Structured Review

Abcam h3k9ac
Depletion of CRL4 DDB2 components DDB2, DDB1 and CUL4A decrease H3K56Ac levels ( A ) DDB2, H3K56Ac, <t>H3K9Ac,</t> H3K27Ac, H3K27Ac and H3K14 levels were examined in LFS-041B cell line and its derivative cell line with re-established DDB2 expression. ( B ) Cytoplasmic and chromatin fractions of LFS-041B and its derivative were obtained through a cellular protein fractionation protocol. Samples containing identical protein amounts from each fraction were examined by Western blotting for DDB2, H3K56Ac and other acetylated histone H3. Actin and Lamin B blots serve as the loading controls. ( C ) NHFs were transiently transfected with control (Ctrl) siRNA or siRNA targeting DDB2, DDB1 or CUL4A. Levels of DDB2, DDB1 and CUL4A as well as H3K56Ac were detected in whole cell Laemmli extracts by Western blotting.
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Images

1) Product Images from "Human CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1"

Article Title: Human CRL4DDB2 ubiquitin ligase preferentially regulates post-repair chromatin restoration of H3K56Ac through recruitment of histone chaperon CAF-1

Journal: Oncotarget

doi: 10.18632/oncotarget.21869

Depletion of CRL4 DDB2 components DDB2, DDB1 and CUL4A decrease H3K56Ac levels ( A ) DDB2, H3K56Ac, H3K9Ac, H3K27Ac, H3K27Ac and H3K14 levels were examined in LFS-041B cell line and its derivative cell line with re-established DDB2 expression. ( B ) Cytoplasmic and chromatin fractions of LFS-041B and its derivative were obtained through a cellular protein fractionation protocol. Samples containing identical protein amounts from each fraction were examined by Western blotting for DDB2, H3K56Ac and other acetylated histone H3. Actin and Lamin B blots serve as the loading controls. ( C ) NHFs were transiently transfected with control (Ctrl) siRNA or siRNA targeting DDB2, DDB1 or CUL4A. Levels of DDB2, DDB1 and CUL4A as well as H3K56Ac were detected in whole cell Laemmli extracts by Western blotting.
Figure Legend Snippet: Depletion of CRL4 DDB2 components DDB2, DDB1 and CUL4A decrease H3K56Ac levels ( A ) DDB2, H3K56Ac, H3K9Ac, H3K27Ac, H3K27Ac and H3K14 levels were examined in LFS-041B cell line and its derivative cell line with re-established DDB2 expression. ( B ) Cytoplasmic and chromatin fractions of LFS-041B and its derivative were obtained through a cellular protein fractionation protocol. Samples containing identical protein amounts from each fraction were examined by Western blotting for DDB2, H3K56Ac and other acetylated histone H3. Actin and Lamin B blots serve as the loading controls. ( C ) NHFs were transiently transfected with control (Ctrl) siRNA or siRNA targeting DDB2, DDB1 or CUL4A. Levels of DDB2, DDB1 and CUL4A as well as H3K56Ac were detected in whole cell Laemmli extracts by Western blotting.

Techniques Used: Expressing, Fractionation, Western Blot, Transfection

2) Product Images from "The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation"

Article Title: The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation

Journal:

doi: 10.1128/EC.00291-12

The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota
Figure Legend Snippet: The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota

Techniques Used: Sequencing

Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed
Figure Legend Snippet: Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed

Techniques Used: In Vivo, Western Blot

3) Product Images from "Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers"

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx1225

Dose-dependent cellular and histone acetylation responses to largazole treatment in HCT116 cells. ( A ) Quantitative analysis of the cell cycle progression by propidium iodide staining using flow cytometry in HCT116 cells treated with the indicted largazole concentration for 25 h. ( B ) Histogram showing the percentages of cells in G1 (red), S (blue), and G2 (yellow) phases of the cell cycle as well as subG1 fraction (green). ( C ) Dose-dependent global changes in indicated histone marks upon largazole exposure for 16 h as determined by immunoblotting with antibodies against each histone mark. Total histone H3 was used as a loading control. ( D and E ) Changes in H3K9ac and H3K27ac induced by largazole according to genomic territories. Pie charts illustrate the distribution of H3K9ac and H3K27ac signals (as determined by FStitch) from ChIP-seq experiments in vehicle (DMSO) treated HCT116 cells. Genomic territories are divided by gene bodies (purple), enhancer regions (green), TSS (blue), intergenic locations (orange) and 3′ ends (red). ( F and G ) The log 2 fold change ratio for increasing H3K9ac and H3K27ac in each genomic territory with various doses of largazole (nM) exposure.
Figure Legend Snippet: Dose-dependent cellular and histone acetylation responses to largazole treatment in HCT116 cells. ( A ) Quantitative analysis of the cell cycle progression by propidium iodide staining using flow cytometry in HCT116 cells treated with the indicted largazole concentration for 25 h. ( B ) Histogram showing the percentages of cells in G1 (red), S (blue), and G2 (yellow) phases of the cell cycle as well as subG1 fraction (green). ( C ) Dose-dependent global changes in indicated histone marks upon largazole exposure for 16 h as determined by immunoblotting with antibodies against each histone mark. Total histone H3 was used as a loading control. ( D and E ) Changes in H3K9ac and H3K27ac induced by largazole according to genomic territories. Pie charts illustrate the distribution of H3K9ac and H3K27ac signals (as determined by FStitch) from ChIP-seq experiments in vehicle (DMSO) treated HCT116 cells. Genomic territories are divided by gene bodies (purple), enhancer regions (green), TSS (blue), intergenic locations (orange) and 3′ ends (red). ( F and G ) The log 2 fold change ratio for increasing H3K9ac and H3K27ac in each genomic territory with various doses of largazole (nM) exposure.

