively. n = 60. See also Supplementary Fig. S6 .

Techniques Used: Immunostaining, Staining

8) Product Images from "Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns"

Article Title: Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns

Journal: Cell reports

doi: 10.1016/j.celrep.2014.02.017

Set1 protein levels correlate with H3K4 methylation A) ChIP of wild-type or H1017K Set1 to PMA1 and RPS13 . Flag-tagged wild type or H1017K Set1 was immunoprecipitated from chromatin and associated DNA analyzed by PCR. Upper panel shows schematic of genes, with bars indicating PCR products. Fold enrichment was calculated by normalization against values for untagged strain. Error bars represent standard error from biological triplicates. B) Flag-Set1 protein and mRNA levels. Extracts from set1 Δ strains transformed with plasmids expressing Flag-tagged wild type Set1 (lane 1), H1017K (lane 2), Y967A (lane 3), K1007/8A (lane 4) or Y1052F (lane 5). Set1 substitutions were immunoprecipitated with M2-agarose beads, resolved by SDS-PAGE and analyzed by immunoblotting for the Flag-tag. Asterisk marks a cross-reacting band that serves as an internal loading control. Separately, 1/10 of the extract used for immunoprecipitation was resolved by SDS-PAGE and analyzed by blotting for TBP (as an input control), H3K4me3, H3K4me2 and Histone H3. At bottom, 1 µg of RNA from the strains was analyzed by RT-PCR with primers specific for SET1 or ADH1 , -RT is a control reaction lacking reverse transcriptase. C) Stabilization of Set1 mutants by wildtype Set1. Flag-tagged wild-type (lanes 2, 4 and 6), H1017K (lanes 3, 5 and 7), ΔNSET (lanes 8 and 11), ΔSET (lanes 9 and 12) or ΔPSET (lanes 10 and 13) Set1 was expressed and immunoprecipitated from set1 Δ (lanes 1–3 and 6–10) or SET1 (lanes 4, 5 and 11–13) strains and analyzed as in part B. D) Flag-Set1 protein levels in RTF1 deletion. Extracts were prepared from set1 Δ (lanes 1 and 2) or set1 Δ, rtf1 Δ (lanes 3 and 4) cells expressing wild type (lanes 1 and 3) or H1017K (lanes 2 and 4) Flag-tagged Set1 and analyzed as in panel B.
Figure Legend Snippet: Set1 protein levels correlate with H3K4 methylation A) ChIP of wild-type or H1017K Set1 to PMA1 and RPS13 . Flag-tagged wild type or H1017K Set1 was immunoprecipitated from chromatin and associated DNA analyzed by PCR. Upper panel shows schematic of genes, with bars indicating PCR products. Fold enrichment was calculated by normalization against values for untagged strain. Error bars represent standard error from biological triplicates. B) Flag-Set1 protein and mRNA levels. Extracts from set1 Δ strains transformed with plasmids expressing Flag-tagged wild type Set1 (lane 1), H1017K (lane 2), Y967A (lane 3), K1007/8A (lane 4) or Y1052F (lane 5). Set1 substitutions were immunoprecipitated with M2-agarose beads, resolved by SDS-PAGE and analyzed by immunoblotting for the Flag-tag. Asterisk marks a cross-reacting band that serves as an internal loading control. Separately, 1/10 of the extract used for immunoprecipitation was resolved by SDS-PAGE and analyzed by blotting for TBP (as an input control), H3K4me3, H3K4me2 and Histone H3. At bottom, 1 µg of RNA from the strains was analyzed by RT-PCR with primers specific for SET1 or ADH1 , -RT is a control reaction lacking reverse transcriptase. C) Stabilization of Set1 mutants by wildtype Set1. Flag-tagged wild-type (lanes 2, 4 and 6), H1017K (lanes 3, 5 and 7), ΔNSET (lanes 8 and 11), ΔSET (lanes 9 and 12) or ΔPSET (lanes 10 and 13) Set1 was expressed and immunoprecipitated from set1 Δ (lanes 1–3 and 6–10) or SET1 (lanes 4, 5 and 11–13) strains and analyzed as in part B. D) Flag-Set1 protein levels in RTF1 deletion. Extracts were prepared from set1 Δ (lanes 1 and 2) or set1 Δ, rtf1 Δ (lanes 3 and 4) cells expressing wild type (lanes 1 and 3) or H1017K (lanes 2 and 4) Flag-tagged Set1 and analyzed as in panel B.

Techniques Used: Methylation, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Transformation Assay, Expressing, SDS Page, FLAG-tag, Reverse Transcription Polymerase Chain Reaction

9) Product Images from "C/EBP? deregulation results in differentiation arrest in acute myeloid leukemia"

Article Title: C/EBP? deregulation results in differentiation arrest in acute myeloid leukemia

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI65102

Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in vivo.
Figure Legend Snippet: Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in vivo.

Techniques Used: In Vivo

DAC treatment restores the C/EBPα-C/EBPγ balance and promotes differentiation of primary human AML samples characterized by C/EBPα silencing and C/EBPγ upregulation in vitro.
Figure Legend Snippet: DAC treatment restores the C/EBPα-C/EBPγ balance and promotes differentiation of primary human AML samples characterized by C/EBPα silencing and C/EBPγ upregulation in vitro.

Techniques Used: In Vitro

C/EBPγ is downregulated with neutrophilic differentiation, whereas constitutive expression of C/EBPγ blocks G-CSF–induced neutrophilic differentiation.
Figure Legend Snippet: C/EBPγ is downregulated with neutrophilic differentiation, whereas constitutive expression of C/EBPγ blocks G-CSF–induced neutrophilic differentiation.

Techniques Used: Expressing

Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in cell culture.
Figure Legend Snippet: Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in cell culture.

Techniques Used: Cell Culture

Downregulation of C/EBPγ in human AML cells restores neutrophilic differentiation in vivo.
Figure Legend Snippet: Downregulation of C/EBPγ in human AML cells restores neutrophilic differentiation in vivo.

Techniques Used: In Vivo

10) Product Images from "Dynamic acetylation of all lysine-4 trimethylated histone H3 is evolutionarily conserved and mediated by p300/CBP"

Article Title: Dynamic acetylation of all lysine-4 trimethylated histone H3 is evolutionarily conserved and mediated by p300/CBP

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1100099108

The most highly acetylated histone H3 in TSA-treated cells is trimethylated at lysine 4. ( A , i ) Purified free histones from untreated (lanes 1–3) or TSA-treated (33 nM, 30 min; lanes 4–6) C3H 10T1/2 cells were immunodepleted with antibodies against H3K4me3 (lanes 2 and 5) or H3K9ac (lanes 3 and 6). Unbound material was resolved by SDS/PAGE and subjected to Western blotting with antibodies against total H3 ( Bottom ), H3K9ac ( Middle ), or H3K4me3 ( Top ). (Lanes 1 and 4: input material before immunodepletion.) ( A , ii ) Blots from A , i were quantified using ImageJ and normalized to total H3. Data (mean of three biological replicates, plotted ± SEM) are presented relative to input under untreated or TSA-treated conditions (lanes 1 and 4 from A , i ). ( B , i ) Purified free histones from control (lanes 1 and 2) or TSA-treated (33 nM, 30 min; lanes 3 and 4) C3H 10T1/2 cells were immunodepleted with anti-H3K4me3 antibody, resolved on acid–urea gels, and subjected to Western blotting using total H3, anti-H3K4me3, or anti-acetyl H3 antibodies as indicated on the Left of each panel. (Lanes 1 and 3: input material; lanes 2 and 4: immunodepleted fraction.) ( B , ii ) Total staining for each modification in blots from B , i was quantified using ImageJ, with normalization to total H3 levels. Data (mean of three biological replicates, plotted ± SEM) are presented relative to input under untreated or TSA-treated conditions (lanes 1 and 3 from B , i ).
Figure Legend Snippet: The most highly acetylated histone H3 in TSA-treated cells is trimethylated at lysine 4. ( A , i ) Purified free histones from untreated (lanes 1–3) or TSA-treated (33 nM, 30 min; lanes 4–6) C3H 10T1/2 cells were immunodepleted with antibodies against H3K4me3 (lanes 2 and 5) or H3K9ac (lanes 3 and 6). Unbound material was resolved by SDS/PAGE and subjected to Western blotting with antibodies against total H3 ( Bottom ), H3K9ac ( Middle ), or H3K4me3 ( Top ). (Lanes 1 and 4: input material before immunodepletion.) ( A , ii ) Blots from A , i were quantified using ImageJ and normalized to total H3. Data (mean of three biological replicates, plotted ± SEM) are presented relative to input under untreated or TSA-treated conditions (lanes 1 and 4 from A , i ). ( B , i ) Purified free histones from control (lanes 1 and 2) or TSA-treated (33 nM, 30 min; lanes 3 and 4) C3H 10T1/2 cells were immunodepleted with anti-H3K4me3 antibody, resolved on acid–urea gels, and subjected to Western blotting using total H3, anti-H3K4me3, or anti-acetyl H3 antibodies as indicated on the Left of each panel. (Lanes 1 and 3: input material; lanes 2 and 4: immunodepleted fraction.) ( B , ii ) Total staining for each modification in blots from B , i was quantified using ImageJ, with normalization to total H3 levels. Data (mean of three biological replicates, plotted ± SEM) are presented relative to input under untreated or TSA-treated conditions (lanes 1 and 3 from B , i ).

Techniques Used: Purification, SDS Page, Western Blot, Staining, Modification

Dynamic acetylation of all H3K4me3 is conserved in higher eukaryotes. ( A ) Dynamic histone acetylation is mediated by lysine acetyltransferases (KATs) and histone deacetylases (HDACs). TSA inhibits classes I, II, and IV HDACs to promote hyperacetylation of H3K4me3. ( B ) Human WI 38 fibroblasts (lanes 1–6), murine C3H 10T1/2 fibroblasts (lanes 7–12), or Drosophila S2 cells (lanes 13–18) were treated with trichostatin A (TSA) (33 nM; lanes 2–4, 8–10, and 14–16) or nicotinamide (NAM) (20 μM; lanes 5, 6, 11, 12, 17, and 18) as indicated. Histones were separated on acid–urea (AU) gels on which acetylation incrementally retards migration to produce the “ladder” of bands seen. Numbers indicate extent of acetylation, 0 referring to nonacetylated H3. Different exposures are shown for H3K4me3 in mouse and Drosophila ( Top for comparison). The H3K9me2 (fourth panel down) signal in mouse has been overexposed to allow detection of low levels of this modification in Drosophila . (Lanes 1, 7, and 13: control untreated cells.)
Figure Legend Snippet: Dynamic acetylation of all H3K4me3 is conserved in higher eukaryotes. ( A ) Dynamic histone acetylation is mediated by lysine acetyltransferases (KATs) and histone deacetylases (HDACs). TSA inhibits classes I, II, and IV HDACs to promote hyperacetylation of H3K4me3. ( B ) Human WI 38 fibroblasts (lanes 1–6), murine C3H 10T1/2 fibroblasts (lanes 7–12), or Drosophila S2 cells (lanes 13–18) were treated with trichostatin A (TSA) (33 nM; lanes 2–4, 8–10, and 14–16) or nicotinamide (NAM) (20 μM; lanes 5, 6, 11, 12, 17, and 18) as indicated. Histones were separated on acid–urea (AU) gels on which acetylation incrementally retards migration to produce the “ladder” of bands seen. Numbers indicate extent of acetylation, 0 referring to nonacetylated H3. Different exposures are shown for H3K4me3 in mouse and Drosophila ( Top for comparison). The H3K9me2 (fourth panel down) signal in mouse has been overexposed to allow detection of low levels of this modification in Drosophila . (Lanes 1, 7, and 13: control untreated cells.)

Techniques Used: Migration, Modification

11) Product Images from "Contribution of Myocyte Enhancer Factor 2 Family Transcription Factors to BZLF1 Expression in Epstein-Barr Virus Reactivation from Latency"

Article Title: Contribution of Myocyte Enhancer Factor 2 Family Transcription Factors to BZLF1 Expression in Epstein-Barr Virus Reactivation from Latency

Journal: Journal of Virology

doi: 10.1128/JVI.01002-13

Library screening identified factors involved in BZLF1 promoter activation. (A) HEK293T cells were transfected with 10 ng of the reporter plasmid pZp-luc, 1 ng of pCMV-RL, and 100 ng of expression plasmids for the indicated genes. (B) As in panel A, HEK293T
Figure Legend Snippet: Library screening identified factors involved in BZLF1 promoter activation. (A) HEK293T cells were transfected with 10 ng of the reporter plasmid pZp-luc, 1 ng of pCMV-RL, and 100 ng of expression plasmids for the indicated genes. (B) As in panel A, HEK293T

Techniques Used: Library Screening, Activation Assay, Transfection, Plasmid Preparation, Expressing

Effects of b-Zip, SP1/KLF, and MEF2 family transcription factors on the BZLF1 promoter. (A) b-Zip family transcription factor activation of the BZLF1 promoter. HEK293T cells were transfected with 10 ng of the reporter plasmid pZp-luc or its derivatives,
Figure Legend Snippet: Effects of b-Zip, SP1/KLF, and MEF2 family transcription factors on the BZLF1 promoter. (A) b-Zip family transcription factor activation of the BZLF1 promoter. HEK293T cells were transfected with 10 ng of the reporter plasmid pZp-luc or its derivatives,

Techniques Used: Activation Assay, Transfection, Plasmid Preparation

12) Product Images from "Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch"

Article Title: Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch

Journal: Chromosoma

doi: 10.1007/s00412-010-0258-9

The GRC and meiotic recombination. a Spread nuclei immunostained for DAPI ( blue ), γH2AX ( green ) and SYCP3 ( red ). After metaphase I, the GRC becomes strongly positive for γH2AX. The GRC is boxed . Bar represents 5 μm. Sec spc secondary spermatocyte. b Spermatocyte spread nucleus immunostained for SYCP3 ( red ), RAD51 ( green ) and DAPI ( blue ). The GRC is boxed . Most RAD51 foci localise on axial elements of the SC. The bottom image represents a magnification of the area of the boxed GRC. Few small RAD51 foci are visible on the GRC. Bar represents 10 μm. c Spermatocyte spread nucleus immunostained for SYCP3 ( red ), MLH1 ( green ) and DAPI ( blue ). The area of the GRC is boxed . The bottom image represents a magnification of the area of the boxed GRC, and shows the presence of a MLH1 focus on the GRC. Bar represents 10 μm
Figure Legend Snippet: The GRC and meiotic recombination. a Spread nuclei immunostained for DAPI ( blue ), γH2AX ( green ) and SYCP3 ( red ). After metaphase I, the GRC becomes strongly positive for γH2AX. The GRC is boxed . Bar represents 5 μm. Sec spc secondary spermatocyte. b Spermatocyte spread nucleus immunostained for SYCP3 ( red ), RAD51 ( green ) and DAPI ( blue ). The GRC is boxed . Most RAD51 foci localise on axial elements of the SC. The bottom image represents a magnification of the area of the boxed GRC. Few small RAD51 foci are visible on the GRC. Bar represents 10 μm. c Spermatocyte spread nucleus immunostained for SYCP3 ( red ), MLH1 ( green ) and DAPI ( blue ). The area of the GRC is boxed . The bottom image represents a magnification of the area of the boxed GRC, and shows the presence of a MLH1 focus on the GRC. Bar represents 10 μm

Techniques Used: Size-exclusion Chromatography

13) Product Images from "Identification of linc-NeD125, a novel long non coding RNA that hosts miR-125b-1 and negatively controls proliferation of human neuroblastoma cells"

Article Title: Identification of linc-NeD125, a novel long non coding RNA that hosts miR-125b-1 and negatively controls proliferation of human neuroblastoma cells

Journal: RNA Biology

doi: 10.1080/15476286.2015.1096488

Phylogenetic analysis inferred from multiple sequence alignment of mature linc-NeD125. ( A ) The dendrogram shows grouping of 18 placental mammal species based on the Neighbor Joining (NJ) agglomeration method applied to the distance matrix of percent divergence
Figure Legend Snippet: Phylogenetic analysis inferred from multiple sequence alignment of mature linc-NeD125. ( A ) The dendrogram shows grouping of 18 placental mammal species based on the Neighbor Joining (NJ) agglomeration method applied to the distance matrix of percent divergence

Techniques Used: Sequencing

Role of linc-NeD125 in apoptosis. ( A ) Left panel: qRT-PCR analysis of Bcl - 2 and Bax mRNA levels in linc-NeD125 and GFP overexpressing BE(2)-C cell lines upon 3 days of doxycycline-treatment. –Dox samples were set as 1. Gapdh mRNA was used as a
Figure Legend Snippet: Role of linc-NeD125 in apoptosis. ( A ) Left panel: qRT-PCR analysis of Bcl - 2 and Bax mRNA levels in linc-NeD125 and GFP overexpressing BE(2)-C cell lines upon 3 days of doxycycline-treatment. –Dox samples were set as 1. Gapdh mRNA was used as a

Techniques Used: Quantitative RT-PCR

Linc-NeD125 transcriptional regulation. ( A ) Identification of linc-NeD125 core promoter by luciferase assay. Upper panel: the luciferase-based reporter construct, containing the 1.5-kb region upstream of the linc-NeD125 TSS (−1500/+1), and its
Figure Legend Snippet: Linc-NeD125 transcriptional regulation. ( A ) Identification of linc-NeD125 core promoter by luciferase assay. Upper panel: the luciferase-based reporter construct, containing the 1.5-kb region upstream of the linc-NeD125 TSS (−1500/+1), and its

Techniques Used: Luciferase, Construct

For figure legend, see page . Figure 7 (see previous page). Role of Linc-NeD125 in cell growth. ( A ) Assessment of linc-NeD125 overexpression in BE(2)-C stable cell lines upon doxycycline treatment. qRT-PCR analysis of linc-NeD125 levels in untreated (-Dox)
Figure Legend Snippet: For figure legend, see page . Figure 7 (see previous page). Role of Linc-NeD125 in cell growth. ( A ) Assessment of linc-NeD125 overexpression in BE(2)-C stable cell lines upon doxycycline treatment. qRT-PCR analysis of linc-NeD125 levels in untreated (-Dox)