Techniques Used: Staining, Flow Cytometry, Cytometry, Concentration Assay, Chromatin Immunoprecipitation

Dose dependent largazole effects on the epigenetic features of distal enhancer elements. ( A and B ) Screen shots from Genome Browser (UCSC) showing ChIP-seq and associated signal determined by FStitch (black rectangles) from HCT116 cells targeting H3K27ac (orange) starting with untreated cells (DMSO) at the bottom and followed by eight increasing largazole dose treatments on top (4.7–300 nM). ChIP-seq signal accumulation for p300 (purple) ( 23 ), total RNAPII (green), H3K4me1 (yellow), and H3K4me2 (pink) is shown for untreated HCT116 cells and for those treated with either 75 nM or 300 nM largazole concentrations (insets to the right). GRO-seq data from unstimulated HCT116 cells illustrate the presence of nascent transcripts resulting from the plus (red) and negative strand (blue) ( 24 ). ( C ) and ( D ) Schematic diagram shown illustrates the features used to identify isolated enhancers (IE) for genomic regions displaying both H3K27ac and H3K4me1 signal (determined by FStitch and MACS2 respectively). Only enhancers elements (green) located with a minimal distance of ±10 kb from neighboring H3K27ac/H3K4me1 locations from canonical ( n = 8667) and poised ( n = 3505) enhancers were used for further cluster analyses. ( E and F ) Largazole induces both the decommission and activation of transcriptional enhancers in a dose dependent manner. Shown are the fraction of IE regions with H3K27ac (left) and H3K9ac (right) signal (FStitch calls) along a ±10 kb distance centered on overlapping peak regions. Peak center locations are indicated by black triangles. Nine ChIP-seq experiments are illustrated with vehicle (DMSO) at the bottom and followed by increasing doses of largazole treatments to a maximum of 300 nM at the top. The fraction of IE elements with significant signal (FStitch) for each histone acetylation marks is illustrated by the heat-color scale: all regions (red); half of regions (green); no regions with signal detected (dark blue).
Figure Legend Snippet: Dose dependent largazole effects on the epigenetic features of distal enhancer elements. ( A and B ) Screen shots from Genome Browser (UCSC) showing ChIP-seq and associated signal determined by FStitch (black rectangles) from HCT116 cells targeting H3K27ac (orange) starting with untreated cells (DMSO) at the bottom and followed by eight increasing largazole dose treatments on top (4.7–300 nM). ChIP-seq signal accumulation for p300 (purple) ( 23 ), total RNAPII (green), H3K4me1 (yellow), and H3K4me2 (pink) is shown for untreated HCT116 cells and for those treated with either 75 nM or 300 nM largazole concentrations (insets to the right). GRO-seq data from unstimulated HCT116 cells illustrate the presence of nascent transcripts resulting from the plus (red) and negative strand (blue) ( 24 ). ( C ) and ( D ) Schematic diagram shown illustrates the features used to identify isolated enhancers (IE) for genomic regions displaying both H3K27ac and H3K4me1 signal (determined by FStitch and MACS2 respectively). Only enhancers elements (green) located with a minimal distance of ±10 kb from neighboring H3K27ac/H3K4me1 locations from canonical ( n = 8667) and poised ( n = 3505) enhancers were used for further cluster analyses. ( E and F ) Largazole induces both the decommission and activation of transcriptional enhancers in a dose dependent manner. Shown are the fraction of IE regions with H3K27ac (left) and H3K9ac (right) signal (FStitch calls) along a ±10 kb distance centered on overlapping peak regions. Peak center locations are indicated by black triangles. Nine ChIP-seq experiments are illustrated with vehicle (DMSO) at the bottom and followed by increasing doses of largazole treatments to a maximum of 300 nM at the top. The fraction of IE elements with significant signal (FStitch) for each histone acetylation marks is illustrated by the heat-color scale: all regions (red); half of regions (green); no regions with signal detected (dark blue).

Techniques Used: Chromatin Immunoprecipitation, Isolation, Activation Assay

Distinct H3K9 and H3K27 hyperacetylation responsive patterns upon largazole treatment. ( A ) A representative genomic snapshot of H3K27ac peaks illustrating different responses of gene bodies to newly acetylated histones. The signal initiates from the TSS (red dotted rectangle) of the FAT1 gene (orange panel) and spreads into the coding region or as in the case of CYP4V2 (purple panel), the preexisting acetylated TSS remains unmodified throughout all largazole dose treatments. Genes that do not show H3K9 or –K27 acetylation at the TSS (green panel) under basal conditions do not associate with the two histone marks as a result of largazole treatment. ( B ) Number of gene regions associated with the three response categories for H3K27ac (left) and H3K9ac (right). ( C ) Venn diagram showing the number of genes that exhibit new association with H3K27ac (green), H3K9ac (purple), and those that display both acetylation marks.
Figure Legend Snippet: Distinct H3K9 and H3K27 hyperacetylation responsive patterns upon largazole treatment. ( A ) A representative genomic snapshot of H3K27ac peaks illustrating different responses of gene bodies to newly acetylated histones. The signal initiates from the TSS (red dotted rectangle) of the FAT1 gene (orange panel) and spreads into the coding region or as in the case of CYP4V2 (purple panel), the preexisting acetylated TSS remains unmodified throughout all largazole dose treatments. Genes that do not show H3K9 or –K27 acetylation at the TSS (green panel) under basal conditions do not associate with the two histone marks as a result of largazole treatment. ( B ) Number of gene regions associated with the three response categories for H3K27ac (left) and H3K9ac (right). ( C ) Venn diagram showing the number of genes that exhibit new association with H3K27ac (green), H3K9ac (purple), and those that display both acetylation marks.

Techniques Used:

4) Product Images from "The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation"

Article Title: The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation

Journal:

doi: 10.1128/EC.00291-12

The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota
Figure Legend Snippet: The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota

Techniques Used: Sequencing

Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed
Figure Legend Snippet: Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed

Techniques Used: In Vivo, Western Blot

5) Product Images from "The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation"

Article Title: The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation

Journal:

doi: 10.1128/EC.00291-12

The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota
Figure Legend Snippet: The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota

Techniques Used: Sequencing

Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed
Figure Legend Snippet: Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed

Techniques Used: In Vivo, Western Blot

6) Product Images from "The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation"

Article Title: The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation

Journal:

doi: 10.1128/EC.00291-12

The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota
Figure Legend Snippet: The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota

Techniques Used: Sequencing

Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed
Figure Legend Snippet: Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed

Techniques Used: In Vivo, Western Blot

7) Product Images from "Regulation of the Human Telomerase Gene TERT by Telomere Position Effect—Over Long Distances (TPE-OLD): Implications for Aging and Cancer"

Article Title: Regulation of the Human Telomerase Gene TERT by Telomere Position Effect—Over Long Distances (TPE-OLD): Implications for Aging and Cancer

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2000016

Chromatin looping and epigenetic modifications (i.e., histone modifications and DNA methylation) determine permissiveness of the h TERT locus. ( A ) Bisulfite DNA methylation sequencing analysis of the h TERT proximal promoter region from -720bp to +90bp. Genomic DNA of BJ clones with different lengths of telomeres was modified and PCR-amplified. Each amplicon was TA-cloned for bacterial amplification and sequenced. Percentage of CpG methylation of the hTERT promoter is indicated in two BJ cell clones with different lengths of telomeres. ( B ) Illustration of genomic locus containing TERT . Black arrows indicate approximate location of primers in the promoters of the indicated genes. Chromatin immunoprecipitation was performed with BJ cells at PD34 and PD74. Six antibodies against H3K4me3, H3K9ac, H3K9me3, H3K27me3, H3 total, and IgG were used to pull down chromatin extracts, and the promoter regions of h TERT , SLC6A18 , SLC6A19 and CLPTM1L were analyzed by ddPCR. Data are presented as means and standard errors of biological and technical duplicates. Student’s paired t tests comparing young and old determined significance (* = p
Figure Legend Snippet: Chromatin looping and epigenetic modifications (i.e., histone modifications and DNA methylation) determine permissiveness of the h TERT locus. ( A ) Bisulfite DNA methylation sequencing analysis of the h TERT proximal promoter region from -720bp to +90bp. Genomic DNA of BJ clones with different lengths of telomeres was modified and PCR-amplified. Each amplicon was TA-cloned for bacterial amplification and sequenced. Percentage of CpG methylation of the hTERT promoter is indicated in two BJ cell clones with different lengths of telomeres. ( B ) Illustration of genomic locus containing TERT . Black arrows indicate approximate location of primers in the promoters of the indicated genes. Chromatin immunoprecipitation was performed with BJ cells at PD34 and PD74. Six antibodies against H3K4me3, H3K9ac, H3K9me3, H3K27me3, H3 total, and IgG were used to pull down chromatin extracts, and the promoter regions of h TERT , SLC6A18 , SLC6A19 and CLPTM1L were analyzed by ddPCR. Data are presented as means and standard errors of biological and technical duplicates. Student’s paired t tests comparing young and old determined significance (* = p

Techniques Used: DNA Methylation Assay, Sequencing, Clone Assay, Modification, Polymerase Chain Reaction, Amplification, CpG Methylation Assay, Chromatin Immunoprecipitation

8) Product Images from "The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation"

Article Title: The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation

Journal:

doi: 10.1128/EC.00291-12

The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota
Figure Legend Snippet: The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota

Techniques Used: Sequencing

Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed
Figure Legend Snippet: Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed

Techniques Used: In Vivo, Western Blot

9) Product Images from "The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation"

Article Title: The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation

Journal:

doi: 10.1128/EC.00291-12

The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota
Figure Legend Snippet: The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota

Techniques Used: Sequencing

Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed
Figure Legend Snippet: Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed

Techniques Used: In Vivo, Western Blot

10) Product Images from "Mediator subunit MED25 links the jasmonate receptor to transcriptionally active chromatin"

Article Title: Mediator subunit MED25 links the jasmonate receptor to transcriptionally active chromatin

Journal:

doi: 10.1073/pnas.1710885114

Enrichment of H3K9ac on the chromatin of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and the PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) ChIP-qPCR shows the enrichment of H3K9ac on the chromatin of JAZ8 and ERF1 . The
Figure Legend Snippet: Enrichment of H3K9ac on the chromatin of JAZ8 and ERF1 . ( A ) Schematic diagrams of JAZ8 , ERF1 , and the PCR amplicons indicated as letters A–D used for ChIP-qPCR. ( B ) ChIP-qPCR shows the enrichment of H3K9ac on the chromatin of JAZ8 and ERF1 . The

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

Depletion of HAC1 impairs JA-responsive gene expression and reduces H3K9ac accumulation on the promoters of JAZ8 and ERF1 . ( A ) qRT-PCR showing JA-Ile–induced expression of indicated genes in WT and hac1-4 . WT and hac1-4 plants were treated with
Figure Legend Snippet: Depletion of HAC1 impairs JA-responsive gene expression and reduces H3K9ac accumulation on the promoters of JAZ8 and ERF1 . ( A ) qRT-PCR showing JA-Ile–induced expression of indicated genes in WT and hac1-4 . WT and hac1-4 plants were treated with

Techniques Used: Expressing, Quantitative RT-PCR

11) Product Images from "Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers"

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx1225

Dose-dependent cellular and histone acetylation responses to largazole treatment in HCT116 cells. ( A ) Quantitative analysis of the cell cycle progression by propidium iodide staining using flow cytometry in HCT116 cells treated with the indicted largazole concentration for 25 h. ( B ) Histogram showing the percentages of cells in G1 (red), S (blue), and G2 (yellow) phases of the cell cycle as well as subG1 fraction (green). ( C ) Dose-dependent global changes in indicated histone marks upon largazole exposure for 16 h as determined by immunoblotting with antibodies against each histone mark. Total histone H3 was used as a loading control. ( D and E ) Changes in H3K9ac and H3K27ac induced by largazole according to genomic territories. Pie charts illustrate the distribution of H3K9ac and H3K27ac signals (as determined by FStitch) from ChIP-seq experiments in vehicle (DMSO) treated HCT116 cells. Genomic territories are divided by gene bodies (purple), enhancer regions (green), TSS (blue), intergenic locations (orange) and 3′ ends (red). ( F and G ) The log 2 fold change ratio for increasing H3K9ac and H3K27ac in each genomic territory with various doses of largazole (nM) exposure.
Figure Legend Snippet: Dose-dependent cellular and histone acetylation responses to largazole treatment in HCT116 cells. ( A ) Quantitative analysis of the cell cycle progression by propidium iodide staining using flow cytometry in HCT116 cells treated with the indicted largazole concentration for 25 h. ( B ) Histogram showing the percentages of cells in G1 (red), S (blue), and G2 (yellow) phases of the cell cycle as well as subG1 fraction (green). ( C ) Dose-dependent global changes in indicated histone marks upon largazole exposure for 16 h as determined by immunoblotting with antibodies against each histone mark. Total histone H3 was used as a loading control. ( D and E ) Changes in H3K9ac and H3K27ac induced by largazole according to genomic territories. Pie charts illustrate the distribution of H3K9ac and H3K27ac signals (as determined by FStitch) from ChIP-seq experiments in vehicle (DMSO) treated HCT116 cells. Genomic territories are divided by gene bodies (purple), enhancer regions (green), TSS (blue), intergenic locations (orange) and 3′ ends (red). ( F and G ) The log 2 fold change ratio for increasing H3K9ac and H3K27ac in each genomic territory with various doses of largazole (nM) exposure.

Techniques Used: Staining, Flow Cytometry, Cytometry, Concentration Assay, Chromatin Immunoprecipitation