Techniques Used: Polyacrylamide Gel Electrophoresis, Over Expression, Stable Transfection, Quantitative RT-PCR

Molecular characterization of linc-NeD125. ( A ) Linc-NeD125 sub-cellular localization. Proliferating (-RA) or 6-days RA-treated (+RA) BE(2)-C cells were fractionated into nuclear (gray bars) and cytoplasmic (black bars) fractions and linc-NeD125 transcript
Figure Legend Snippet: Molecular characterization of linc-NeD125. ( A ) Linc-NeD125 sub-cellular localization. Proliferating (-RA) or 6-days RA-treated (+RA) BE(2)-C cells were fractionated into nuclear (gray bars) and cytoplasmic (black bars) fractions and linc-NeD125 transcript

Techniques Used:

Linc-NeD125 post-transcriptional regulation. ( A ) Untreated (-RA, left panel) or 4 days RA-treated (+RA, right panel) BE(2)-C cells were maintained in the presence of Actinomycin D (ActD). Linc-NeD125 transcript was analyzed by qRT-PCR at the indicated
Figure Legend Snippet: Linc-NeD125 post-transcriptional regulation. ( A ) Untreated (-RA, left panel) or 4 days RA-treated (+RA, right panel) BE(2)-C cells were maintained in the presence of Actinomycin D (ActD). Linc-NeD125 transcript was analyzed by qRT-PCR at the indicated

Techniques Used: Quantitative RT-PCR

14) Product Images from "Regulation of hippocampal H3 histone methylation by acute and chronic stress"

Article Title: Regulation of hippocampal H3 histone methylation by acute and chronic stress

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0911143106

Levels of H3K9me1 immunoreactivity after acute stress in the DG ( A ) and CA1 ( B ). *, P
Figure Legend Snippet: Levels of H3K9me1 immunoreactivity after acute stress in the DG ( A ) and CA1 ( B ). *, P

Techniques Used:

15) Product Images from "Characterization of Drosophila melanogaster JmjC+N histone demethylases"

Article Title: Characterization of Drosophila melanogaster JmjC+N histone demethylases

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn098

dJMJD2(1)/CG15835 demethylates H3K9me3 and H3K36me3. (A) A CG15835-Flag fusion protein was over-expressed in Drosophila S2 cells and transfected cells were stained with αFlag (shown in green), and αH3K4me3, αH3K9me3, αH3K27me3 or αH3K36me3 antibodies (shown in red). DNA was stained with DAPI (shown in blue). Arrows indicate cells over-expressing CG15835. (B) Similar experiments as those described in (A), but with a mutated CG15835 form carrying a single-point mutation at the catalytic JmjC domain, H195A, which perturbs co-ordination of Fe(II) and impairs enzymatic activity.
Figure Legend Snippet: dJMJD2(1)/CG15835 demethylates H3K9me3 and H3K36me3. (A) A CG15835-Flag fusion protein was over-expressed in Drosophila S2 cells and transfected cells were stained with αFlag (shown in green), and αH3K4me3, αH3K9me3, αH3K27me3 or αH3K36me3 antibodies (shown in red). DNA was stained with DAPI (shown in blue). Arrows indicate cells over-expressing CG15835. (B) Similar experiments as those described in (A), but with a mutated CG15835 form carrying a single-point mutation at the catalytic JmjC domain, H195A, which perturbs co-ordination of Fe(II) and impairs enzymatic activity.

Techniques Used: Transfection, Staining, Expressing, Mutagenesis, Activity Assay

dJARID1/Lid demethylates H3K4me3. (A) A Lid-Flag fusion protein was over-expressed in Drosophila S2-cells and transfected cells were stained with αFlag (shown in green), and αH3K4me3, αH3K9me3, αH3K27me3 or αH3K36me3 antibodies (shown in red). DNA was stained with DAPI (shown in blue). Arrows indicate cells over-expressing dJARID1/Lid. (B) Polytene chromosomes from control wild-type larvae (+/+) and homozygous lid 12367 /lid 12367 mutant larvae stained with αH3K4me3 are shown. DNA was stained with DAPI.
Figure Legend Snippet: dJARID1/Lid demethylates H3K4me3. (A) A Lid-Flag fusion protein was over-expressed in Drosophila S2-cells and transfected cells were stained with αFlag (shown in green), and αH3K4me3, αH3K9me3, αH3K27me3 or αH3K36me3 antibodies (shown in red). DNA was stained with DAPI (shown in blue). Arrows indicate cells over-expressing dJARID1/Lid. (B) Polytene chromosomes from control wild-type larvae (+/+) and homozygous lid 12367 /lid 12367 mutant larvae stained with αH3K4me3 are shown. DNA was stained with DAPI.

Techniques Used: Transfection, Staining, Expressing, Mutagenesis

Over-expression of dJMJD2(1)/CG15835 induces spreading of HP1 into euchromatin. (A) S2 cells transfected with CG15835-Flag were stained with αFlag (shown in green) and αHP1 antibodies (shown in red). DNA was stained with DAPI (shown in blue). In untransfected cells, HP1 localizes mostly to a single intense spot in the nuclei (small arrow heads), which corresponds to the heterochromatic chromocentre. Cells over-expressing CG15835 (big arrow heads) show a diffuse nuclear distribution of HP1 with no specific enrichment at the chromocentre. (B) Polytene chromosomes from UAS GAL4 -CG15835-Flag; act5C -GAL4 larvae, where CG15835 is ubiquitously expressed (see text for details) (panels dJMJD2(1)/CG15835), and control wild-type larvae (panels +/+), were stained with specific αHP1 antibodies. DNA was stained with DAPI. Arrows indicate the position of the heterochromatic chromocentre.
Figure Legend Snippet: Over-expression of dJMJD2(1)/CG15835 induces spreading of HP1 into euchromatin. (A) S2 cells transfected with CG15835-Flag were stained with αFlag (shown in green) and αHP1 antibodies (shown in red). DNA was stained with DAPI (shown in blue). In untransfected cells, HP1 localizes mostly to a single intense spot in the nuclei (small arrow heads), which corresponds to the heterochromatic chromocentre. Cells over-expressing CG15835 (big arrow heads) show a diffuse nuclear distribution of HP1 with no specific enrichment at the chromocentre. (B) Polytene chromosomes from UAS GAL4 -CG15835-Flag; act5C -GAL4 larvae, where CG15835 is ubiquitously expressed (see text for details) (panels dJMJD2(1)/CG15835), and control wild-type larvae (panels +/+), were stained with specific αHP1 antibodies. DNA was stained with DAPI. Arrows indicate the position of the heterochromatic chromocentre.

Techniques Used: Over Expression, Transfection, Staining, Expressing

dJMJD2(1)/CG15835 localizes at euchromatin and regulates H3K36me3. (A) Polytene chromosomes from UAS GAL4 -CG15835-Flag; act5C -GAL4 larvae, where CG15835-Flag is ubiquitously expressed, (panels dJMJD2(1)/CG15835), and control wild-type larvae (panels +/+), were stained with αH3K9me3 antibodies. (B) Similar experiments as those described in (A), but polytene chromosomes were stained αH3K36me3 antibodies. (C) Polytene chromosomes from UAS GAL4 -CG15835-Flag; act5C -GAL4 larvae were stained with αFlag antibodies to determine the localization of the CG15835-Flag fused protein. DNA was stained with DAPI. Arrows indicate the position of the heterochromatic chromocentre.
Figure Legend Snippet: dJMJD2(1)/CG15835 localizes at euchromatin and regulates H3K36me3. (A) Polytene chromosomes from UAS GAL4 -CG15835-Flag; act5C -GAL4 larvae, where CG15835-Flag is ubiquitously expressed, (panels dJMJD2(1)/CG15835), and control wild-type larvae (panels +/+), were stained with αH3K9me3 antibodies. (B) Similar experiments as those described in (A), but polytene chromosomes were stained αH3K36me3 antibodies. (C) Polytene chromosomes from UAS GAL4 -CG15835-Flag; act5C -GAL4 larvae were stained with αFlag antibodies to determine the localization of the CG15835-Flag fused protein. DNA was stained with DAPI. Arrows indicate the position of the heterochromatic chromocentre.

Techniques Used: Staining

16) Product Images from "Two novel NAC transcription factors regulate gene expression and flowering time by associating with the histone demethylase JMJ14"

Article Title: Two novel NAC transcription factors regulate gene expression and flowering time by associating with the histone demethylase JMJ14

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku1382

Effect of jmj14 and NAC050/052-RNAi on H3K4me3 as determined by ChIP-seq. (A and B) Venn diagrams showing the overlap between the H3K4me3 hypermethylated genes in jmj14 and the upregulated (A) or downregulated genes (B) in jmj14 and NAC050/052-RNAi plants. (C) Box plot showing the effect of jmj14 and NAC050/052-RNAi on gene expression for 494 co-upregulated genes in jmj14 and nac050/052-RNAi . (D) H3K4me3 of the 494 overlapping upregulated genes in jmj14 and NAC050/052-RNAi plants was plotted for the transcription regions along with the 1-kb upstream and downstream flanking regions. The y -axis indicates the normalized reads number. (E) H3K4me3 hypermethylated genes identified by ChIP-seq were confirmed by ChIP-PCR in the WT, jmj14 and NAC050/052-RNAi plants. Diagrams show positions of all DNA fragments amplified in the ChIP-PCR assay. The hypermethylated sites include AT1G11540-A, AT2G18193-A and AT5G27940-A . The sites that are adjacent to the H3K4me3 hypermethylated regions were used as negative controls. These sites are AT1G11540-B, AT2G18193-B, AT5G27940-B and AT5G27940-C . Two-week-old seedlings were used for ChIP-seq and ChIP-PCR.
Figure Legend Snippet: Effect of jmj14 and NAC050/052-RNAi on H3K4me3 as determined by ChIP-seq. (A and B) Venn diagrams showing the overlap between the H3K4me3 hypermethylated genes in jmj14 and the upregulated (A) or downregulated genes (B) in jmj14 and NAC050/052-RNAi plants. (C) Box plot showing the effect of jmj14 and NAC050/052-RNAi on gene expression for 494 co-upregulated genes in jmj14 and nac050/052-RNAi . (D) H3K4me3 of the 494 overlapping upregulated genes in jmj14 and NAC050/052-RNAi plants was plotted for the transcription regions along with the 1-kb upstream and downstream flanking regions. The y -axis indicates the normalized reads number. (E) H3K4me3 hypermethylated genes identified by ChIP-seq were confirmed by ChIP-PCR in the WT, jmj14 and NAC050/052-RNAi plants. Diagrams show positions of all DNA fragments amplified in the ChIP-PCR assay. The hypermethylated sites include AT1G11540-A, AT2G18193-A and AT5G27940-A . The sites that are adjacent to the H3K4me3 hypermethylated regions were used as negative controls. These sites are AT1G11540-B, AT2G18193-B, AT5G27940-B and AT5G27940-C . Two-week-old seedlings were used for ChIP-seq and ChIP-PCR.

Techniques Used: Chromatin Immunoprecipitation, Expressing, Polymerase Chain Reaction, Amplification

17) Product Images from "Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch"

Article Title: Meiotic silencing and fragmentation of the male germline restricted chromosome in zebra finch

Journal: Chromosoma

doi: 10.1007/s00412-010-0258-9

The GRC throughout meiotic prophase in zebra finch spermatocytes. a Overview of the different stages of meiotic prophase I of zebra finch spermatocytes. The top panel shows the DAPI staining of the different nuclei, the lower panel shows the corresponding nuclei stained for SYCP3 ( red ), the bottom panel shows the same nuclei stained for both SYCP3 ( red ) and γH2AX ( green ). The position of the GRC is indicated by a box . Bar represents 10 μm. b , c Spermatocyte spread nuclei immunostained for SYCP3 ( red ) and SYCP1 ( green ). The GRC is boxed . Enlargement and the schematic drawing of GRC are shown. No ( b ) or a small fragment ( c ) of SYCP1 signal is present on the GRC. Bar represents 5 μm
Figure Legend Snippet: The GRC throughout meiotic prophase in zebra finch spermatocytes. a Overview of the different stages of meiotic prophase I of zebra finch spermatocytes. The top panel shows the DAPI staining of the different nuclei, the lower panel shows the corresponding nuclei stained for SYCP3 ( red ), the bottom panel shows the same nuclei stained for both SYCP3 ( red ) and γH2AX ( green ). The position of the GRC is indicated by a box . Bar represents 10 μm. b , c Spermatocyte spread nuclei immunostained for SYCP3 ( red ) and SYCP1 ( green ). The GRC is boxed . Enlargement and the schematic drawing of GRC are shown. No ( b ) or a small fragment ( c ) of SYCP1 signal is present on the GRC. Bar represents 5 μm

Techniques Used: Staining

The GRC and meiotic recombination. a Spread nuclei immunostained for DAPI ( blue ), γH2AX ( green ) and SYCP3 ( red ). After metaphase I, the GRC becomes strongly positive for γH2AX. The GRC is boxed . Bar represents 5 μm. Sec spc secondary spermatocyte. b Spermatocyte spread nucleus immunostained for SYCP3 ( red ), RAD51 ( green ) and DAPI ( blue ). The GRC is boxed . Most RAD51 foci localise on axial elements of the SC. The bottom image represents a magnification of the area of the boxed GRC. Few small RAD51 foci are visible on the GRC. Bar represents 10 μm. c Spermatocyte spread nucleus immunostained for SYCP3 ( red ), MLH1 ( green ) and DAPI ( blue ). The area of the GRC is boxed . The bottom image represents a magnification of the area of the boxed GRC, and shows the presence of a MLH1 focus on the GRC. Bar represents 10 μm
Figure Legend Snippet: The GRC and meiotic recombination. a Spread nuclei immunostained for DAPI ( blue ), γH2AX ( green ) and SYCP3 ( red ). After metaphase I, the GRC becomes strongly positive for γH2AX. The GRC is boxed . Bar represents 5 μm. Sec spc secondary spermatocyte. b Spermatocyte spread nucleus immunostained for SYCP3 ( red ), RAD51 ( green ) and DAPI ( blue ). The GRC is boxed . Most RAD51 foci localise on axial elements of the SC. The bottom image represents a magnification of the area of the boxed GRC. Few small RAD51 foci are visible on the GRC. Bar represents 10 μm. c Spermatocyte spread nucleus immunostained for SYCP3 ( red ), MLH1 ( green ) and DAPI ( blue ). The area of the GRC is boxed . The bottom image represents a magnification of the area of the boxed GRC, and shows the presence of a MLH1 focus on the GRC. Bar represents 10 μm

Techniques Used: Size-exclusion Chromatography

The GRC is silenced during early meiotic prophase. a – d Preleptotene spermatocyte spread nuclei. Bar represents 5 μm. a Top panel shows early and late preleptotene nuclei stained for SYCP3 ( red ) and DAPI ( blue ). The DAPI dense GRC localisation in late preleptotene stages is indicated with an asterisk ; the lower panel shows only the DAPI staining. The early preleptotene nucleus shows no heterochromatic area, indicating that the DDB has not yet formed. The formation of a few SYCP3 axial element fragments in all nuclei marks the preleptotene stage. b The top panel shows a mid and late preleptotene nucleus stained for SYCP3 ( red ) and DAPI ( blue ). The asterisk indicates the DAPI dense GRC. The lower panel shows the same nuclei, but immunostained for γH2AX ( green ), indicative of the presence of the first DNA double strand breaks that more or less colocalise with SYCP3. c , d The top panel shows early and late preleptotene stages with DAPI staining. The asterisk indicates the DAPI dense GRC. The lower panel shows the corresponding nuclei stained for H3K9me3 ( c ) and macroH2A ( d ). Early preleptotene nuclei lack intense H3K9me3 ( c ) and macroH2A ( d ) staining, and no DDB is observed. e The top panel shows DAPI staining of the different nuclei, the middle panel shows the corresponding nuclei stained for H3K9me3 ( red ) and the bottom panel shows the merge. In preleptotene, a faint staining is present throughout the nucleus. In all consecutive meiotic prophase stadia, the GRC is positive for H3K9me3. Sec spc secondary spermatocyte. Bar represents 5 μm
Figure Legend Snippet: The GRC is silenced during early meiotic prophase. a – d Preleptotene spermatocyte spread nuclei. Bar represents 5 μm. a Top panel shows early and late preleptotene nuclei stained for SYCP3 ( red ) and DAPI ( blue ). The DAPI dense GRC localisation in late preleptotene stages is indicated with an asterisk ; the lower panel shows only the DAPI staining. The early preleptotene nucleus shows no heterochromatic area, indicating that the DDB has not yet formed. The formation of a few SYCP3 axial element fragments in all nuclei marks the preleptotene stage. b The top panel shows a mid and late preleptotene nucleus stained for SYCP3 ( red ) and DAPI ( blue ). The asterisk indicates the DAPI dense GRC. The lower panel shows the same nuclei, but immunostained for γH2AX ( green ), indicative of the presence of the first DNA double strand breaks that more or less colocalise with SYCP3. c , d The top panel shows early and late preleptotene stages with DAPI staining. The asterisk indicates the DAPI dense GRC. The lower panel shows the corresponding nuclei stained for H3K9me3 ( c ) and macroH2A ( d ). Early preleptotene nuclei lack intense H3K9me3 ( c ) and macroH2A ( d ) staining, and no DDB is observed. e The top panel shows DAPI staining of the different nuclei, the middle panel shows the corresponding nuclei stained for H3K9me3 ( red ) and the bottom panel shows the merge. In preleptotene, a faint staining is present throughout the nucleus. In all consecutive meiotic prophase stadia, the GRC is positive for H3K9me3. Sec spc secondary spermatocyte. Bar represents 5 μm

Techniques Used: Staining, Size-exclusion Chromatography

18) Product Images from "Characterisation of maturation of photoreceptor cell subtypes during zebrafish retinal development"

Article Title: Characterisation of maturation of photoreceptor cell subtypes during zebrafish retinal development

Journal: Biology Open

doi: 10.1242/bio.036632

Polarity proteins show differential expression at the initial stages of PRC maturation. Confocal images of the PRC layer in retinal sections of Tg(rasGFP) embryos showing the plasma membrane in green and the respective polarity protein. (A) Crb2a and PrkC antibody staining of embryos at 51 hpf in magenta. (B) PrkC (magenta) and Crb2a (cyan) antibody staining of embryos at 59 hpf. (C) Crb2b antibody staining of embryos at 59 hpf and 63 hpf in magenta. Dashed lines mark the level of the OLM and arrowheads highlight antibody staining. Scale bars: 5 µm.
Figure Legend Snippet: Polarity proteins show differential expression at the initial stages of PRC maturation. Confocal images of the PRC layer in retinal sections of Tg(rasGFP) embryos showing the plasma membrane in green and the respective polarity protein. (A) Crb2a and PrkC antibody staining of embryos at 51 hpf in magenta. (B) PrkC (magenta) and Crb2a (cyan) antibody staining of embryos at 59 hpf. (C) Crb2b antibody staining of embryos at 59 hpf and 63 hpf in magenta. Dashed lines mark the level of the OLM and arrowheads highlight antibody staining. Scale bars: 5 µm.

Techniques Used: Expressing, Staining

19) Product Images from "AOF1 is a histone H3K4 demethylase possessing demethylase activity-independent repression function"

Article Title: AOF1 is a histone H3K4 demethylase possessing demethylase activity-independent repression function

Journal: Cell research

doi: 10.1038/cr.2010.12

AOF1 acts as a mono- and di-H3K4 demethylase in cells. (A) Diagram showing point and deletion mutants of AOF1. (B) Flag-tagged AOF1 and its mutants were transfected into HeLa cells and the demethylase activity was detected by immunofuorescence using various methylated H3-specifc antibodies. LSD1 served as a positive control for H3K4me2 demethylase activity. Arrows mark the cells in which the proteins of interest were expressed. Note that reduced levels of H3K4me1 and H3K4me2 were observed in cells expressing the wild-type Flag-AOF1 but not in cells expressing AOF1 K667A mutant and deletion mutants.
Figure Legend Snippet: AOF1 acts as a mono- and di-H3K4 demethylase in cells. (A) Diagram showing point and deletion mutants of AOF1. (B) Flag-tagged AOF1 and its mutants were transfected into HeLa cells and the demethylase activity was detected by immunofuorescence using various methylated H3-specifc antibodies. LSD1 served as a positive control for H3K4me2 demethylase activity. Arrows mark the cells in which the proteins of interest were expressed. Note that reduced levels of H3K4me1 and H3K4me2 were observed in cells expressing the wild-type Flag-AOF1 but not in cells expressing AOF1 K667A mutant and deletion mutants.

Techniques Used: Transfection, Activity Assay, Methylation, Positive Control, Expressing, Mutagenesis

20) Product Images from "Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem"

Article Title: Genome-wide mapping of histone H3 lysine 4 trimethylation in Eucalyptus grandis developing xylem

Journal: BMC Plant Biology

doi: 10.1186/s12870-015-0499-0

Association of H3K4me3 secondary cell wall candidate genes in E. grandis . (a) Cellulose and xylan biosynthesis. (b) Phenylpropanoid biosynthesis (b) . Genes enriched (orange dots) or unenriched (black dots) for H3K4me3 were plotted by absolute transcript abundance in DSX tissue ( y- axis; median FPKM value of 89,300 indicated) and relative transcript abundance in DSX tissue compared to shoot tips, young leaves, mature leaves, flowers, roots and phloem ( x- axis; expected value of 0.142 indicated). The full gene lists are presented in Additional file 3 : Table S5, Table S6.
Figure Legend Snippet: Association of H3K4me3 secondary cell wall candidate genes in E. grandis . (a) Cellulose and xylan biosynthesis. (b) Phenylpropanoid biosynthesis (b) . Genes enriched (orange dots) or unenriched (black dots) for H3K4me3 were plotted by absolute transcript abundance in DSX tissue ( y- axis; median FPKM value of 89,300 indicated) and relative transcript abundance in DSX tissue compared to shoot tips, young leaves, mature leaves, flowers, roots and phloem ( x- axis; expected value of 0.142 indicated). The full gene lists are presented in Additional file 3 : Table S5, Table S6.

Techniques Used:

21) Product Images from "Hepatocyte-specific loss of GPS2 in mice reduces non-alcoholic steatohepatitis via activation of PPARα"

Article Title: Hepatocyte-specific loss of GPS2 in mice reduces non-alcoholic steatohepatitis via activation of PPARα

Journal: Nature Communications

doi: 10.1038/s41467-019-09524-z

GPS2 cooperates with NCOR to repress target gene expression. a , b ChIP-seq tracks representing H3K27ac at ( a ) Pdk4 and ( b ) Cyp4a14 loci in Gps2 , Ncor and Smrt LKO and respective WT mouse livers. Percentage of increase represents logFC of each peak and is calculated from biological duplicates (for Gps2 LKO versus WT) or triplicates (for Ncor or Smrt LKO versus WT). c , d ChIP-seq tracks representing NCOR, GPS2, PPARα, and Pol II recruitment in Pparα KO, Ncor LKO, Gps2 LKO and respective WT livers at ( c ) Pdk4 and ( d ) Cyp4a14 loci. Percentage of increase represents logFC of each peak and is calculated from biological triplicates. e Boxplot representing average logFC of co-localized GPS2 peaks in Ncor LKO and Pparα KO versus WT livers ( n = 3 in each group). f , g MA plots showing the average logFC of GPS2 peaks between ( f ) Ncor LKO versus WT; and ( g ) Pparα KO versus WT livers, against average logCPM. UP, UN, and DN represents upregulated, unchanged, and downregulated peaks, respectively. All data are represented as mean ± s.e.m. * P
Figure Legend Snippet: GPS2 cooperates with NCOR to repress target gene expression. a , b ChIP-seq tracks representing H3K27ac at ( a ) Pdk4 and ( b ) Cyp4a14 loci in Gps2 , Ncor and Smrt LKO and respective WT mouse livers. Percentage of increase represents logFC of each peak and is calculated from biological duplicates (for Gps2 LKO versus WT) or triplicates (for Ncor or Smrt LKO versus WT). c , d ChIP-seq tracks representing NCOR, GPS2, PPARα, and Pol II recruitment in Pparα KO, Ncor LKO, Gps2 LKO and respective WT livers at ( c ) Pdk4 and ( d ) Cyp4a14 loci. Percentage of increase represents logFC of each peak and is calculated from biological triplicates. e Boxplot representing average logFC of co-localized GPS2 peaks in Ncor LKO and Pparα KO versus WT livers ( n = 3 in each group). f , g MA plots showing the average logFC of GPS2 peaks between ( f ) Ncor LKO versus WT; and ( g ) Pparα KO versus WT livers, against average logCPM. UP, UN, and DN represents upregulated, unchanged, and downregulated peaks, respectively. All data are represented as mean ± s.e.m. * P

Techniques Used: Expressing, Chromatin Immunoprecipitation

GPS2 expression associates with NASH and fibrosis in human. a Flowchart representing the analysis of microarray results in the human liver biopsies. b Heatmap representing the rho correlation coefficient of GPS2 and other core subunits with NASH-regulated genes in human NASH liver biopsies. c , d Correlation analysis of GPS2 mRNA level with ( c ) TGFB and ( d ) TIMP1 in human NAFLD liver samples (GSE49541), n = 72, non-parametric Spearman’s test. e GPS2 mRNA level in non-fibrosis and fibrosis liver biopsies in the NASH subjects, n (NASH non-fibrosis) = 74, n (NASH fibrosis) = 24, unpaired nonparametric Mann–Whitney test. f GPS2 mRNA level in NASH after weight loss in paired obese subjects, n = 54, paired nonparametric Wilcoxon matched-pairs signed rank test. g Heatmap representing the rho correlation coefficient of GPS2 and PPARA with NASH-regulated genes in human NASH liver biospies ( n = 104). h Model illustrating the causal relationship between GPS2 expression and PPARα function in hepatocytes in the context of fatty liver disease. All data are represented as mean ± s.e.m. * P
Figure Legend Snippet: GPS2 expression associates with NASH and fibrosis in human. a Flowchart representing the analysis of microarray results in the human liver biopsies. b Heatmap representing the rho correlation coefficient of GPS2 and other core subunits with NASH-regulated genes in human NASH liver biopsies. c , d Correlation analysis of GPS2 mRNA level with ( c ) TGFB and ( d ) TIMP1 in human NAFLD liver samples (GSE49541), n = 72, non-parametric Spearman’s test. e GPS2 mRNA level in non-fibrosis and fibrosis liver biopsies in the NASH subjects, n (NASH non-fibrosis) = 74, n (NASH fibrosis) = 24, unpaired nonparametric Mann–Whitney test. f GPS2 mRNA level in NASH after weight loss in paired obese subjects, n = 54, paired nonparametric Wilcoxon matched-pairs signed rank test. g Heatmap representing the rho correlation coefficient of GPS2 and PPARA with NASH-regulated genes in human NASH liver biospies ( n = 104). h Model illustrating the causal relationship between GPS2 expression and PPARα function in hepatocytes in the context of fatty liver disease. All data are represented as mean ± s.e.m. * P

Techniques Used: Expressing, Microarray, MANN-WHITNEY

GPS2 represses PPARα-regulated promoters and enhancers. a GPS2, H3K4me3, H3K27ac, and H3K4me1 ChIP-seq peaks are aligned to the GPS2 peak center in a window of ±3 kb. b , c ChIP-seq tracks of GPS2, H3K27ac, and H3K4me3 peaks at ( b ) Pdk4 and ( c ) Cyp4a14 loci, percentage of increase (LKO versus WT) represents logFC of each peak and is calculated from biological duplicates. d , e ChIP-seq tracks of GPS2, PPARα (vehicle and GW7647 treated, GSE61817) peaks at ( d ) Pdk4 and ( e ) Cyp4a14 loci. f Venn diagram representing the overlapped GPS2 and PPARα peaks detected at least twice in mouse liver. g Further ChIP qPCR validation of H3K27ac at Pdk4 promoter (upper left), Pdk4 enhancer (upper right), Cyp4a14 promoter (lower left) and control (lower right) locus in WT, LKO, PKO, and PGKO livers, n = 5 in WT and LKO groups, n = 4 in PKO group and n = 3 in PGKO group, one-way ANOVA followed by Tukey’s test. All data are represented as mean ± s.e.m.* P
Figure Legend Snippet: GPS2 represses PPARα-regulated promoters and enhancers. a GPS2, H3K4me3, H3K27ac, and H3K4me1 ChIP-seq peaks are aligned to the GPS2 peak center in a window of ±3 kb. b , c ChIP-seq tracks of GPS2, H3K27ac, and H3K4me3 peaks at ( b ) Pdk4 and ( c ) Cyp4a14 loci, percentage of increase (LKO versus WT) represents logFC of each peak and is calculated from biological duplicates. d , e ChIP-seq tracks of GPS2, PPARα (vehicle and GW7647 treated, GSE61817) peaks at ( d ) Pdk4 and ( e ) Cyp4a14 loci. f Venn diagram representing the overlapped GPS2 and PPARα peaks detected at least twice in mouse liver. g Further ChIP qPCR validation of H3K27ac at Pdk4 promoter (upper left), Pdk4 enhancer (upper right), Cyp4a14 promoter (lower left) and control (lower right) locus in WT, LKO, PKO, and PGKO livers, n = 5 in WT and LKO groups, n = 4 in PKO group and n = 3 in PGKO group, one-way ANOVA followed by Tukey’s test. All data are represented as mean ± s.e.m.* P

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

GPS2 depletion causes PPARα activation. a Heatmap representing the top 1000 significant (sorted based on adj P value) gene expression in CD-fed WT and LKO livers. b KEGG pathway analysis of significantly up-regulated and down-regulated genes in LKO mice. c , d QPCR analysis of mRNA expression in WT and LKO mice upon ( c ) fasting, n = 5 in each group; and ( d ) PPARα agonist (GW7647) treatment, n = 5 in the vehicle groups, n = 7 in the GW7647-treated groups, one-way ANOVA followed by Tukey’s test. e Motif analysis of GPS2-occupied regions in mouse liver. f Co-immunoprecipitation of GPS2 domains with PPARα in 293 cells (* represents non-specific bands). g QPCR analysis of GPS2-regulated genes and ( h ) fed and fasted serum ketone bodies in PPARα KO (PKO) and liver Gps2 / Pparα double KO (PGKO) mice livers, n = 3 in PKO and n = 4 in PGKO, non-parametric Mann–Whitney test. All data are represented as mean ± s.e.m. * P
Figure Legend Snippet: GPS2 depletion causes PPARα activation. a Heatmap representing the top 1000 significant (sorted based on adj P value) gene expression in CD-fed WT and LKO livers. b KEGG pathway analysis of significantly up-regulated and down-regulated genes in LKO mice. c , d QPCR analysis of mRNA expression in WT and LKO mice upon ( c ) fasting, n = 5 in each group; and ( d ) PPARα agonist (GW7647) treatment, n = 5 in the vehicle groups, n = 7 in the GW7647-treated groups, one-way ANOVA followed by Tukey’s test. e Motif analysis of GPS2-occupied regions in mouse liver. f Co-immunoprecipitation of GPS2 domains with PPARα in 293 cells (* represents non-specific bands). g QPCR analysis of GPS2-regulated genes and ( h ) fed and fasted serum ketone bodies in PPARα KO (PKO) and liver Gps2 / Pparα double KO (PGKO) mice livers, n = 3 in PKO and n = 4 in PGKO, non-parametric Mann–Whitney test. All data are represented as mean ± s.e.m. * P

Techniques Used: Activation Assay, Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, MANN-WHITNEY

22) Product Images from "Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin"

Article Title: Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddr506

H3K27m3 and OCT4 expression in undifferentiated hESCs. Shown are representative immunofluorescence images of the HSF-6 hESC line at passage 51, ( A ) DAPI, ( B ) OCT4, ( C ) H3K27me3 (arrow indicating focal dot in some nuclei), ( D ) Merger of (A) and (C). (
Figure Legend Snippet: H3K27m3 and OCT4 expression in undifferentiated hESCs. Shown are representative immunofluorescence images of the HSF-6 hESC line at passage 51, ( A ) DAPI, ( B ) OCT4, ( C ) H3K27me3 (arrow indicating focal dot in some nuclei), ( D ) Merger of (A) and (C). (

Techniques Used: Expressing, Immunofluorescence

Epigenetic status of the X chromosome with differentiation or serial passaging. ( A ) Six-day differentiation of HSF-6 (7), HSF-6 (8) and HSF-6 (10). OCT4+ and H3K27me3+ nuclei were counted at days 0, 3 and 6 of differentiation in each subline ( n = 200
Figure Legend Snippet: Epigenetic status of the X chromosome with differentiation or serial passaging. ( A ) Six-day differentiation of HSF-6 (7), HSF-6 (8) and HSF-6 (10). OCT4+ and H3K27me3+ nuclei were counted at days 0, 3 and 6 of differentiation in each subline ( n = 200

Techniques Used: Passaging

Reprogramming of class II hESCs to class I. ( A ) Experimental design for analysis of HSF-6 (8), HSF-6 (10) and HSF-6 (S9) over 22 passages. ( B ) Western blot of purified histones blotted for H3K27me3, H3K4me3 and the loading control histone H3 in HSF-6
Figure Legend Snippet: Reprogramming of class II hESCs to class I. ( A ) Experimental design for analysis of HSF-6 (8), HSF-6 (10) and HSF-6 (S9) over 22 passages. ( B ) Western blot of purified histones blotted for H3K27me3, H3K4me3 and the loading control histone H3 in HSF-6

Techniques Used: Western Blot, Purification

Reprogramming of new hESC lines. ( A and B ) Differentiation of UCLA3 for 12 days. (A) Quantification of H3K27me3 foci (class II) at day 0 ( n = 599), day 5 ( n = 259) and day 12 ( n = 259). (B) Representative image of acquired H3K27me3 foci at day 12 (white
Figure Legend Snippet: Reprogramming of new hESC lines. ( A and B ) Differentiation of UCLA3 for 12 days. (A) Quantification of H3K27me3 foci (class II) at day 0 ( n = 599), day 5 ( n = 259) and day 12 ( n = 259). (B) Representative image of acquired H3K27me3 foci at day 12 (white

Techniques Used:

23) Product Images from "Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells"

Article Title: Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20160120

EBNA3C specifically targets regulatory elements and epigenetically controls AID expression. (A) University of California, Santa Cruz (UCSC) genome browser overview of AID genomic locus ( AICDA ) showing model-based analysis of ChIP-Seq (MACS) peaks for EBNA3C-TAP, mouse sequence alignment, and primer locations for ChIP-qPCR. Anti-Flag ChIP-Seq signals from EBNA3C-TAP–tagged and untagged wild-type LCLs were displayed in the Savant genome browser and are shown for the same genomic coordinates. (B–F) ChIP-qPCR for H3K4me3 (B), H3K9ac (C), H3K27ac (D), acetyltransferase p300 (E), and RBPJ (F) on samples from 3CHT A13 time course at locations across the AID locus at GAPDH or myoglobin as indicated. Cells were grown in the absence (−HT) or presence of HT (+HT), and numbers indicate the day of harvest. ChIP values represent enrichment relative to input ± SD of triplicate qPCR reactions for ChIP and input of each sample. These are representative results of two biological replicates.
Figure Legend Snippet: EBNA3C specifically targets regulatory elements and epigenetically controls AID expression. (A) University of California, Santa Cruz (UCSC) genome browser overview of AID genomic locus ( AICDA ) showing model-based analysis of ChIP-Seq (MACS) peaks for EBNA3C-TAP, mouse sequence alignment, and primer locations for ChIP-qPCR. Anti-Flag ChIP-Seq signals from EBNA3C-TAP–tagged and untagged wild-type LCLs were displayed in the Savant genome browser and are shown for the same genomic coordinates. (B–F) ChIP-qPCR for H3K4me3 (B), H3K9ac (C), H3K27ac (D), acetyltransferase p300 (E), and RBPJ (F) on samples from 3CHT A13 time course at locations across the AID locus at GAPDH or myoglobin as indicated. Cells were grown in the absence (−HT) or presence of HT (+HT), and numbers indicate the day of harvest. ChIP values represent enrichment relative to input ± SD of triplicate qPCR reactions for ChIP and input of each sample. These are representative results of two biological replicates.