Dose dependent largazole effects on the epigenetic features of distal enhancer elements. ( A and B ) Screen shots from Genome Browser (UCSC) showing ChIP-seq and associated signal determined by FStitch (black rectangles) from HCT116 cells targeting H3K27ac (orange) starting with untreated cells (DMSO) at the bottom and followed by eight increasing largazole dose treatments on top (4.7–300 nM). ChIP-seq signal accumulation for p300 (purple) ( 23 ), total RNAPII (green), H3K4me1 (yellow), and H3K4me2 (pink) is shown for untreated HCT116 cells and for those treated with either 75 nM or 300 nM largazole concentrations (insets to the right). GRO-seq data from unstimulated HCT116 cells illustrate the presence of nascent transcripts resulting from the plus (red) and negative strand (blue) ( 24 ). ( C ) and ( D ) Schematic diagram shown illustrates the features used to identify isolated enhancers (IE) for genomic regions displaying both H3K27ac and H3K4me1 signal (determined by FStitch and MACS2 respectively). Only enhancers elements (green) located with a minimal distance of ±10 kb from neighboring H3K27ac/H3K4me1 locations from canonical ( n = 8667) and poised ( n = 3505) enhancers were used for further cluster analyses. ( E and F ) Largazole induces both the decommission and activation of transcriptional enhancers in a dose dependent manner. Shown are the fraction of IE regions with H3K27ac (left) and H3K9ac (right) signal (FStitch calls) along a ±10 kb distance centered on overlapping peak regions. Peak center locations are indicated by black triangles. Nine ChIP-seq experiments are illustrated with vehicle (DMSO) at the bottom and followed by increasing doses of largazole treatments to a maximum of 300 nM at the top. The fraction of IE elements with significant signal (FStitch) for each histone acetylation marks is illustrated by the heat-color scale: all regions (red); half of regions (green); no regions with signal detected (dark blue).
Figure Legend Snippet: Dose dependent largazole effects on the epigenetic features of distal enhancer elements. ( A and B ) Screen shots from Genome Browser (UCSC) showing ChIP-seq and associated signal determined by FStitch (black rectangles) from HCT116 cells targeting H3K27ac (orange) starting with untreated cells (DMSO) at the bottom and followed by eight increasing largazole dose treatments on top (4.7–300 nM). ChIP-seq signal accumulation for p300 (purple) ( 23 ), total RNAPII (green), H3K4me1 (yellow), and H3K4me2 (pink) is shown for untreated HCT116 cells and for those treated with either 75 nM or 300 nM largazole concentrations (insets to the right). GRO-seq data from unstimulated HCT116 cells illustrate the presence of nascent transcripts resulting from the plus (red) and negative strand (blue) ( 24 ). ( C ) and ( D ) Schematic diagram shown illustrates the features used to identify isolated enhancers (IE) for genomic regions displaying both H3K27ac and H3K4me1 signal (determined by FStitch and MACS2 respectively). Only enhancers elements (green) located with a minimal distance of ±10 kb from neighboring H3K27ac/H3K4me1 locations from canonical ( n = 8667) and poised ( n = 3505) enhancers were used for further cluster analyses. ( E and F ) Largazole induces both the decommission and activation of transcriptional enhancers in a dose dependent manner. Shown are the fraction of IE regions with H3K27ac (left) and H3K9ac (right) signal (FStitch calls) along a ±10 kb distance centered on overlapping peak regions. Peak center locations are indicated by black triangles. Nine ChIP-seq experiments are illustrated with vehicle (DMSO) at the bottom and followed by increasing doses of largazole treatments to a maximum of 300 nM at the top. The fraction of IE elements with significant signal (FStitch) for each histone acetylation marks is illustrated by the heat-color scale: all regions (red); half of regions (green); no regions with signal detected (dark blue).

Techniques Used: Chromatin Immunoprecipitation, Isolation, Activation Assay

Distinct H3K9 and H3K27 hyperacetylation responsive patterns upon largazole treatment. ( A ) A representative genomic snapshot of H3K27ac peaks illustrating different responses of gene bodies to newly acetylated histones. The signal initiates from the TSS (red dotted rectangle) of the FAT1 gene (orange panel) and spreads into the coding region or as in the case of CYP4V2 (purple panel), the preexisting acetylated TSS remains unmodified throughout all largazole dose treatments. Genes that do not show H3K9 or –K27 acetylation at the TSS (green panel) under basal conditions do not associate with the two histone marks as a result of largazole treatment. ( B ) Number of gene regions associated with the three response categories for H3K27ac (left) and H3K9ac (right). ( C ) Venn diagram showing the number of genes that exhibit new association with H3K27ac (green), H3K9ac (purple), and those that display both acetylation marks.
Figure Legend Snippet: Distinct H3K9 and H3K27 hyperacetylation responsive patterns upon largazole treatment. ( A ) A representative genomic snapshot of H3K27ac peaks illustrating different responses of gene bodies to newly acetylated histones. The signal initiates from the TSS (red dotted rectangle) of the FAT1 gene (orange panel) and spreads into the coding region or as in the case of CYP4V2 (purple panel), the preexisting acetylated TSS remains unmodified throughout all largazole dose treatments. Genes that do not show H3K9 or –K27 acetylation at the TSS (green panel) under basal conditions do not associate with the two histone marks as a result of largazole treatment. ( B ) Number of gene regions associated with the three response categories for H3K27ac (left) and H3K9ac (right). ( C ) Venn diagram showing the number of genes that exhibit new association with H3K27ac (green), H3K9ac (purple), and those that display both acetylation marks.

Techniques Used:

12) Product Images from "Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane"

Article Title: Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane

Journal: PLoS ONE

doi: 10.1371/journal.pone.0134586

Distribution patterns of histone post-translational modifications in sugarcane. (A) Immunoblot analysis of global histone H3 modifications in sugarcane tissues. (B) Sub-nuclear localization of H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac. (C) Chromatin distribution of sugarcane and Arabidopsis; white arrows show DAPI densely stained regions in sugarcane, representing heterochromatic blocks. In Arabidopsis, the chromocenters are well defined regions of heterochromatin (yellow arrows). (D) H3T3ph (red signals) does not co-localize with actively transcribed regions rich in RNA Polymerase II (green signals). Instead, it appears to be associated with silent chromatin; DAPI densely stained regions (grey nucleus, blue arrows) coincide with H3T3ph brighter foci (red nucleus, blue arrows), whereas weaker/absent H3T3ph regions (red nucleus, orange arrows) coincide with the less condensed chromatin poorly stained with DAPI (grey nucleus, orange arrows). Bars = 5 μm.
Figure Legend Snippet: Distribution patterns of histone post-translational modifications in sugarcane. (A) Immunoblot analysis of global histone H3 modifications in sugarcane tissues. (B) Sub-nuclear localization of H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac. (C) Chromatin distribution of sugarcane and Arabidopsis; white arrows show DAPI densely stained regions in sugarcane, representing heterochromatic blocks. In Arabidopsis, the chromocenters are well defined regions of heterochromatin (yellow arrows). (D) H3T3ph (red signals) does not co-localize with actively transcribed regions rich in RNA Polymerase II (green signals). Instead, it appears to be associated with silent chromatin; DAPI densely stained regions (grey nucleus, blue arrows) coincide with H3T3ph brighter foci (red nucleus, blue arrows), whereas weaker/absent H3T3ph regions (red nucleus, orange arrows) coincide with the less condensed chromatin poorly stained with DAPI (grey nucleus, orange arrows). Bars = 5 μm.

Techniques Used: Staining

13) Product Images from "Characterisation of the epigenetic changes during human gonadal primordial germ cells reprogramming"

Article Title: Characterisation of the epigenetic changes during human gonadal primordial germ cells reprogramming

Journal:

doi: 10.1002/stem.2422

Chromatin changes of H3K9ac in human fetal PGCs.
Figure Legend Snippet: Chromatin changes of H3K9ac in human fetal PGCs.

Techniques Used:

14) Product Images from "Differential Acetylation of Histone H3 at the Regulatory Region of OsDREB1b Promoter Facilitates Chromatin Remodelling and Transcription Activation during Cold Stress"

Article Title: Differential Acetylation of Histone H3 at the Regulatory Region of OsDREB1b Promoter Facilitates Chromatin Remodelling and Transcription Activation during Cold Stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0100343

Alteration of histone H3 modifications during cold stress. Relative change in Histone H3 acetylation (H3K9ac, H3K14ac, and H3K27ac) during cold stress at (A) region Ia (−232 to −40) and region III (+157 to +307) (B) region Ib (−415 to −246) and region II (−610 to −440) of OsDREB1b gene. (C) Relative change in histone modifications at promoter and upstream region of OsDREB2a during cold stress. Samples were analysed by real time PCR except (A). The mean values for each region were normalised to Actin promoter values. Error bar represent standard error (SE) where number of independent experiments (n) = 3. The significance of the results were analysed by student’s t test and the significant changes (P≤0.05) were marked by *. (C) Western blot showing H3K9ac, H3K14ac and H3K27ac signal in whole cell extract isolated from control and cold stress treated rice seedlings.
Figure Legend Snippet: Alteration of histone H3 modifications during cold stress. Relative change in Histone H3 acetylation (H3K9ac, H3K14ac, and H3K27ac) during cold stress at (A) region Ia (−232 to −40) and region III (+157 to +307) (B) region Ib (−415 to −246) and region II (−610 to −440) of OsDREB1b gene. (C) Relative change in histone modifications at promoter and upstream region of OsDREB2a during cold stress. Samples were analysed by real time PCR except (A). The mean values for each region were normalised to Actin promoter values. Error bar represent standard error (SE) where number of independent experiments (n) = 3. The significance of the results were analysed by student’s t test and the significant changes (P≤0.05) were marked by *. (C) Western blot showing H3K9ac, H3K14ac and H3K27ac signal in whole cell extract isolated from control and cold stress treated rice seedlings.

Techniques Used: IA, Real-time Polymerase Chain Reaction, Western Blot, Isolation

15) Product Images from "Systematic analysis of chromatin state dynamics in nine human cell types"

Article Title: Systematic analysis of chromatin state dynamics in nine human cell types