Techniques Used: Expressing, Chromatin Immunoprecipitation, Magnetic Cell Separation, Sequencing, Real-time Polymerase Chain Reaction

24) Product Images from "Soma influences GSC progeny differentiation via the cell adhesion-mediated steroid-let-7-Wingless signaling cascade that regulates chromatin dynamics"

Article Title: Soma influences GSC progeny differentiation via the cell adhesion-mediated steroid-let-7-Wingless signaling cascade that regulates chromatin dynamics

Journal: Biology Open

doi: 10.1242/bio.201410553

Ecdysone signaling cell non-autonomously regulates GSC progeny chromatin dynamics. (A) Control and ecdysone signaling mutant germaria are compared. In controls, spectrosome-containing germline cells (SpGCs = GSCs+pre-CBs+pro-CBs+CBs) and fusome-containing differentiating cysts are proportionally distributed and ECs form squamous epithelium. In the ecdysone-deficient germarium, supernumerary SpGCs and columnar epithelium-like ECs appear. (B) Table with germline cell markers in wildtype and ecdysone signaling or bam mutant germaria. (C) The limbo-GCs in ecdysone signaling mutants (pre-CBs) do not show H2Bub1 staining ( OregonR and ecd 1ts 4 days at 29°C). (D) Bre1 is required for H2Bub1 modification and, similarly to steroid signaling, affects the efficiency of early germline differentiation (please see supplementary material Fig. S2 ). Bre1 genetically interacts with EcR , resulting in the decreased differentiation index (Ratio: Cysts/SpGCs; Bre1 P1549 /+ , EcR Q50st /+ , EcRQ 50st /+; Bre1 P1549 /+ , supplementary material Table S1 ). (E) The Δ bam (bam EY04821 /bam EY03755 ) supernumerary SpGCs (pro-CBs) are positive for H2Bub1. (F) Perturbation of ecdysone signaling via EcR overexpression ( hsEcR.B1/+ , 2×1 h heat shock at 37°C) leads to a differentiation delay in the GSC progeny, and forced expression of bam ( hsbam/+ , 2×1 h heat shock at 37°C) causes GSC loss by differentiation. Overexpression of both proteins ( hsEcR.B1/+ ; hsbam/+ , 2×1 h heat shock at 37°C) overcomes the differentiation delay and leads to the increased differentiation ratio (Ratio: Cysts/SpGCs, supplementary material Table S1 ). Ecdysone signaling temporally acts upstream of the chromatin modification H2Bub1 and the germline differentiation factor Bam. (G) Similarly, soma-specific ecdysone signaling perturbation ( ptc ts > ab: ptcGal4/+ ; tubGal80 ts /UASab , 16 days at 29°C) leads to the appearance of supernumerary pre-CBs negative for H2Bub1 and delayed differentiation ( supplementary material Table S2 ). Germaria are stained with H2Bub1 (red, C,E,G), Lamin C (LC red, D,F) to visualize TFs and CpCs and Adducin (Add red, D,F) to mark spectrosomes and fusomes. Nuclei are stained with DAPI (blue, C–G). CpCs are outlined in yellow (C–G), SpGCs are outlined in white (C,G) or indicated with arrows (D). p-values were calculated using the two tailed Student's t-test and error bars represent S.E.M. *p
Figure Legend Snippet: Ecdysone signaling cell non-autonomously regulates GSC progeny chromatin dynamics. (A) Control and ecdysone signaling mutant germaria are compared. In controls, spectrosome-containing germline cells (SpGCs = GSCs+pre-CBs+pro-CBs+CBs) and fusome-containing differentiating cysts are proportionally distributed and ECs form squamous epithelium. In the ecdysone-deficient germarium, supernumerary SpGCs and columnar epithelium-like ECs appear. (B) Table with germline cell markers in wildtype and ecdysone signaling or bam mutant germaria. (C) The limbo-GCs in ecdysone signaling mutants (pre-CBs) do not show H2Bub1 staining ( OregonR and ecd 1ts 4 days at 29°C). (D) Bre1 is required for H2Bub1 modification and, similarly to steroid signaling, affects the efficiency of early germline differentiation (please see supplementary material Fig. S2 ). Bre1 genetically interacts with EcR , resulting in the decreased differentiation index (Ratio: Cysts/SpGCs; Bre1 P1549 /+ , EcR Q50st /+ , EcRQ 50st /+; Bre1 P1549 /+ , supplementary material Table S1 ). (E) The Δ bam (bam EY04821 /bam EY03755 ) supernumerary SpGCs (pro-CBs) are positive for H2Bub1. (F) Perturbation of ecdysone signaling via EcR overexpression ( hsEcR.B1/+ , 2×1 h heat shock at 37°C) leads to a differentiation delay in the GSC progeny, and forced expression of bam ( hsbam/+ , 2×1 h heat shock at 37°C) causes GSC loss by differentiation. Overexpression of both proteins ( hsEcR.B1/+ ; hsbam/+ , 2×1 h heat shock at 37°C) overcomes the differentiation delay and leads to the increased differentiation ratio (Ratio: Cysts/SpGCs, supplementary material Table S1 ). Ecdysone signaling temporally acts upstream of the chromatin modification H2Bub1 and the germline differentiation factor Bam. (G) Similarly, soma-specific ecdysone signaling perturbation ( ptc ts > ab: ptcGal4/+ ; tubGal80 ts /UASab , 16 days at 29°C) leads to the appearance of supernumerary pre-CBs negative for H2Bub1 and delayed differentiation ( supplementary material Table S2 ). Germaria are stained with H2Bub1 (red, C,E,G), Lamin C (LC red, D,F) to visualize TFs and CpCs and Adducin (Add red, D,F) to mark spectrosomes and fusomes. Nuclei are stained with DAPI (blue, C–G). CpCs are outlined in yellow (C–G), SpGCs are outlined in white (C,G) or indicated with arrows (D). p-values were calculated using the two tailed Student's t-test and error bars represent S.E.M. *p

Techniques Used: Mutagenesis, Staining, Modification, Over Expression, Expressing, Two Tailed Test

Wg signaling cell autonomously influences the germline differentiation speed. (A) The defects caused by heat-shock induced overexpression of usp DN or EcR DN (increased number of SpGCs and decreased Cysts/SpGCs ratio) can be significantly alleviated by reducing the dose of DE-Cad or arm ( hs-Gal4-usp.LBD/+ , hs-Gal4-EcR.LBD/+ , hs-Gal4-usp.LBD/shg E187 , hs-Gal4-usp.LBD/arm 2 and hs-Gal4-EcR.LBD/arm 2 , see supplementary material Table S2 ). (B) Scheme shows the presumable consequences of Wg signaling perturbation on the germline differentiation speed. (C,C′) Downregulation of Wg signaling activity in the germline ( nos > fz RNAi : NGT40/fz RNAi ;nanosGAL4/+ and nos > arm RNAi : NGT40/arm RNAi ;nanosGAL4/+ ) increases the number of SpGCs delayed in differentiation, marked by the presence of the spectrosomes (dot-like Adducin (Add)-positive structures) (see supplementary material Tables S1, S5 ). (E,E′) Similarly, germline Bre1 mutant cysts (marked by the absence of GFP, hsFlp; FRT 2A Bre1 P1549 /FRT 2A GFP ) show delayed differentiation (marked by arrows). Additionally, 10% of germaria with Bre1 mutant germline cysts contain dying cysts, which was not observed in control (see supplementary material Table S6 ). (D,D′) In contrast, upregulation of Wg signaling activity in the germline ( nos > pan RNAi : NGT40/pan RNAi ;nanosGAL4/+ and nos > arm: NGT40/UASarm;nanosGAL4/+ ) leads to premature germline differentiation, 8–16-cell cysts are observed already in region 1 of the germarium (see supplementary material Tables S1, S5 ). (F,F′) The same is observed in sgg germline clones ( FRT 101 sgg D127 /FRT 101 GFP; hsFlp/+ , clones are marked by the absence of GFP). Note that sgg clonal germline cells containing a spherical spectrosome (pink arrowhead) and 16-cell cysts (yellow arrowhead) are found side by side ( supplementary material Table S6 ). (G,G′,H,H′) Similar defects in H2Bub1 modification pattern are observed in supernumerary pre-CBs caused by either germline-specific Wg or soma-specific ecdysone signaling perturbations ( Fig. 1G ). Germaria are stained with LaminC (LC red, A,C–F) to visualize TFs and CpCs and Adducin (Add red, A, C–F) to mark spectrosomes and fusomes. Vasa marks germline (green, A,C,D). Absence of GFP (green, E,F) marks clonal mutant cells. Monoubiquitination of H2B is shown (red, G,H). Nuclei are marked with DAPI (blue, A,C–H). White dashed lines mark GSCs (D,F), differentiation delayed GCs (E) or GSCs and additional SpGCs (G,H). Yellow dashed lines depict differentiating cysts (C,D) or clonal mutant differentiating cysts (E,F). p-values were calculated using the two tailed Student's t-test and error bars represent S.E.M. *p
Figure Legend Snippet: Wg signaling cell autonomously influences the germline differentiation speed. (A) The defects caused by heat-shock induced overexpression of usp DN or EcR DN (increased number of SpGCs and decreased Cysts/SpGCs ratio) can be significantly alleviated by reducing the dose of DE-Cad or arm ( hs-Gal4-usp.LBD/+ , hs-Gal4-EcR.LBD/+ , hs-Gal4-usp.LBD/shg E187 , hs-Gal4-usp.LBD/arm 2 and hs-Gal4-EcR.LBD/arm 2 , see supplementary material Table S2 ). (B) Scheme shows the presumable consequences of Wg signaling perturbation on the germline differentiation speed. (C,C′) Downregulation of Wg signaling activity in the germline ( nos > fz RNAi : NGT40/fz RNAi ;nanosGAL4/+ and nos > arm RNAi : NGT40/arm RNAi ;nanosGAL4/+ ) increases the number of SpGCs delayed in differentiation, marked by the presence of the spectrosomes (dot-like Adducin (Add)-positive structures) (see supplementary material Tables S1, S5 ). (E,E′) Similarly, germline Bre1 mutant cysts (marked by the absence of GFP, hsFlp; FRT 2A Bre1 P1549 /FRT 2A GFP ) show delayed differentiation (marked by arrows). Additionally, 10% of germaria with Bre1 mutant germline cysts contain dying cysts, which was not observed in control (see supplementary material Table S6 ). (D,D′) In contrast, upregulation of Wg signaling activity in the germline ( nos > pan RNAi : NGT40/pan RNAi ;nanosGAL4/+ and nos > arm: NGT40/UASarm;nanosGAL4/+ ) leads to premature germline differentiation, 8–16-cell cysts are observed already in region 1 of the germarium (see supplementary material Tables S1, S5 ). (F,F′) The same is observed in sgg germline clones ( FRT 101 sgg D127 /FRT 101 GFP; hsFlp/+ , clones are marked by the absence of GFP). Note that sgg clonal germline cells containing a spherical spectrosome (pink arrowhead) and 16-cell cysts (yellow arrowhead) are found side by side ( supplementary material Table S6 ). (G,G′,H,H′) Similar defects in H2Bub1 modification pattern are observed in supernumerary pre-CBs caused by either germline-specific Wg or soma-specific ecdysone signaling perturbations ( Fig. 1G ). Germaria are stained with LaminC (LC red, A,C–F) to visualize TFs and CpCs and Adducin (Add red, A, C–F) to mark spectrosomes and fusomes. Vasa marks germline (green, A,C,D). Absence of GFP (green, E,F) marks clonal mutant cells. Monoubiquitination of H2B is shown (red, G,H). Nuclei are marked with DAPI (blue, A,C–H). White dashed lines mark GSCs (D,F), differentiation delayed GCs (E) or GSCs and additional SpGCs (G,H). Yellow dashed lines depict differentiating cysts (C,D) or clonal mutant differentiating cysts (E,F). p-values were calculated using the two tailed Student's t-test and error bars represent S.E.M. *p

Techniques Used: Over Expression, Activity Assay, Mutagenesis, Clone Assay, Modification, Staining, Two Tailed Test

25) Product Images from "Omeprazole Blocks STAT6 Binding to the Eotaxin-3 Promoter in Eosinophilic Esophagitis Cells"

Article Title: Omeprazole Blocks STAT6 Binding to the Eotaxin-3 Promoter in Eosinophilic Esophagitis Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0050037

Omeprazole (Ome) decreases IL-4-stimulated RNA Pol II binding to the endogenous eotaxin-3 promoter in (A) EoE1-T and (B) EoE2-T cells. Data are the means ± SEM of 3 separate experiments. **, p≤0.01 compared to IL-4 stimulation; ***, p≤0.001 compared to IL-4 stimulation Isotype matched IgG served as a control. Representative experiment demonstrating that omeprazole reduces the levels of IL-4 stimulated H3K4me3 bound to the endogenous eotaxin-3 promoter in (C) EoE1-T and (D) EoE2-T cells. Isotype matched IgG served as a control. Depicted is one of 3 separate experiments. M, marker.
Figure Legend Snippet: Omeprazole (Ome) decreases IL-4-stimulated RNA Pol II binding to the endogenous eotaxin-3 promoter in (A) EoE1-T and (B) EoE2-T cells. Data are the means ± SEM of 3 separate experiments. **, p≤0.01 compared to IL-4 stimulation; ***, p≤0.001 compared to IL-4 stimulation Isotype matched IgG served as a control. Representative experiment demonstrating that omeprazole reduces the levels of IL-4 stimulated H3K4me3 bound to the endogenous eotaxin-3 promoter in (C) EoE1-T and (D) EoE2-T cells. Isotype matched IgG served as a control. Depicted is one of 3 separate experiments. M, marker.

Techniques Used: Binding Assay, Marker

26) Product Images from "The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor"

Article Title: The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor

Journal: Nature Communications

doi: 10.1038/s41467-019-09310-x

JMJ13 is an H3K27me3 demethylase in vivo. a Schematic representation of GFP-tagged JMJ13 and JMJ13-H293A-E295A-GFP constructs. HD, helical domain. b , c Over-expression of JMJ13-GFP reduces the levels of H3K27me3 but not H3K27me2 and H3K27me1 in vivo. d , e Over-expression of JMJ13-H293A-E295A-GFP has no effect on H3K27 methylation. In b , d , the white arrows point to the transfected nuclei stained by methylation-specific histone antibodies (red, right panels), DAPI (blue, left panels), and the GFP signal from the JMJ13-GFP or JMJ13-H293A-E295A-GFP (green, middle panels), respectively. Scale bars, 2 μm. In c , e , more than 20 pairs of transfected nuclei versus non-transfected nuclei in the same field of view were observed and quantifications statistical analyzed. Error bars represent mean ± SE. Student’s t test was used to calculate the P value between JMJ13-GFP and WT. **** P value
Figure Legend Snippet: JMJ13 is an H3K27me3 demethylase in vivo. a Schematic representation of GFP-tagged JMJ13 and JMJ13-H293A-E295A-GFP constructs. HD, helical domain. b , c Over-expression of JMJ13-GFP reduces the levels of H3K27me3 but not H3K27me2 and H3K27me1 in vivo. d , e Over-expression of JMJ13-H293A-E295A-GFP has no effect on H3K27 methylation. In b , d , the white arrows point to the transfected nuclei stained by methylation-specific histone antibodies (red, right panels), DAPI (blue, left panels), and the GFP signal from the JMJ13-GFP or JMJ13-H293A-E295A-GFP (green, middle panels), respectively. Scale bars, 2 μm. In c , e , more than 20 pairs of transfected nuclei versus non-transfected nuclei in the same field of view were observed and quantifications statistical analyzed. Error bars represent mean ± SE. Student’s t test was used to calculate the P value between JMJ13-GFP and WT. **** P value

Techniques Used: In Vivo, Construct, Over Expression, Methylation, Transfection, Staining

27) Product Images from "Modifications at K31 on the lateral surface of histone H4 contribute to genome structure and expression in apicomplexan parasites"

Article Title: Modifications at K31 on the lateral surface of histone H4 contribute to genome structure and expression in apicomplexan parasites

Journal: eLife

doi: 10.7554/eLife.29391

Chromosomal projection of H4K31ac, H3K9me3 and H3K4me3 occupancies in P. falciparum . The full set of chromosomes is represented in this circular plot.
Figure Legend Snippet: Chromosomal projection of H4K31ac, H3K9me3 and H3K4me3 occupancies in P. falciparum . The full set of chromosomes is represented in this circular plot.

Techniques Used:

PTM distribution and gene expression in T. gondii . Genome-wide PTM occupancy profiles at peri-ATG regions are plotted for the gene groups ranked by ( a ) their mRNA levels, ( b ) H3K14ac, ( c ) H3K4me3, ( d ) H3K4me1, and ( e ) H3K9me3 are shown. The y-axis shows the average tag count of the enrichment. The vertical dashed line indicates the position of the ATG.
Figure Legend Snippet: PTM distribution and gene expression in T. gondii . Genome-wide PTM occupancy profiles at peri-ATG regions are plotted for the gene groups ranked by ( a ) their mRNA levels, ( b ) H3K14ac, ( c ) H3K4me3, ( d ) H3K4me1, and ( e ) H3K9me3 are shown. The y-axis shows the average tag count of the enrichment. The vertical dashed line indicates the position of the ATG.

Techniques Used: Expressing, Genome Wide

Chromosomal projection of H4K31me1, H3K9me3 and HP1 occupancies in P. falciparum. The full set of chromosomes is represented in this circular plot, in which centromeric regions are marked by black arrows.
Figure Legend Snippet: Chromosomal projection of H4K31me1, H3K9me3 and HP1 occupancies in P. falciparum. The full set of chromosomes is represented in this circular plot, in which centromeric regions are marked by black arrows.