Journal: Nature

doi: 10.1038/nature09906

Chromatin state discovery and characterization a, Top: Profiles for nine chromatin marks (grayscale) are shown across the wntless (WLS) gene in four cell types, and summarized in a single chromatin state annotation track for each (colored according to b ). WLS is poised in ES cells, repressed in GM12878 cells, and transcribed in HUVEC and NHLF. Its TSS switches accordingly between poised (purple), repressed (grey) and active (red) promoter states; enhancer regions within the gene body become strongly activated (orange, yellow); and its gene body changes from low signal (white) to transcribed (green). These chromatin state changes summarize coordinated changes in many chromatin marks; for example, H3K27me3, H3K4me3 and H3K4me2 jointly mark a poised promoter, while loss of H3K27me3 and gain of H3K27ac and H3K9ac mark promoter activation. Bottom: Nine chromatin state tracks, one per cell type, in a 900kb region centered at WLS summarize 90 chromatin tracks in directly-interpretable dynamic annotations, showing activation and repression patterns for 6 genes and hundreds of regulatory regions, including enhancer states. b, Chromatin states learned jointly across cell types by a multivariate HMM. Table shows emission parameters learned de novo based on genome-wide recurrent combinations of chromatin marks. Each entry denotes the frequency with which a given mark is found at genomic positions corresponding to the chromatin state. c, Genome coverage, functional enrichments, and candidate annotations for each chromatin state. Blue shading indicates intensity, scaled by column. d, Box plot depicts enhancer activity for predicted regulatory elements. 250bp-long sequences corresponding to strong or weak/poised HepG2 enhancer elements, or GM12878-specific strong enhancer elements were inserted upstream of a luciferase gene and transfected into HepG2 cells. Reporter activity was measured in relative light units. Robust activity is seen for strong enhancers in the matched cell type, but not for weak/poised enhancers or for strong enhancers specific to a different cell type. Box-and-whiskers indicate 5 th , 25 th , 50 th , 75 th and 95 th percentiles.
Figure Legend Snippet: Chromatin state discovery and characterization a, Top: Profiles for nine chromatin marks (grayscale) are shown across the wntless (WLS) gene in four cell types, and summarized in a single chromatin state annotation track for each (colored according to b ). WLS is poised in ES cells, repressed in GM12878 cells, and transcribed in HUVEC and NHLF. Its TSS switches accordingly between poised (purple), repressed (grey) and active (red) promoter states; enhancer regions within the gene body become strongly activated (orange, yellow); and its gene body changes from low signal (white) to transcribed (green). These chromatin state changes summarize coordinated changes in many chromatin marks; for example, H3K27me3, H3K4me3 and H3K4me2 jointly mark a poised promoter, while loss of H3K27me3 and gain of H3K27ac and H3K9ac mark promoter activation. Bottom: Nine chromatin state tracks, one per cell type, in a 900kb region centered at WLS summarize 90 chromatin tracks in directly-interpretable dynamic annotations, showing activation and repression patterns for 6 genes and hundreds of regulatory regions, including enhancer states. b, Chromatin states learned jointly across cell types by a multivariate HMM. Table shows emission parameters learned de novo based on genome-wide recurrent combinations of chromatin marks. Each entry denotes the frequency with which a given mark is found at genomic positions corresponding to the chromatin state. c, Genome coverage, functional enrichments, and candidate annotations for each chromatin state. Blue shading indicates intensity, scaled by column. d, Box plot depicts enhancer activity for predicted regulatory elements. 250bp-long sequences corresponding to strong or weak/poised HepG2 enhancer elements, or GM12878-specific strong enhancer elements were inserted upstream of a luciferase gene and transfected into HepG2 cells. Reporter activity was measured in relative light units. Robust activity is seen for strong enhancers in the matched cell type, but not for weak/poised enhancers or for strong enhancers specific to a different cell type. Box-and-whiskers indicate 5 th , 25 th , 50 th , 75 th and 95 th percentiles.

Techniques Used: Activation Assay, Genome Wide, Functional Assay, Activity Assay, Luciferase, Transfection

16) Product Images from "Calorie restriction protects against experimental abdominal aortic aneurysms in mice"

Article Title: Calorie restriction protects against experimental abdominal aortic aneurysms in mice

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20151794

CR attenuates AngII-induced MMP2 expression through VSMC-SIRT1–dependent H3K9 deacetylation in the Mmp2 promoter. (A, left) Western blots of H3K9 acetylation (H3K9ac), H3, H4K16ac, and H4 in aortas of Apoe −/− mice and SVKO; Apoe −/− mice. (Right) Densitometry was quantified and normalized to the AL-Con group. n = 4 per group. (B and C) ChIP of H3K9ac (B) and H4K16ac (C) on the Mmp2 promoter in Apoe −/− mouse aortas. Four regions were detected: −919 ∼ −784, −669 ∼ −485, −404 ∼ −261, and −260 ∼ −105 bp. n = 4 per group. (D) ChIP assays of H3K9ac on the Mmp2 promoter upon saline (Con) or AngII infusion. H3K9ac in the regions of −919 to −784, −669 to −485, −404 to −261, and −260 to −105 bp of the Mmp2 promoter in the aortas of Apoe −/− and SVKO; Apoe −/− mice is shown. n = 3 per group. (E) ChIP of H4K16ac on the Mmp2 promoter in aortic tissues of AL-Con, CR-Con, AL-AngII, and CR-AngII Apoe −/− mice. n = 3 per group. (F) Western blotting examination of aortic SIRT1 expression upon saline (Con) or AngII infusion for 4 wk in Apoe −/− mice. The quantification of Western blots is provided. n = 3 per group. (G and H) SIRT1 deacetylase activity (G) and NAD + /NADH ratio (H) in the aortas of Apoe −/− mice in the indicated groups were examined. n = 6 per group. (I) SIRT1 enrichment in the Mmp2 promoter was markedly higher than normal IgG in the aortas of AL-Con Apoe −/− mice. n = 3 per group. (J) SIRT1 enrichment in the regions of −919 to −784, −669 to −485, −404 to −261, and −260 to −105 bp of the Mmp2 promoter in the aortas of Apoe −/− mice in the indicated groups. n = 3 per group. Three independent experiments were performed for ChIP assays. All values are shown as the means ± SEM. *, P
Figure Legend Snippet: CR attenuates AngII-induced MMP2 expression through VSMC-SIRT1–dependent H3K9 deacetylation in the Mmp2 promoter. (A, left) Western blots of H3K9 acetylation (H3K9ac), H3, H4K16ac, and H4 in aortas of Apoe −/− mice and SVKO; Apoe −/− mice. (Right) Densitometry was quantified and normalized to the AL-Con group. n = 4 per group. (B and C) ChIP of H3K9ac (B) and H4K16ac (C) on the Mmp2 promoter in Apoe −/− mouse aortas. Four regions were detected: −919 ∼ −784, −669 ∼ −485, −404 ∼ −261, and −260 ∼ −105 bp. n = 4 per group. (D) ChIP assays of H3K9ac on the Mmp2 promoter upon saline (Con) or AngII infusion. H3K9ac in the regions of −919 to −784, −669 to −485, −404 to −261, and −260 to −105 bp of the Mmp2 promoter in the aortas of Apoe −/− and SVKO; Apoe −/− mice is shown. n = 3 per group. (E) ChIP of H4K16ac on the Mmp2 promoter in aortic tissues of AL-Con, CR-Con, AL-AngII, and CR-AngII Apoe −/− mice. n = 3 per group. (F) Western blotting examination of aortic SIRT1 expression upon saline (Con) or AngII infusion for 4 wk in Apoe −/− mice. The quantification of Western blots is provided. n = 3 per group. (G and H) SIRT1 deacetylase activity (G) and NAD + /NADH ratio (H) in the aortas of Apoe −/− mice in the indicated groups were examined. n = 6 per group. (I) SIRT1 enrichment in the Mmp2 promoter was markedly higher than normal IgG in the aortas of AL-Con Apoe −/− mice. n = 3 per group. (J) SIRT1 enrichment in the regions of −919 to −784, −669 to −485, −404 to −261, and −260 to −105 bp of the Mmp2 promoter in the aortas of Apoe −/− mice in the indicated groups. n = 3 per group. Three independent experiments were performed for ChIP assays. All values are shown as the means ± SEM. *, P