Techniques Used:

28) Product Images from "TET2 promotes histone O-GlcNAcylation during gene transcription"

Article Title: TET2 promotes histone O-GlcNAcylation during gene transcription

Journal: Nature

doi: 10.1038/nature11742

TET2 enhanced histone glycosylation a , Down-regulation of TET2 impairs H2B GlcNAcylation in ES cells. H2B GlcNAcylation was examined by IP with anti-H2B antibody and Western blot with anti-GlcNAc antibody (RL2) or anti-H2B S112 GlcNAc antibody. Histogram shows the relative level of H2B S112 GlcNAc in TET2 down-regulated cells compared to that in control shRNA treated cells. b , Up-regulation of wild type TET2 or TET2 enzymatic dead mutant (H1382Y/D1384A) induced H2B S112 GlcNAcylation in 293 cells. c , The interaction between OGT and TET2 is important for H2B GlcNAcylation and H2B S112G GlcNAcylation. Wild type OGT, the enzymatic dead mutant of OGT (G482S) and the D2 mutant were expressed in 293T cells. H2B GlcNAcylation and H2B S112 GlcNAcylation were examined. d , TET2 facilitated OGT-dependent histone glycosylation in mono-nucleosome but not in recombinant core histones. Tritium-labeled GlcNAc was incorporated into the histones in the in vitro GlcNAcylation assay. All error bars denote s.d., n=3.
Figure Legend Snippet: TET2 enhanced histone glycosylation a , Down-regulation of TET2 impairs H2B GlcNAcylation in ES cells. H2B GlcNAcylation was examined by IP with anti-H2B antibody and Western blot with anti-GlcNAc antibody (RL2) or anti-H2B S112 GlcNAc antibody. Histogram shows the relative level of H2B S112 GlcNAc in TET2 down-regulated cells compared to that in control shRNA treated cells. b , Up-regulation of wild type TET2 or TET2 enzymatic dead mutant (H1382Y/D1384A) induced H2B S112 GlcNAcylation in 293 cells. c , The interaction between OGT and TET2 is important for H2B GlcNAcylation and H2B S112G GlcNAcylation. Wild type OGT, the enzymatic dead mutant of OGT (G482S) and the D2 mutant were expressed in 293T cells. H2B GlcNAcylation and H2B S112 GlcNAcylation were examined. d , TET2 facilitated OGT-dependent histone glycosylation in mono-nucleosome but not in recombinant core histones. Tritium-labeled GlcNAc was incorporated into the histones in the in vitro GlcNAcylation assay. All error bars denote s.d., n=3.

Techniques Used: Western Blot, shRNA, Mutagenesis, Recombinant, Labeling, In Vitro

TET2 regulates H2B S112 GlcNAc and gene transcription a , Venn diagram shows a significant overlap between OGT, H2B S112 GlcNAc and TET2 target genes. b , Mean distribution of tags at gene TSS (± 4 kb). c , Examples of OGT, H2B S112 GlcNAc and TET2 ChIP-seq results in mouse ES cells. d , ChIP-qPCR was performed to examine TET2, OGT and H2B S112 GlcNAc in control, TET2 knockdown or OGT knockdown cells (shCon, shTET2 and shOGT). e , The expression of TET2 and OGT common target genes is higher than average gene expression in ES cells. Data analysis was explained in the Material and Method. Boxplots show median, 25 th and 75 th percentile expression levels in ES cells. p
Figure Legend Snippet: TET2 regulates H2B S112 GlcNAc and gene transcription a , Venn diagram shows a significant overlap between OGT, H2B S112 GlcNAc and TET2 target genes. b , Mean distribution of tags at gene TSS (± 4 kb). c , Examples of OGT, H2B S112 GlcNAc and TET2 ChIP-seq results in mouse ES cells. d , ChIP-qPCR was performed to examine TET2, OGT and H2B S112 GlcNAc in control, TET2 knockdown or OGT knockdown cells (shCon, shTET2 and shOGT). e , The expression of TET2 and OGT common target genes is higher than average gene expression in ES cells. Data analysis was explained in the Material and Method. Boxplots show median, 25 th and 75 th percentile expression levels in ES cells. p

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

29) Product Images from "The endoglycosidase heparanase enters the nucleus of T lymphocytes and modulates H3 methylation at actively transcribed genes via the interplay with key chromatin modifying enzymes"

Article Title: The endoglycosidase heparanase enters the nucleus of T lymphocytes and modulates H3 methylation at actively transcribed genes via the interplay with key chromatin modifying enzymes

Journal: Transcription

doi: 10.4161/trns.19998

Figure 6. Nuclear heparanase may control H3 methylation profiles by recruiting LSD1 and excluding MLL from gene promoters in T cells. (A) LSD1 ChIP assays were performed on Jurkat T cells transfected with heparanase RNAi3 or a negative siRNA control
Figure Legend Snippet: Figure 6. Nuclear heparanase may control H3 methylation profiles by recruiting LSD1 and excluding MLL from gene promoters in T cells. (A) LSD1 ChIP assays were performed on Jurkat T cells transfected with heparanase RNAi3 or a negative siRNA control

Techniques Used: Methylation, Chromatin Immunoprecipitation, Transfection

30) Product Images from "Bmi1 promotes erythroid development through regulating ribosome biogenesis"

Article Title: Bmi1 promotes erythroid development through regulating ribosome biogenesis

Journal: Stem cells (Dayton, Ohio)

doi: 10.1002/stem.1896

Bmi1 associates with the promoter of some ribosomal protein genes and enhances their expression (A) Representative Ring1b and control IgG ChIP-seq profiles of loci occupied by Ring1b in murine L8057 megakaryoblastic cells. It appears that Ring1b associates with the transcription start site (TSS) of multiple ribosomal protein genes. (B) BMI1 and RING1b associates with the promoter of some ribosomal protein genes in vivo . Chromatin-bound DNA from K562 cells were immunoprecipitated with a BMI1-antibody, a RING1b-antibody or with normal mouse IgG. qPCR amplification was performed on corresponding templates using primers for RPS14, RPS19, RPL5, RPL11 and RPL23 genes. (C) Chromatin-bound DNA from K562 cells were immunoprecipitated with a BMI1-antibody, a RING1B-antibody, a H3K9ac-antibody, a H3K4me3-antibody, a H3K27me3-antibody or with normal mouse IgG. qPCR amplification was performed on corresponding templates using primers for RPS19 gene. (D) Knockdown of BMI1 decreases the activation of the RPL11 promoter. K562 cells with reduced BMI1 expression were transfected with RPL11 promoter-driven luciferase plasmid. Luciferase activity was assayed 24 hr after transfection. Values are means ± SD, n=3, *p
Figure Legend Snippet: Bmi1 associates with the promoter of some ribosomal protein genes and enhances their expression (A) Representative Ring1b and control IgG ChIP-seq profiles of loci occupied by Ring1b in murine L8057 megakaryoblastic cells. It appears that Ring1b associates with the transcription start site (TSS) of multiple ribosomal protein genes. (B) BMI1 and RING1b associates with the promoter of some ribosomal protein genes in vivo . Chromatin-bound DNA from K562 cells were immunoprecipitated with a BMI1-antibody, a RING1b-antibody or with normal mouse IgG. qPCR amplification was performed on corresponding templates using primers for RPS14, RPS19, RPL5, RPL11 and RPL23 genes. (C) Chromatin-bound DNA from K562 cells were immunoprecipitated with a BMI1-antibody, a RING1B-antibody, a H3K9ac-antibody, a H3K4me3-antibody, a H3K27me3-antibody or with normal mouse IgG. qPCR amplification was performed on corresponding templates using primers for RPS19 gene. (D) Knockdown of BMI1 decreases the activation of the RPL11 promoter. K562 cells with reduced BMI1 expression were transfected with RPL11 promoter-driven luciferase plasmid. Luciferase activity was assayed 24 hr after transfection. Values are means ± SD, n=3, *p

Techniques Used: Expressing, Chromatin Immunoprecipitation, In Vivo, Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay

31) Product Images from "The Conserved PHD1-PHD2 Domain of ZFP-1/AF10 Is a Discrete Functional Module Essential for Viability in Caenorhabditis elegans"

Article Title: The Conserved PHD1-PHD2 Domain of ZFP-1/AF10 Is a Discrete Functional Module Essential for Viability in Caenorhabditis elegans

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.01462-12

ZFP-1 localizes to promoters. The figure shows snapshots of two genomic loci, with tacks illustrating ChIP-chip signals from (top to bottom) ZFP-1, H3K4me1, H3K4me2, and H3K4me3, based on available modENCODE data.
Figure Legend Snippet: ZFP-1 localizes to promoters. The figure shows snapshots of two genomic loci, with tacks illustrating ChIP-chip signals from (top to bottom) ZFP-1, H3K4me1, H3K4me2, and H3K4me3, based on available modENCODE data.

Techniques Used: Chromatin Immunoprecipitation

H3K4me3 is severely depleted in lsy-15 ( tm3463 ) embryos. (A) Western blot detecting H3K4me3 in wild-type compared to lsy-15 ( tm3463 ) embryos. H3 served as a loading control. (B) Same as panel A, showing H3K4me1, H3K4me2, and H3, with Ponceau staining as
Figure Legend Snippet: H3K4me3 is severely depleted in lsy-15 ( tm3463 ) embryos. (A) Western blot detecting H3K4me3 in wild-type compared to lsy-15 ( tm3463 ) embryos. H3 served as a loading control. (B) Same as panel A, showing H3K4me1, H3K4me2, and H3, with Ponceau staining as

Techniques Used: Western Blot, Staining

32) Product Images from "Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells"

Article Title: Cell-specific occupancy of an extended repertoire of CREM and CREB binding loci in male germ cells

Journal: BMC Genomics

doi: 10.1186/1471-2164-11-530

Correlative analysis of CREB and CREM binding site occupancy . A. Association of CREB, pol II and H3K4me3 in GC1-spg cells. Comparison of pol II and H3K4me3 tag density in the region of +/- 5 kb around the CREB-occupied loci. Clustering analysis identifies 3 classes, A; CREB loci with low or no pol II and H3K4me3, B-C; CREB loci with high pol II and H3K4me3 corresponding to transcription on the sense and anti-sense strands, respectively. B. Comparison of CREM tag density in the region of +/- 5 kb around the CREB-occupied loci. Clustering identifies 2 groups A; CREB loci with low or no CREM and B; CREB loci with high CREM. C . Venn diagramme comparing common and cell-specific CREB/CREM occupancy of binding sites using the CREB data in panel B and the equivalent analysis of the CREM occupied loci (data not shown).
Figure Legend Snippet: Correlative analysis of CREB and CREM binding site occupancy . A. Association of CREB, pol II and H3K4me3 in GC1-spg cells. Comparison of pol II and H3K4me3 tag density in the region of +/- 5 kb around the CREB-occupied loci. Clustering analysis identifies 3 classes, A; CREB loci with low or no pol II and H3K4me3, B-C; CREB loci with high pol II and H3K4me3 corresponding to transcription on the sense and anti-sense strands, respectively. B. Comparison of CREM tag density in the region of +/- 5 kb around the CREB-occupied loci. Clustering identifies 2 groups A; CREB loci with low or no CREM and B; CREB loci with high CREM. C . Venn diagramme comparing common and cell-specific CREB/CREM occupancy of binding sites using the CREB data in panel B and the equivalent analysis of the CREM occupied loci (data not shown).

Techniques Used: Binding Assay

Specific occupancy of target promoters by CREM and CREB . A-D . Graphic representation of ChIP-seq results as. wig format files in UCSC web browser at the indicated loci. The same format has been used for Figs. 6 and 7 as well as Additional file 1 , Figs. S4 and 5. The ChIP-seq results for CREB and CREM in GC1-spg and testis haploid cells respectively are shown along with results for H3K4me3 in GC1-spg cells (K4-GC1) and testis (K4-T). In addition, the mammalian conservation track from the UCSC browser is also included. CREM and CREB binding peaks are indicated by arrows.
Figure Legend Snippet: Specific occupancy of target promoters by CREM and CREB . A-D . Graphic representation of ChIP-seq results as. wig format files in UCSC web browser at the indicated loci. The same format has been used for Figs. 6 and 7 as well as Additional file 1 , Figs. S4 and 5. The ChIP-seq results for CREB and CREM in GC1-spg and testis haploid cells respectively are shown along with results for H3K4me3 in GC1-spg cells (K4-GC1) and testis (K4-T). In addition, the mammalian conservation track from the UCSC browser is also included. CREM and CREB binding peaks are indicated by arrows.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay

33) Product Images from "Polycomb-Like 3 Promotes Polycomb Repressive Complex 2 Binding to CpG Islands and Embryonic Stem Cell Self-Renewal"

Article Title: Polycomb-Like 3 Promotes Polycomb Repressive Complex 2 Binding to CpG Islands and Embryonic Stem Cell Self-Renewal

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1002576

Pcl3 is a component of PRC2. (A) Protein levels of Suz12 and Suz12-TAP were measured in wild type, Suz12 Gt/+ , Suz12 Rev/+ , and Suz12 Suz12TAP/+ cell lines by immunoblot. (B) Proteins detected by mass spectrometry that specifically co-purified with Suz12-TAP, their symbol, unique hits, and percent coverage. (C) Pcl3-V5 binds to Suz12-TAP. Suz12 Suz12TAP/+ ESCs were transfected with empty vector, Pcl3-V5, or Mks1-V5 (control), and lysates were immunoprecipitated with FlagM2 and probed with anti-V5. (D) Pcl3-V5 binds Suz12, Ezh2, and Eed. Lysates from ESCs transfected with empty vector, Pcl3-V5, and Mks1-V5 (control) vectors were subjected to immunoprecipitation with anti-V5. Samples were then probed with anti-Suz12, anti-Ezh3, and anti-Eed. All westerns and co-immunoprecipitations were performed three times.
Figure Legend Snippet: Pcl3 is a component of PRC2. (A) Protein levels of Suz12 and Suz12-TAP were measured in wild type, Suz12 Gt/+ , Suz12 Rev/+ , and Suz12 Suz12TAP/+ cell lines by immunoblot. (B) Proteins detected by mass spectrometry that specifically co-purified with Suz12-TAP, their symbol, unique hits, and percent coverage. (C) Pcl3-V5 binds to Suz12-TAP. Suz12 Suz12TAP/+ ESCs were transfected with empty vector, Pcl3-V5, or Mks1-V5 (control), and lysates were immunoprecipitated with FlagM2 and probed with anti-V5. (D) Pcl3-V5 binds Suz12, Ezh2, and Eed. Lysates from ESCs transfected with empty vector, Pcl3-V5, and Mks1-V5 (control) vectors were subjected to immunoprecipitation with anti-V5. Samples were then probed with anti-Suz12, anti-Ezh3, and anti-Eed. All westerns and co-immunoprecipitations were performed three times.

Techniques Used: Mass Spectrometry, Purification, Transfection, Plasmid Preparation, Immunoprecipitation

Pcl2 and Pcl3 localize to CpG islands. (A) The 500 bp central regions of Pcl3 ChIP-seq peaks were scanned for enriched motifs by using a 9th order Markov background dependence model [86] . Two examples of 10- and 14-mer enriched motifs are shown. (B–C) Smoothed scatter plots of maximum position specific-scoring matrix (PSSM) scores for the two motifs and CpG density are shown for (B) Suz12 binding sites depleted upon Pcl3 knockdown overlapping with Pcl2 and Pcl3 and (C) Suz12 binding sites unaffected upon Pcl3 knockdown and that do not overlap with Pcl2 and Pcl3. (D) Shown are the decision boundaries of a support vector machine classifier using these three features, where the purple regions correspond to Suz12 co-localizing with Pcl2 and Pcl3. The predictor had a cross validation accuracy of 75%. (E) A model of Pcl3 and Pcl2 regulation of PRC2 binding and activity. In wild type ESCs, Pcl3 promotes PRC2 binding and H3K27me3. Pcl2 antagonizes Pcl3-mediated Suz12 binding at sites bound by both but promotes PRC2 function at sites solely regulated by Pcl2. Knockdown of Pcl3 causes decreased PRC2 binding and H3K27me3. Pcl2 does not compensate at Pcl2 and Pcl3 targets and continues to inhibit or promote PRC2 function depending on the gene.
Figure Legend Snippet: Pcl2 and Pcl3 localize to CpG islands. (A) The 500 bp central regions of Pcl3 ChIP-seq peaks were scanned for enriched motifs by using a 9th order Markov background dependence model [86] . Two examples of 10- and 14-mer enriched motifs are shown. (B–C) Smoothed scatter plots of maximum position specific-scoring matrix (PSSM) scores for the two motifs and CpG density are shown for (B) Suz12 binding sites depleted upon Pcl3 knockdown overlapping with Pcl2 and Pcl3 and (C) Suz12 binding sites unaffected upon Pcl3 knockdown and that do not overlap with Pcl2 and Pcl3. (D) Shown are the decision boundaries of a support vector machine classifier using these three features, where the purple regions correspond to Suz12 co-localizing with Pcl2 and Pcl3. The predictor had a cross validation accuracy of 75%. (E) A model of Pcl3 and Pcl2 regulation of PRC2 binding and activity. In wild type ESCs, Pcl3 promotes PRC2 binding and H3K27me3. Pcl2 antagonizes Pcl3-mediated Suz12 binding at sites bound by both but promotes PRC2 function at sites solely regulated by Pcl2. Knockdown of Pcl3 causes decreased PRC2 binding and H3K27me3. Pcl2 does not compensate at Pcl2 and Pcl3 targets and continues to inhibit or promote PRC2 function depending on the gene.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Plasmid Preparation, Activity Assay

Pcl3 requires Tudor domain residues for function. (A) Schematic of Pcl3 architecture including Tudor and PHD domains. Size of domains are indicated by amino acid number, but schematic is not to scale. Orange boxes indicate mutated residues. (B) Sequence alignment of the Tudor domain of human and mouse Polycomb-like homologues. Bottom graph indicates the degree of conservation at each residue. Green circles indicate putative histone binding sites. Orange squares indicate mutated residues. (C) Mutant Pcl3-TAP protein is detected at similar levels to wild type Pcl3-TAP by immunoprecipitating and probing with anti-FlagM2. Mutant Pcl3-TAP can bind Suz12 as well as wild type Pcl3-TAP by co-immunoprecipitation. (D) Pcl3-TAP W48A;Y48A and Pcl3-TAP F72S;D74S do not support H3K27me3 whereas Pcl3-TAP N75S;Y78S does. Immunoblot displaying H3K27me3 levels in histones purified from cells expressing Pcl3 shRNA, Pcl3 shRNA with Pcl3-TAP, and Pcl3 shRNA with mutant Pcl3-TAP. Histone H3 and α-tubulin were used as loading controls. Each lane represents a different clone. All westerns and immunoprecipitations were performed 3–4 times with at least two clones from each mutant.
Figure Legend Snippet: Pcl3 requires Tudor domain residues for function. (A) Schematic of Pcl3 architecture including Tudor and PHD domains. Size of domains are indicated by amino acid number, but schematic is not to scale. Orange boxes indicate mutated residues. (B) Sequence alignment of the Tudor domain of human and mouse Polycomb-like homologues. Bottom graph indicates the degree of conservation at each residue. Green circles indicate putative histone binding sites. Orange squares indicate mutated residues. (C) Mutant Pcl3-TAP protein is detected at similar levels to wild type Pcl3-TAP by immunoprecipitating and probing with anti-FlagM2. Mutant Pcl3-TAP can bind Suz12 as well as wild type Pcl3-TAP by co-immunoprecipitation. (D) Pcl3-TAP W48A;Y48A and Pcl3-TAP F72S;D74S do not support H3K27me3 whereas Pcl3-TAP N75S;Y78S does. Immunoblot displaying H3K27me3 levels in histones purified from cells expressing Pcl3 shRNA, Pcl3 shRNA with Pcl3-TAP, and Pcl3 shRNA with mutant Pcl3-TAP. Histone H3 and α-tubulin were used as loading controls. Each lane represents a different clone. All westerns and immunoprecipitations were performed 3–4 times with at least two clones from each mutant.

Techniques Used: Sequencing, Binding Assay, Mutagenesis, Immunoprecipitation, Purification, Expressing, shRNA, Clone Assay

Pcl3 and Pcl2 participate in separate PRC2 complexes but overlap at many PRC2 binding sites. (A) Pcl2 and Pcl1 mRNA levels are not significantly different between scramble and Pcl3 shRNA cells. Graph represents average expression from three different experiments in 2–6 different clones and assayed in quadruplet. Error bars indicate standard deviation. (B) Co-immunoprecipitation showing that immunoprecipitated Suz12-TAP associates with Pcl2 whereas immunoprecipitated Pcl3-TAP does not. Pcl3-TAP does bind Suz12 as seen in the lower blot. α-tubulin was used as a loading control. Reciprocal co-immunoprecipitations showing a Pcl2-specific association with Suz12-TAP but not an association between Pcl2 and Pcl3-TAP. β-actin was used as a loading control. Each blot is representative of three different experiments. (C) Graph depicts chromosome-wise distributions of Pcl2 co-localization with sites depleted of Suz12 following Pcl3 knockdown (blue). Regions containing only Suz12 depletion and only Pcl2 binding are indicated in cyan and orange respectively. (D) Decreased Suz12 binding upon Pcl3 knockdown occurs at 86% of targets bound by Suz12 and Pcl2 (Fisher test p-value
Figure Legend Snippet: Pcl3 and Pcl2 participate in separate PRC2 complexes but overlap at many PRC2 binding sites. (A) Pcl2 and Pcl1 mRNA levels are not significantly different between scramble and Pcl3 shRNA cells. Graph represents average expression from three different experiments in 2–6 different clones and assayed in quadruplet. Error bars indicate standard deviation. (B) Co-immunoprecipitation showing that immunoprecipitated Suz12-TAP associates with Pcl2 whereas immunoprecipitated Pcl3-TAP does not. Pcl3-TAP does bind Suz12 as seen in the lower blot. α-tubulin was used as a loading control. Reciprocal co-immunoprecipitations showing a Pcl2-specific association with Suz12-TAP but not an association between Pcl2 and Pcl3-TAP. β-actin was used as a loading control. Each blot is representative of three different experiments. (C) Graph depicts chromosome-wise distributions of Pcl2 co-localization with sites depleted of Suz12 following Pcl3 knockdown (blue). Regions containing only Suz12 depletion and only Pcl2 binding are indicated in cyan and orange respectively. (D) Decreased Suz12 binding upon Pcl3 knockdown occurs at 86% of targets bound by Suz12 and Pcl2 (Fisher test p-value

Techniques Used: Binding Assay, shRNA, Expressing, Clone Assay, Standard Deviation, Immunoprecipitation

Pcl3 regulates Suz12, but not complex stability. (A) PRC2 components associate in the absence of Pcl3 based on co-immunoprecipitation. β-actin used as a loading control. (B) Protein levels of Suz12, Ezh2, and Eed in scramble and Pcl3 shRNA clones as measured by immunoblot. Experiments were performed with 3–6 scramble and Pcl3 shRNA clones each. (C) Expression levels of Suz12 , Ezh2 , and Eed in scramble and Pcl3 shRNA measured by qRT-PCR. (D) Suz12 mRNA levels measured by qRT-PCR in wild type and Pcl3 overexpressing cells. (E) Suz12 protein levels in wild type cells and wild type cells overexpressing Pcl3-TAP . All immunoblot and co-immunoprecipitation experiments were performed 2–5 times. All expression analysis represents three experiments assayed in quadruplicate. Error bars represent standard deviation, and asterisk indicates statistical significance of p
Figure Legend Snippet: Pcl3 regulates Suz12, but not complex stability. (A) PRC2 components associate in the absence of Pcl3 based on co-immunoprecipitation. β-actin used as a loading control. (B) Protein levels of Suz12, Ezh2, and Eed in scramble and Pcl3 shRNA clones as measured by immunoblot. Experiments were performed with 3–6 scramble and Pcl3 shRNA clones each. (C) Expression levels of Suz12 , Ezh2 , and Eed in scramble and Pcl3 shRNA measured by qRT-PCR. (D) Suz12 mRNA levels measured by qRT-PCR in wild type and Pcl3 overexpressing cells. (E) Suz12 protein levels in wild type cells and wild type cells overexpressing Pcl3-TAP . All immunoblot and co-immunoprecipitation experiments were performed 2–5 times. All expression analysis represents three experiments assayed in quadruplicate. Error bars represent standard deviation, and asterisk indicates statistical significance of p

Techniques Used: Immunoprecipitation, shRNA, Clone Assay, Expressing, Quantitative RT-PCR, Standard Deviation

Pcl3 affects Suz12 binding at a subset of PRC2 targets. (A) Graph depicts chromosome-wise distributions of decreased Suz12 and H3K27me3 ChIP-seq reads upon Pcl3 knockdown ( Pcl3 KD) relative to scramble control. Many regions display both Suz12 and H3K27me3 depletion (blue). Independently decreased Suz12 occupancy and H3K27me3 are marked in cyan and orange, respectively. ChIP-seq peaks were called at a Skellam distribution p-value cutoff of 10 −7 . Fisher test p-value was 3.2×10 −41 for the overlap of H3K27me3 with Suz12 sites. (B) Reduced Suz12 and H3K27me3 ChIP-seq read density upon Pcl3 knockdown is correlated with gene density. Pearson correlation is 0.40 for Suz12 and 0.26 for H3K27me3. (C) 65% of PRC2 targets show decreased Suz12 binding upon Pcl3 knockdown, particularly bivalent genes (Fisher test p = 4.9×10 −250 ). (D) Binding of Pcl3 and Suz12, and histone mark profiles of H3K27me3 at the Hoxa3 gene and in the region surrounding mir-196a1 . Units are number of reads/250 bp. (E) Log fold-changes in Suz12 and H3K27me3 ChIP-seq read density at specific genes in Pcl 3 knockdown cells compared to scramble control. (F) Levels of Suz12 binding and H3K27me3 in Suz12 Suz12TAP/+ cells expressing either scramble or Pcl3 shRNA assessed by FlagM2 or H3K27me3 ChIP-qRT-PCR. ChIP-qRT-PCR experiments were performed at 3–4 times and assayed in quadruplicate. Error bars indicate standard deviation. Decreases are statistically significant, p
Figure Legend Snippet: Pcl3 affects Suz12 binding at a subset of PRC2 targets. (A) Graph depicts chromosome-wise distributions of decreased Suz12 and H3K27me3 ChIP-seq reads upon Pcl3 knockdown ( Pcl3 KD) relative to scramble control. Many regions display both Suz12 and H3K27me3 depletion (blue). Independently decreased Suz12 occupancy and H3K27me3 are marked in cyan and orange, respectively. ChIP-seq peaks were called at a Skellam distribution p-value cutoff of 10 −7 . Fisher test p-value was 3.2×10 −41 for the overlap of H3K27me3 with Suz12 sites. (B) Reduced Suz12 and H3K27me3 ChIP-seq read density upon Pcl3 knockdown is correlated with gene density. Pearson correlation is 0.40 for Suz12 and 0.26 for H3K27me3. (C) 65% of PRC2 targets show decreased Suz12 binding upon Pcl3 knockdown, particularly bivalent genes (Fisher test p = 4.9×10 −250 ). (D) Binding of Pcl3 and Suz12, and histone mark profiles of H3K27me3 at the Hoxa3 gene and in the region surrounding mir-196a1 . Units are number of reads/250 bp. (E) Log fold-changes in Suz12 and H3K27me3 ChIP-seq read density at specific genes in Pcl 3 knockdown cells compared to scramble control. (F) Levels of Suz12 binding and H3K27me3 in Suz12 Suz12TAP/+ cells expressing either scramble or Pcl3 shRNA assessed by FlagM2 or H3K27me3 ChIP-qRT-PCR. ChIP-qRT-PCR experiments were performed at 3–4 times and assayed in quadruplicate. Error bars indicate standard deviation. Decreases are statistically significant, p

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Expressing, shRNA, Quantitative RT-PCR, Standard Deviation

Pcl3 promotes PRC2 function. (A) Immunoblot showing levels of H3K27me3 in multiple clones of scramble and Pcl3 shRNA ESCs and EBs. (B) H3K27me3 levels in Suz12 and Pcl3 siRNA treated cells. (C) Increased levels of H3K27me3 as measured by immunoblot in cells overexpressing Pcl3. (D) Immunoblot of H3K27me3, H2AK119Ub, H3K9me3, H3K4me3, and H3K27ac levels in histones from scramble and Pcl3 shRNA-expressing cells. (E) Pcl3-TAP resistant to Pcl3 shRNA was reintroduced into Pcl3 shRNA cells, immunoprecipitated, and detected with anti-FlagM2. Suz12 Suz12TAP/+ cells were used as a positive control. (F) Pcl3-TAP binds Suz12, Eed, and Ezh2. Lysates from scramble and Pcl3 shRNA cells containing Pcl3-TAP were immunoprecipitated with FlagM2 and immunoblotted for Suz12, Eed, and Ezh2. (G) qRT-PCR shows partial rescue of Pcl3 expression in Pcl3 shRNA clones expressing Pcl3-TAP . Error bars indicate standard deviation. Graph represents average expression from 3–6 different clones in three experiments assayed in quadruplet. (H) Immunoblot showing restoration of H3K27me3 levels in Pcl3 shRNA cells transduced with Pcl3-TAP. Histone H3 and α-tubulin were used as loading controls. All westerns and immunoprecipitations were performed three or more times with 2–6 clones.
Figure Legend Snippet: Pcl3 promotes PRC2 function. (A) Immunoblot showing levels of H3K27me3 in multiple clones of scramble and Pcl3 shRNA ESCs and EBs. (B) H3K27me3 levels in Suz12 and Pcl3 siRNA treated cells. (C) Increased levels of H3K27me3 as measured by immunoblot in cells overexpressing Pcl3. (D) Immunoblot of H3K27me3, H2AK119Ub, H3K9me3, H3K4me3, and H3K27ac levels in histones from scramble and Pcl3 shRNA-expressing cells. (E) Pcl3-TAP resistant to Pcl3 shRNA was reintroduced into Pcl3 shRNA cells, immunoprecipitated, and detected with anti-FlagM2. Suz12 Suz12TAP/+ cells were used as a positive control. (F) Pcl3-TAP binds Suz12, Eed, and Ezh2. Lysates from scramble and Pcl3 shRNA cells containing Pcl3-TAP were immunoprecipitated with FlagM2 and immunoblotted for Suz12, Eed, and Ezh2. (G) qRT-PCR shows partial rescue of Pcl3 expression in Pcl3 shRNA clones expressing Pcl3-TAP . Error bars indicate standard deviation. Graph represents average expression from 3–6 different clones in three experiments assayed in quadruplet. (H) Immunoblot showing restoration of H3K27me3 levels in Pcl3 shRNA cells transduced with Pcl3-TAP. Histone H3 and α-tubulin were used as loading controls. All westerns and immunoprecipitations were performed three or more times with 2–6 clones.

Techniques Used: Clone Assay, shRNA, Expressing, Immunoprecipitation, Positive Control, Quantitative RT-PCR, Standard Deviation, Transduction

Pcl3 localizes to PRC2 targets. (A) Heatmaps showing Pcl3 and Suz12 ChIP-seq read density in counts per 100 bp around Pcl3 peak centers. Approximately 44% of Suz12 targets are bound by Pcl3-TAP. Each row corresponds to a Pcl3 ChIP-seq peak with rows ranked by Pcl3 peak significance (assessed by the Skellam distribution p-values). (B) Graph of −log p-values indicating that the most significant Suz12 ChIP-seq peaks overlap with Pcl3, while regions containing less significant Suz12 ChIP-seq peaks are not bound by Pcl3. Wilcoxon p-value
Figure Legend Snippet: Pcl3 localizes to PRC2 targets. (A) Heatmaps showing Pcl3 and Suz12 ChIP-seq read density in counts per 100 bp around Pcl3 peak centers. Approximately 44% of Suz12 targets are bound by Pcl3-TAP. Each row corresponds to a Pcl3 ChIP-seq peak with rows ranked by Pcl3 peak significance (assessed by the Skellam distribution p-values). (B) Graph of −log p-values indicating that the most significant Suz12 ChIP-seq peaks overlap with Pcl3, while regions containing less significant Suz12 ChIP-seq peaks are not bound by Pcl3. Wilcoxon p-value

Techniques Used: Chromatin Immunoprecipitation

34) Product Images from "KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis"

Article Title: KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.238

KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of H3K4me3 expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P
Figure Legend Snippet: KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of H3K4me3 expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P

Techniques Used: Quantitative RT-PCR, Western Blot, Infection, ALP Assay, Staining, Activity Assay, Expressing, Immunostaining, Isolation, Mouse Assay

KDM5A inhibited Runx2 expression in MSC by removal of H3K4me3 marks. ( a ) Western blot analysis of p-Smad1/5/8, Smad1 and Smad4 in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. ( b ) Quantitative analysis of p-Smad1 expression. Smad1 was used as internal control. ( c ) Immunostaining of p-Smad1/5/8 (green) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. Scale bar, 20 μ m. ( d ) Schematics of Runx2 promoter denoting ChIP-PCR amplified region (−1105 bp to −1065 bp) encompassing the SMAD binding element and the control region 6-kb upstream of the transcription start site (−6173 bp to −6034 bp). ( e ) Western blot analysis of H3K4me3 in MSCs after 0, 3, 7 and 14 days BMP2 treatment. ( f ) Occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment. ( g ) SMAD5 occupancy at the Runx2 promoter after BMP2 treatment. ( h ) Western blot analysis of H3K4me3 in MSCs of sham and OVX mice. ( i ) Occupancy of H3K4me3 at the Runx2 promoter in MSCs of sham and OVX mice following BMP2 treatment. ( j ) Knockdown of Kdm5a increased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. ( k ) Overexpression Kdm5a decreased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P
Figure Legend Snippet: KDM5A inhibited Runx2 expression in MSC by removal of H3K4me3 marks. ( a ) Western blot analysis of p-Smad1/5/8, Smad1 and Smad4 in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. ( b ) Quantitative analysis of p-Smad1 expression. Smad1 was used as internal control. ( c ) Immunostaining of p-Smad1/5/8 (green) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. Scale bar, 20 μ m. ( d ) Schematics of Runx2 promoter denoting ChIP-PCR amplified region (−1105 bp to −1065 bp) encompassing the SMAD binding element and the control region 6-kb upstream of the transcription start site (−6173 bp to −6034 bp). ( e ) Western blot analysis of H3K4me3 in MSCs after 0, 3, 7 and 14 days BMP2 treatment. ( f ) Occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment. ( g ) SMAD5 occupancy at the Runx2 promoter after BMP2 treatment. ( h ) Western blot analysis of H3K4me3 in MSCs of sham and OVX mice. ( i ) Occupancy of H3K4me3 at the Runx2 promoter in MSCs of sham and OVX mice following BMP2 treatment. ( j ) Knockdown of Kdm5a increased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. ( k ) Overexpression Kdm5a decreased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

Techniques Used: Expressing, Western Blot, Infection, Plasmid Preparation, Immunostaining, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Binding Assay, Mouse Assay, Over Expression

KDM5A overexpression impaired osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after 0, 3, 7 and 14 days osteogenic induction. ( c ) qRT-PCR analysis and ( d ) western blot analysis of Kdm5a in MSCs after infected with lentiviral vector (MSC/V) and lentiviral-Kdm5a (MSC/KDM5A). ( e ) Western blot analysis of H3K4me3, H3K9me3 and H3K27me3 in MSCs with overexpression of Kdm5a. ( f ) ALP activity o f MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction were detected with ALP staining and quantified. ( g ) Mineralized nodules formed by MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 14 days of osteogenic induction were detected with Alizarin red staining and quantified. ( h ) qRT-PCR analysis and ( i ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. ( j ) Immunostaining of RUNX2 (red) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. Scale bar, 20 μ m. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P
Figure Legend Snippet: KDM5A overexpression impaired osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after 0, 3, 7 and 14 days osteogenic induction. ( c ) qRT-PCR analysis and ( d ) western blot analysis of Kdm5a in MSCs after infected with lentiviral vector (MSC/V) and lentiviral-Kdm5a (MSC/KDM5A). ( e ) Western blot analysis of H3K4me3, H3K9me3 and H3K27me3 in MSCs with overexpression of Kdm5a. ( f ) ALP activity o f MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction were detected with ALP staining and quantified. ( g ) Mineralized nodules formed by MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 14 days of osteogenic induction were detected with Alizarin red staining and quantified. ( h ) qRT-PCR analysis and ( i ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. ( j ) Immunostaining of RUNX2 (red) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. Scale bar, 20 μ m. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot, Infection, Plasmid Preparation, ALP Assay, Activity Assay, Staining, Expressing, Immunostaining

35) Product Images from "Characterization of STAT6 Target Genes in Human B Cells and Lung Epithelial Cells"

Article Title: Characterization of STAT6 Target Genes in Human B Cells and Lung Epithelial Cells

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsr025

Status of histone modifications and pol II binding in the proximal regions of STAT6 target TSSs. (A) Distribution of the averaged ChIP Seq tags for H3K4me3 (top panels), H3Ac (middle panels) and pol II (bottom panels) in Ramos cells. Data from active target genes (STAT6 binding plus TSS induction in Ramos cells) are shown in the left panels, and data from silent target genes (STAT6 binding plus TSS induction in BEAS2B cells but both negative in Ramos cells) are shown in the right panels. Blue, green, red and purple lines indicate the results for the IP (IL-4 (+)), IP (IL-4 (−)), WCE (IL-4 (+)) and WCE (IL-4 (−)) experiments, respectively. On the x -axis, the position of the associated TSS is designated as zero. (B) Results of an analysis similar to that shown in (A) in BEAS2B cells.
Figure Legend Snippet: Status of histone modifications and pol II binding in the proximal regions of STAT6 target TSSs. (A) Distribution of the averaged ChIP Seq tags for H3K4me3 (top panels), H3Ac (middle panels) and pol II (bottom panels) in Ramos cells. Data from active target genes (STAT6 binding plus TSS induction in Ramos cells) are shown in the left panels, and data from silent target genes (STAT6 binding plus TSS induction in BEAS2B cells but both negative in Ramos cells) are shown in the right panels. Blue, green, red and purple lines indicate the results for the IP (IL-4 (+)), IP (IL-4 (−)), WCE (IL-4 (+)) and WCE (IL-4 (−)) experiments, respectively. On the x -axis, the position of the associated TSS is designated as zero. (B) Results of an analysis similar to that shown in (A) in BEAS2B cells.

Techniques Used: Binding Assay, Chromatin Immunoprecipitation

36) Product Images from "Polycomb Repressive Complex 2 Targets Murine Cytomegalovirus Chromatin for Modification and Associates with Viral Replication Centers"

Article Title: Polycomb Repressive Complex 2 Targets Murine Cytomegalovirus Chromatin for Modification and Associates with Viral Replication Centers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029410

Western blot analysis of cellular H3K27me3 levels during MCMV infection. Whole cell lysates were prepared from mock-infected or MCMV-infected fibroblasts at the indicated times. Blots were probed with antibodies against H3K27me3 or total H3.
Figure Legend Snippet: Western blot analysis of cellular H3K27me3 levels during MCMV infection. Whole cell lysates were prepared from mock-infected or MCMV-infected fibroblasts at the indicated times. Blots were probed with antibodies against H3K27me3 or total H3.

Techniques Used: Western Blot, Infection

Measurement of H3K27me3 enrichment at the MIE locus of MCMV. (A) Graphical illustration of the MIE locus of MCMV. Solid black boxes represent regions probed for H3K27me3 H3K4me3 enrichment. Regions denoted in figure: (1) Represents the Enhancer 2 region, (2) the transcriptional start site (TSS) of the IE1-3 transcriptional unit and (3) represents Exon 1 within the ORF of the IE1-3 locus. +1 transcriptional start sites are indicated with black arrows. Enhancer regions are marked in hatched boxes. IE1-3 exons are depicted as gray arrows. (B) H3K27me3 is enriched at the MIE locus at pre-IE times during MCMV infection. ChIPs using anti-H3K27me3 antibody were analyzed by Q-PCR using primer/probe sets specific for the indicated loci at 1.5, 3, 6 and 12 hpi as indicated. Each graph displays the mean value and S.E.M. for each queried locus, with data from three independent ChIPs. The results are presented as the IgG subtracted %Input of the queried locus normalized to the IgG subtracted β-Actin %Input. Samples with values that vary significantly from the negative control, Dlx1,are indicated by asterisks (* P value
Figure Legend Snippet: Measurement of H3K27me3 enrichment at the MIE locus of MCMV. (A) Graphical illustration of the MIE locus of MCMV. Solid black boxes represent regions probed for H3K27me3 H3K4me3 enrichment. Regions denoted in figure: (1) Represents the Enhancer 2 region, (2) the transcriptional start site (TSS) of the IE1-3 transcriptional unit and (3) represents Exon 1 within the ORF of the IE1-3 locus. +1 transcriptional start sites are indicated with black arrows. Enhancer regions are marked in hatched boxes. IE1-3 exons are depicted as gray arrows. (B) H3K27me3 is enriched at the MIE locus at pre-IE times during MCMV infection. ChIPs using anti-H3K27me3 antibody were analyzed by Q-PCR using primer/probe sets specific for the indicated loci at 1.5, 3, 6 and 12 hpi as indicated. Each graph displays the mean value and S.E.M. for each queried locus, with data from three independent ChIPs. The results are presented as the IgG subtracted %Input of the queried locus normalized to the IgG subtracted β-Actin %Input. Samples with values that vary significantly from the negative control, Dlx1,are indicated by asterisks (* P value

Techniques Used: Infection, Polymerase Chain Reaction, Negative Control

Validation of ChIPs for (A) H3K27me3 and (B) H3K4me3. Chromatin from uninfected mouse fibroblasts was immunoprecipitated using anti-H3K27me3 antibody. The specificity of the ChIPs was assessed by measuring the %Input by Q-PCR at HoxC11, Dlx1 and β-Actin. Each graph displays the mean value and S.E.M. for each queried locus, with data from four independent ChIPs. *** P value
Figure Legend Snippet: Validation of ChIPs for (A) H3K27me3 and (B) H3K4me3. Chromatin from uninfected mouse fibroblasts was immunoprecipitated using anti-H3K27me3 antibody. The specificity of the ChIPs was assessed by measuring the %Input by Q-PCR at HoxC11, Dlx1 and β-Actin. Each graph displays the mean value and S.E.M. for each queried locus, with data from four independent ChIPs. *** P value

Techniques Used: Immunoprecipitation, Polymerase Chain Reaction

Co-immunofluorescence assay for PRC2 enrichment within MCMV replication compartments. At 18 hpi, mock-infected or MCMV-infected fibroblasts were fixed and incubated with antibodies against M44 and (A) EZH2, (B) SUZ12 or (C) H3K27me3. (D) Purified rabbit IgG served as an isotype control. The first column of panels displays DAPI staining for nuclei. The second column of panels displays M44 staining, marking MCMV replication compartments. The third column of panels displays staining EZH2, SUZ12, H3K27me3 or isotype IgG. The fourth column of panels displays a merged image of the first three channels. The fifth column of panels displays a high-magnification image of the merged image from the fourth column. All images are 40×, unless otherwise indicated.
Figure Legend Snippet: Co-immunofluorescence assay for PRC2 enrichment within MCMV replication compartments. At 18 hpi, mock-infected or MCMV-infected fibroblasts were fixed and incubated with antibodies against M44 and (A) EZH2, (B) SUZ12 or (C) H3K27me3. (D) Purified rabbit IgG served as an isotype control. The first column of panels displays DAPI staining for nuclei. The second column of panels displays M44 staining, marking MCMV replication compartments. The third column of panels displays staining EZH2, SUZ12, H3K27me3 or isotype IgG. The fourth column of panels displays a merged image of the first three channels. The fifth column of panels displays a high-magnification image of the merged image from the fourth column. All images are 40×, unless otherwise indicated.

Techniques Used: Immunofluorescence, Infection, Incubation, Purification, Staining

37) Product Images from "Integrative Analysis from the Epigenome to Translatome Uncovers Patterns of Dominant Nuclear Regulation during Transient Stress [OPEN]"

Article Title: Integrative Analysis from the Epigenome to Translatome Uncovers Patterns of Dominant Nuclear Regulation during Transient Stress [OPEN]

Journal: The Plant Cell

doi: 10.1105/tpc.19.00463

Multiscale Chromatin and RNA Gene Regulatory Analyses of Control (Normoxic) and Hypoxic Arabidopsis Seedlings. (A) Schematic of the experiments performed. Seven-day–old seedlings were subjected to control (normoxic; 2NS, 9NS), hypoxic stress (2HS, 9HS), or reoxygenation after 2HS (R) conditions. 2NS is ZT16, and 9NS is ZT1, with 1-h extended darkness. Vertical arrows indicate time of harvest. ChIP was performed to evaluate genomic regions bound by Ser2P, the Histone 2 variant H2A.Z, modified Histone 3 (H3K4me3, H3K27me3, H3K9ac, and H3K14ac), and the ERF TF, HRE2. INTACT purified nuclei were used for the ATAC and purification of nRNA. Ribosome-associated mRNA was obtained by TRAP. (B) Individual replicate samples of nRNA, polyA RNA, and TRAP mRNA [TRAP] were compared by t-SNE. (C) to (E) Distributions of histone modifications/variants across genic regions (C) for the core HRG s ( n = 49; D) and cytosolic RP s ( n = 246; E). Read distributions (RPKM * 1,000) are plotted from 1 kb upstream to 1 kb downstream of gene units defined by the TSS and TES.
Figure Legend Snippet: Multiscale Chromatin and RNA Gene Regulatory Analyses of Control (Normoxic) and Hypoxic Arabidopsis Seedlings. (A) Schematic of the experiments performed. Seven-day–old seedlings were subjected to control (normoxic; 2NS, 9NS), hypoxic stress (2HS, 9HS), or reoxygenation after 2HS (R) conditions. 2NS is ZT16, and 9NS is ZT1, with 1-h extended darkness. Vertical arrows indicate time of harvest. ChIP was performed to evaluate genomic regions bound by Ser2P, the Histone 2 variant H2A.Z, modified Histone 3 (H3K4me3, H3K27me3, H3K9ac, and H3K14ac), and the ERF TF, HRE2. INTACT purified nuclei were used for the ATAC and purification of nRNA. Ribosome-associated mRNA was obtained by TRAP. (B) Individual replicate samples of nRNA, polyA RNA, and TRAP mRNA [TRAP] were compared by t-SNE. (C) to (E) Distributions of histone modifications/variants across genic regions (C) for the core HRG s ( n = 49; D) and cytosolic RP s ( n = 246; E). Read distributions (RPKM * 1,000) are plotted from 1 kb upstream to 1 kb downstream of gene units defined by the TSS and TES.

Techniques Used: Chromatin Immunoprecipitation, Variant Assay, Modification, Purification

38) Product Images from "KDM5 histone demethylase activity links cellular transcriptomic heterogeneity to therapeutic resistance"

Article Title: KDM5 histone demethylase activity links cellular transcriptomic heterogeneity to therapeutic resistance

Journal: Cancer cell

doi: 10.1016/j.ccell.2018.10.014

KDM5 activity and H3K4me3 peak broadness. (A) H3K4me3 and H3K4me2 peak width plotted against peak height before and at different time points (day 0–14) after treatment with KDM5-C70 inhibitor. Mean values are shown as dotted lines. Shaded areas indicate interquartile range. (B) Gene tracks depicting KDM5B and H3K4me3 signal at selected genomic loci. X-axis shows position along the chromosome with gene structures drawn below, whereas y-axis shows genomic occupancy in units of reads per million reads (RPM). (C) Correlation between promoter H3K4me3 peak broadness changes and changes in percent of cells expressing the corresponding gene in KDM5C70-treated cells. Enrichment analysis of H3K4me3 width increase in C70 is performed against the genes with increased percent of expressing cells in C70 for all genes or genes without expression change. H3K4me3 width changes are calculated as the average width changes across all six cell lines. *** fdr
Figure Legend Snippet: KDM5 activity and H3K4me3 peak broadness. (A) H3K4me3 and H3K4me2 peak width plotted against peak height before and at different time points (day 0–14) after treatment with KDM5-C70 inhibitor. Mean values are shown as dotted lines. Shaded areas indicate interquartile range. (B) Gene tracks depicting KDM5B and H3K4me3 signal at selected genomic loci. X-axis shows position along the chromosome with gene structures drawn below, whereas y-axis shows genomic occupancy in units of reads per million reads (RPM). (C) Correlation between promoter H3K4me3 peak broadness changes and changes in percent of cells expressing the corresponding gene in KDM5C70-treated cells. Enrichment analysis of H3K4me3 width increase in C70 is performed against the genes with increased percent of expressing cells in C70 for all genes or genes without expression change. H3K4me3 width changes are calculated as the average width changes across all six cell lines. *** fdr

Techniques Used: Activity Assay, Expressing

39) Product Images from "C/EBP? deregulation results in differentiation arrest in acute myeloid leukemia"

Article Title: C/EBP? deregulation results in differentiation arrest in acute myeloid leukemia

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI65102

Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in vivo.
Figure Legend Snippet: Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in vivo.

Techniques Used: In Vivo

DAC treatment restores the C/EBPα-C/EBPγ balance and promotes differentiation of primary human AML samples characterized by C/EBPα silencing and C/EBPγ upregulation in vitro.
Figure Legend Snippet: DAC treatment restores the C/EBPα-C/EBPγ balance and promotes differentiation of primary human AML samples characterized by C/EBPα silencing and C/EBPγ upregulation in vitro.

Techniques Used: In Vitro

C/EBPα interacts with the Cebpg promoter and mediates Cebpg repression.
Figure Legend Snippet: C/EBPα interacts with the Cebpg promoter and mediates Cebpg repression.

Techniques Used:

Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in cell culture.
Figure Legend Snippet: Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in cell culture.

Techniques Used: Cell Culture

CEBPG RNA is upregulated in the absence of CEBPA in a subset of human AML and in murine C/EBPα-deficient hematopoietic stem/progenitor cells.
Figure Legend Snippet: CEBPG RNA is upregulated in the absence of CEBPA in a subset of human AML and in murine C/EBPα-deficient hematopoietic stem/progenitor cells.

Techniques Used:

40) Product Images from "Histone H4K20 tri‐methylation at late‐firing origins ensures timely heterochromatin replication"

Article Title: Histone H4K20 tri‐methylation at late‐firing origins ensures timely heterochromatin replication

Journal: The EMBO Journal

doi: 10.15252/embj.201796541

H4K20 mutation affects S‐phase progression and prevents DNA re‐replication induced by PR‐Set7 stabilization Immunoblot analysis of histone H4 and FLAG‐tagged histone H4 protein levels in FLAG‐H4 WT and FLAG‐H4 K20A U2OS cells and subjected to biochemical fractionation: Cytosolic (S1) and nuclear (S2) are soluble supernatants and P3 is the chromatin‐enriched fraction. MEK1 was used as a control of soluble components and HCF‐1 protein was used for control of chromatin fraction. Immunoblot analysis of PR‐Set7 and the levels of acetylation and methylation of endogenous H4 and FLAG‐tagged H4 in FLAG‐H4 WT and FLAG‐H4 K20A U2OS cells. FACS analysis of DNA content and FLAG signal in FLAG‐H4 WT or FLAG‐H4 K20A cells. DNA content was analyzed according to the low (gate 1) and high (gate 2) levels of FLAG‐tagged histone H4 proteins. FACS analysis of DNA content in cells expressing similar levels (gate 1) of FLAG‐tagged histone H4 WT and H4 K20A upon expression of the PR‐Set7 PIPmut and PR‐Set7 PIPmut+SETmut mutants. Quantitation of re‐replicating parental (No FLAG), FLAG‐H4 WT , and H4 K20A cells upon PR‐Set7 PIPmut expression. Data are means ± SD, n = 3. (*) Statistical significance with P
Figure Legend Snippet: H4K20 mutation affects S‐phase progression and prevents DNA re‐replication induced by PR‐Set7 stabilization Immunoblot analysis of histone H4 and FLAG‐tagged histone H4 protein levels in FLAG‐H4 WT and FLAG‐H4 K20A U2OS cells and subjected to biochemical fractionation: Cytosolic (S1) and nuclear (S2) are soluble supernatants and P3 is the chromatin‐enriched fraction. MEK1 was used as a control of soluble components and HCF‐1 protein was used for control of chromatin fraction. Immunoblot analysis of PR‐Set7 and the levels of acetylation and methylation of endogenous H4 and FLAG‐tagged H4 in FLAG‐H4 WT and FLAG‐H4 K20A U2OS cells. FACS analysis of DNA content and FLAG signal in FLAG‐H4 WT or FLAG‐H4 K20A cells. DNA content was analyzed according to the low (gate 1) and high (gate 2) levels of FLAG‐tagged histone H4 proteins. FACS analysis of DNA content in cells expressing similar levels (gate 1) of FLAG‐tagged histone H4 WT and H4 K20A upon expression of the PR‐Set7 PIPmut and PR‐Set7 PIPmut+SETmut mutants. Quantitation of re‐replicating parental (No FLAG), FLAG‐H4 WT , and H4 K20A cells upon PR‐Set7 PIPmut expression. Data are means ± SD, n = 3. (*) Statistical significance with P

Techniques Used: Mutagenesis, Fractionation, Methylation, FACS, Expressing, Quantitation Assay

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Immunodetection:

Article Title: Histone acetyltransferase inhibitor CPTH6 preferentially targets lung cancer stem-like cells
Article Snippet: .. Immunodetection was performed using antibodies directed to: H3 histone (abcam), H3 acetylated histone (Millipore, Billerica, MA, USA), α-tubulin (DM1A) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), acetyl-α-tubulin (K40) (Sigma-Aldrich), γH2AX (Ser139) (Millipore), PARP cleaved (Millipore), PARP (Santa Cruz Biotechnology) β-actin (Sigma-Aldrich), HSP72/73 (Calbiochem, San Diego, CA, USA), anti-mouse or anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugated antibodies (Cell Signaling; Amersham Biosciences, Freiburg, Germany). .. Antibody binding was visualized by enhanced chemiluminescence method (Amersham Biosciences) according to manufacturer's specification and recorded on autoradiographic film (Amersham Biosciences).

Immunostaining:

Article Title: UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119 *
Article Snippet: .. The antibodies used for immunoblotting and immunostaining experiments include the mouse antibody to ubiquitin (clone P4G7, MMS-258R, Covance), actin (SC-1616, Santa Cruz), Myc (clone 4A6, 05–724, Millipore), FLAG M2 (F3165, Sigma), mouse, rabbit, and goat antibodies to UbE2E1 (611218, BD, A-630, Boston Biochem and SC-475478, Santa Cruz), USP7 (A300-033, Bethyl), Histone H2A (07–146, Millipore), ubiquitin-Histone H2A Lys-119 (clone D27C4, 8240, Cell Signaling), p16 (Neomarkers), UbcH5/UbE2D (A-615, Boston Biochem), Ring1A (clone D2P4D, 13069, Cell Signaling), CBX2 (ab80044, ABCAM), BMI1 (5856, Cell Signaling), Ring1B (clone D22F2, 5694, Cell Signaling and Active Motif), and H3K4me2/3 (ab6000, ABCAM). ..

other:

Article Title: Super-low Dose Endotoxin Pre-conditioning Exacerbates Sepsis Mortality
Article Snippet: Anti-citrullinated histone H3 antibody was from Abcam (Cambridge, MA) and the anti-citrullinated Histone H4 antibody was from Millipore (Billerica, MA).

Article Title: UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119 *
Article Snippet: A., Endoh M., Appanah R., Nesterova T. B., Silva J., Otte A. P., Vidal M., Koseki H., and Brockdorff N. (2004) Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X inactivation .

Western Blot:

Article Title: Histone deacetylase inhibitors induce growth arrest and differentiation in uveal melanoma
Article Snippet: .. Western blotting was performed as previously described ( ) using anti-BAP1 (1:250; Santa Cruz), anti-alpha-tubulin (loading control, 1:1000; Sigma), anti-acetyl-histone H3Lys9 (1:200; Cell signaling), anti-histone H3 (1:200; Cell signaling), anti-ubiquityl-histone H2A (1:100; Millipore), and anti-histone H2A (1:200; Millipore) antibodies. .. Histone proteins for western blotting were extracted from cells according to previously published protocol ( ).

Article Title: Chromosome alignment maintenance requires the MAP RECQL4, mutated in the Rothmund–Thomson syndrome
Article Snippet: .. The following published and commercial antibodies were used: XCAP-G for Western blot at 1 μg/ml , chTOG antibody for Western blot at 1 μg/ml , Xenopus Ndc80/Hec1 antibody at 1 μg/ml for immunofluorescence , human histone H2B antibody (Millipore, used 1:1,000 for Western blotting), BubR1 (Millipore, at 1:500 for immunofluorescence), CREST antibody (Antibody Inc., at 1:200 for immunofluorescence), phospho-histone H2AX (Cell Signaling, at 1:200 for immunofluorescence), α-tubulin (mouse DM1A; Sigma-Aldrich, at 1:200 for immunofluorescence), and γ-tubulin at 1 μg/ml for immunofluorescence ( ). .. Secondary antibodies for immunofluorescence were Alexa-Fluor-488-anti-mouse, Alexa-Fluor-647-anti-human, and Alexa-Fluor-647-anti-rabbit (from Life Technologies, used at 1:1,000).

Binding Assay:

Article Title: Statins inhibit tumor progression via an enhancer of zeste homolog 2-mediated epigenetic alteration in colorectal cancer
Article Snippet: .. The antibodies used were: HDAC3 (Cell Signaling, MA, USA), HDAC5 (Santa-Cruz, Texas, USA), HDAC7 (Santa Cruz), EZH2 (BD Biosciences, CA, USA), sterol regulatory element binding protein 2 (SREBP2; Cayman, Michigan, USA), p27 (BD Biosciences), β-actin (Cell Signaling), Histone1 (Sigma). .. EZH2 suppression by small interfering RNA (siRNA) duplex oligo ribonucleotide EZH2 suppression was performed using siRNA and Stealth RNAi siRNA duplex oligo nucleotides (Invitrogen).

Immunofluorescence:

Article Title: Chromosome alignment maintenance requires the MAP RECQL4, mutated in the Rothmund–Thomson syndrome
Article Snippet: .. The following published and commercial antibodies were used: XCAP-G for Western blot at 1 μg/ml , chTOG antibody for Western blot at 1 μg/ml , Xenopus Ndc80/Hec1 antibody at 1 μg/ml for immunofluorescence , human histone H2B antibody (Millipore, used 1:1,000 for Western blotting), BubR1 (Millipore, at 1:500 for immunofluorescence), CREST antibody (Antibody Inc., at 1:200 for immunofluorescence), phospho-histone H2AX (Cell Signaling, at 1:200 for immunofluorescence), α-tubulin (mouse DM1A; Sigma-Aldrich, at 1:200 for immunofluorescence), and γ-tubulin at 1 μg/ml for immunofluorescence ( ). .. Secondary antibodies for immunofluorescence were Alexa-Fluor-488-anti-mouse, Alexa-Fluor-647-anti-human, and Alexa-Fluor-647-anti-rabbit (from Life Technologies, used at 1:1,000).

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  • 97
    Millipore anti h3k4me3
    KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of <t>H3K4me3</t> expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P
    Anti H3k4me3, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore antibodies against histone h3k4me3
    DNA methylation and <t>H3K4me3</t> in the normal livers of A/J and WSB/EiJ mice. a MeDIP microarray analysis of CpG island methylation in the normal livers of A/J and WSB/EiJ mice. b ChIP-on-chip analysis of H3K4me3 in the normal livers of A/J and WSB/EiL mice. Heat map illustrating significant differences in hepatic CpG island methylation between A/J and WSB/EiJ control mice. Unsupervised hierarchical clustering analysis was performed using one-way ANOVA with p value cut-off at 0.05. The color bar identifies high-methylated or high H3K4me3-enriched ( red ) and low-methylated or low H3K4me3-enriched ( green ) CpG islands
    Antibodies Against Histone H3k4me3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against histone h3k4me3/product/Millipore
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    KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of H3K4me3 expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P

    Journal: Cell Death & Disease

    Article Title: KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

    doi: 10.1038/cddis.2016.238

    Figure Lengend Snippet: KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of H3K4me3 expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P

    Article Snippet: Immunoprecipitation (IP) was carried out overnight with purified anti-H3K4me3 and SMAD5 antibody (Millipore, Bedford, MA, USA) or normal mouse IgG as a negative control.

    Techniques: Quantitative RT-PCR, Western Blot, Infection, ALP Assay, Staining, Activity Assay, Expressing, Immunostaining, Isolation, Mouse Assay

    KDM5A inhibited Runx2 expression in MSC by removal of H3K4me3 marks. ( a ) Western blot analysis of p-Smad1/5/8, Smad1 and Smad4 in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. ( b ) Quantitative analysis of p-Smad1 expression. Smad1 was used as internal control. ( c ) Immunostaining of p-Smad1/5/8 (green) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. Scale bar, 20 μ m. ( d ) Schematics of Runx2 promoter denoting ChIP-PCR amplified region (−1105 bp to −1065 bp) encompassing the SMAD binding element and the control region 6-kb upstream of the transcription start site (−6173 bp to −6034 bp). ( e ) Western blot analysis of H3K4me3 in MSCs after 0, 3, 7 and 14 days BMP2 treatment. ( f ) Occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment. ( g ) SMAD5 occupancy at the Runx2 promoter after BMP2 treatment. ( h ) Western blot analysis of H3K4me3 in MSCs of sham and OVX mice. ( i ) Occupancy of H3K4me3 at the Runx2 promoter in MSCs of sham and OVX mice following BMP2 treatment. ( j ) Knockdown of Kdm5a increased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. ( k ) Overexpression Kdm5a decreased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Journal: Cell Death & Disease

    Article Title: KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

    doi: 10.1038/cddis.2016.238

    Figure Lengend Snippet: KDM5A inhibited Runx2 expression in MSC by removal of H3K4me3 marks. ( a ) Western blot analysis of p-Smad1/5/8, Smad1 and Smad4 in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. ( b ) Quantitative analysis of p-Smad1 expression. Smad1 was used as internal control. ( c ) Immunostaining of p-Smad1/5/8 (green) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. Scale bar, 20 μ m. ( d ) Schematics of Runx2 promoter denoting ChIP-PCR amplified region (−1105 bp to −1065 bp) encompassing the SMAD binding element and the control region 6-kb upstream of the transcription start site (−6173 bp to −6034 bp). ( e ) Western blot analysis of H3K4me3 in MSCs after 0, 3, 7 and 14 days BMP2 treatment. ( f ) Occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment. ( g ) SMAD5 occupancy at the Runx2 promoter after BMP2 treatment. ( h ) Western blot analysis of H3K4me3 in MSCs of sham and OVX mice. ( i ) Occupancy of H3K4me3 at the Runx2 promoter in MSCs of sham and OVX mice following BMP2 treatment. ( j ) Knockdown of Kdm5a increased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. ( k ) Overexpression Kdm5a decreased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Article Snippet: Immunoprecipitation (IP) was carried out overnight with purified anti-H3K4me3 and SMAD5 antibody (Millipore, Bedford, MA, USA) or normal mouse IgG as a negative control.

    Techniques: Expressing, Western Blot, Infection, Plasmid Preparation, Immunostaining, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Binding Assay, Mouse Assay, Over Expression

    KDM5A overexpression impaired osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after 0, 3, 7 and 14 days osteogenic induction. ( c ) qRT-PCR analysis and ( d ) western blot analysis of Kdm5a in MSCs after infected with lentiviral vector (MSC/V) and lentiviral-Kdm5a (MSC/KDM5A). ( e ) Western blot analysis of H3K4me3, H3K9me3 and H3K27me3 in MSCs with overexpression of Kdm5a. ( f ) ALP activity o f MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction were detected with ALP staining and quantified. ( g ) Mineralized nodules formed by MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 14 days of osteogenic induction were detected with Alizarin red staining and quantified. ( h ) qRT-PCR analysis and ( i ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. ( j ) Immunostaining of RUNX2 (red) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. Scale bar, 20 μ m. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Journal: Cell Death & Disease

    Article Title: KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

    doi: 10.1038/cddis.2016.238

    Figure Lengend Snippet: KDM5A overexpression impaired osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after 0, 3, 7 and 14 days osteogenic induction. ( c ) qRT-PCR analysis and ( d ) western blot analysis of Kdm5a in MSCs after infected with lentiviral vector (MSC/V) and lentiviral-Kdm5a (MSC/KDM5A). ( e ) Western blot analysis of H3K4me3, H3K9me3 and H3K27me3 in MSCs with overexpression of Kdm5a. ( f ) ALP activity o f MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction were detected with ALP staining and quantified. ( g ) Mineralized nodules formed by MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 14 days of osteogenic induction were detected with Alizarin red staining and quantified. ( h ) qRT-PCR analysis and ( i ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. ( j ) Immunostaining of RUNX2 (red) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. Scale bar, 20 μ m. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Article Snippet: Immunoprecipitation (IP) was carried out overnight with purified anti-H3K4me3 and SMAD5 antibody (Millipore, Bedford, MA, USA) or normal mouse IgG as a negative control.

    Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Infection, Plasmid Preparation, ALP Assay, Activity Assay, Staining, Expressing, Immunostaining

    Status of histone modifications and pol II binding in the proximal regions of STAT6 target TSSs. (A) Distribution of the averaged ChIP Seq tags for H3K4me3 (top panels), H3Ac (middle panels) and pol II (bottom panels) in Ramos cells. Data from active target genes (STAT6 binding plus TSS induction in Ramos cells) are shown in the left panels, and data from silent target genes (STAT6 binding plus TSS induction in BEAS2B cells but both negative in Ramos cells) are shown in the right panels. Blue, green, red and purple lines indicate the results for the IP (IL-4 (+)), IP (IL-4 (−)), WCE (IL-4 (+)) and WCE (IL-4 (−)) experiments, respectively. On the x -axis, the position of the associated TSS is designated as zero. (B) Results of an analysis similar to that shown in (A) in BEAS2B cells.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Characterization of STAT6 Target Genes in Human B Cells and Lung Epithelial Cells

    doi: 10.1093/dnares/dsr025

    Figure Lengend Snippet: Status of histone modifications and pol II binding in the proximal regions of STAT6 target TSSs. (A) Distribution of the averaged ChIP Seq tags for H3K4me3 (top panels), H3Ac (middle panels) and pol II (bottom panels) in Ramos cells. Data from active target genes (STAT6 binding plus TSS induction in Ramos cells) are shown in the left panels, and data from silent target genes (STAT6 binding plus TSS induction in BEAS2B cells but both negative in Ramos cells) are shown in the right panels. Blue, green, red and purple lines indicate the results for the IP (IL-4 (+)), IP (IL-4 (−)), WCE (IL-4 (+)) and WCE (IL-4 (−)) experiments, respectively. On the x -axis, the position of the associated TSS is designated as zero. (B) Results of an analysis similar to that shown in (A) in BEAS2B cells.

    Article Snippet: Anti-STAT-6 antibody (Santa Cruz, M-20), anti-RNA pol II antibody (Abcam, ab817), anti-H3K4me3 antibody (ab1012) and anti-H3Ac antibody (Millipore, 06–599) were used for the indicated experiments.

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    Relative enrichment of histone 3 lysine 4 methylation of chromatin surrounding the TCH3 locus in the sdg8 mutants. (A) The position of PCR amplicons used to quantify H3K4 methylation associated with the TCH3 locus is schematically represented. Primer sequences are presented in Supplemental Table 2 online. (B,C) The level of H3K4me3 (B) and H3K4me2 (C) surrounding TCH3 chromatin is presented as a ratio mutant/wild type, following normalization using a region of the house-keeping gene, S-ADENOSYL METHIONINE SYNTHASE (SAM). CRTISO was included as a positive control for H3K4 di- or tri-methylation activity. For most amplicons two biological and 3 technical repeats were performed, except for TCH3-exon2 where only a single biological repeat was successful. In all experiments there was an enrichment of at least 10 fold above the no antibody background.

    Journal: Frontiers in Plant Science

    Article Title: A chromatin modifying enzyme, SDG8, is involved in morphological, gene expression, and epigenetic responses to mechanical stimulation

    doi: 10.3389/fpls.2014.00533

    Figure Lengend Snippet: Relative enrichment of histone 3 lysine 4 methylation of chromatin surrounding the TCH3 locus in the sdg8 mutants. (A) The position of PCR amplicons used to quantify H3K4 methylation associated with the TCH3 locus is schematically represented. Primer sequences are presented in Supplemental Table 2 online. (B,C) The level of H3K4me3 (B) and H3K4me2 (C) surrounding TCH3 chromatin is presented as a ratio mutant/wild type, following normalization using a region of the house-keeping gene, S-ADENOSYL METHIONINE SYNTHASE (SAM). CRTISO was included as a positive control for H3K4 di- or tri-methylation activity. For most amplicons two biological and 3 technical repeats were performed, except for TCH3-exon2 where only a single biological repeat was successful. In all experiments there was an enrichment of at least 10 fold above the no antibody background.

    Article Snippet: Antibodies recognizing H3K4me3 (Millipore Cat#04-745) and H3K4me2 (Millipore Cat#07-030) were purchased from Upstate Biotechnology (Charlottesville, VI, USA).

    Techniques: Methylation, Polymerase Chain Reaction, Mutagenesis, Positive Control, Activity Assay

    DNA methylation and H3K4me3 in the normal livers of A/J and WSB/EiJ mice. a MeDIP microarray analysis of CpG island methylation in the normal livers of A/J and WSB/EiJ mice. b ChIP-on-chip analysis of H3K4me3 in the normal livers of A/J and WSB/EiL mice. Heat map illustrating significant differences in hepatic CpG island methylation between A/J and WSB/EiJ control mice. Unsupervised hierarchical clustering analysis was performed using one-way ANOVA with p value cut-off at 0.05. The color bar identifies high-methylated or high H3K4me3-enriched ( red ) and low-methylated or low H3K4me3-enriched ( green ) CpG islands

    Journal: BMC Genomics

    Article Title: Status of hepatic DNA methylome predetermines and modulates the severity of non-alcoholic fatty liver injury in mice

    doi: 10.1186/s12864-016-2617-2

    Figure Lengend Snippet: DNA methylation and H3K4me3 in the normal livers of A/J and WSB/EiJ mice. a MeDIP microarray analysis of CpG island methylation in the normal livers of A/J and WSB/EiJ mice. b ChIP-on-chip analysis of H3K4me3 in the normal livers of A/J and WSB/EiL mice. Heat map illustrating significant differences in hepatic CpG island methylation between A/J and WSB/EiJ control mice. Unsupervised hierarchical clustering analysis was performed using one-way ANOVA with p value cut-off at 0.05. The color bar identifies high-methylated or high H3K4me3-enriched ( red ) and low-methylated or low H3K4me3-enriched ( green ) CpG islands

    Article Snippet: Histone H3 K4 me3 chromatin immunoprecipitation microarray analysis Formaldehyde crosslinking and chromatin immunoprecipitation (ChIP) assays were performed with primary antibodies against histone H3K4me3 (EMD Millipore, Billerica, MA, USA) using a Magna ChIP™ A chromatin immunoprecipitation kit (EMD Millipore).

    Techniques: DNA Methylation Assay, Mouse Assay, Methylated DNA Immunoprecipitation, Microarray, Methylation, Chromatin Immunoprecipitation