Techniques Used: Expressing, Western Blot, Mouse Assay, Chromatin Immunoprecipitation, Histone Deacetylase Assay, Activity Assay

17) Product Images from "Calorie restriction protects against experimental abdominal aortic aneurysms in mice"

Article Title: Calorie restriction protects against experimental abdominal aortic aneurysms in mice

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20151794

CR attenuates AngII-induced MMP2 expression through VSMC-SIRT1–dependent H3K9 deacetylation in the Mmp2 promoter. (A, left) Western blots of H3K9 acetylation (H3K9ac), H3, H4K16ac, and H4 in aortas of Apoe −/− mice and SVKO; Apoe −/− mice. (Right) Densitometry was quantified and normalized to the AL-Con group. n = 4 per group. (B and C) ChIP of H3K9ac (B) and H4K16ac (C) on the Mmp2 promoter in Apoe −/− mouse aortas. Four regions were detected: −919 ∼ −784, −669 ∼ −485, −404 ∼ −261, and −260 ∼ −105 bp. n = 4 per group. (D) ChIP assays of H3K9ac on the Mmp2 promoter upon saline (Con) or AngII infusion. H3K9ac in the regions of −919 to −784, −669 to −485, −404 to −261, and −260 to −105 bp of the Mmp2 promoter in the aortas of Apoe −/− and SVKO; Apoe −/− mice is shown. n = 3 per group. (E) ChIP of H4K16ac on the Mmp2 promoter in aortic tissues of AL-Con, CR-Con, AL-AngII, and CR-AngII Apoe −/− mice. n = 3 per group. (F) Western blotting examination of aortic SIRT1 expression upon saline (Con) or AngII infusion for 4 wk in Apoe −/− mice. The quantification of Western blots is provided. n = 3 per group. (G and H) SIRT1 deacetylase activity (G) and NAD + /NADH ratio (H) in the aortas of Apoe −/− mice in the indicated groups were examined. n = 6 per group. (I) SIRT1 enrichment in the Mmp2 promoter was markedly higher than normal IgG in the aortas of AL-Con Apoe −/− mice. n = 3 per group. (J) SIRT1 enrichment in the regions of −919 to −784, −669 to −485, −404 to −261, and −260 to −105 bp of the Mmp2 promoter in the aortas of Apoe −/− mice in the indicated groups. n = 3 per group. Three independent experiments were performed for ChIP assays. All values are shown as the means ± SEM. *, P
Figure Legend Snippet: CR attenuates AngII-induced MMP2 expression through VSMC-SIRT1–dependent H3K9 deacetylation in the Mmp2 promoter. (A, left) Western blots of H3K9 acetylation (H3K9ac), H3, H4K16ac, and H4 in aortas of Apoe −/− mice and SVKO; Apoe −/− mice. (Right) Densitometry was quantified and normalized to the AL-Con group. n = 4 per group. (B and C) ChIP of H3K9ac (B) and H4K16ac (C) on the Mmp2 promoter in Apoe −/− mouse aortas. Four regions were detected: −919 ∼ −784, −669 ∼ −485, −404 ∼ −261, and −260 ∼ −105 bp. n = 4 per group. (D) ChIP assays of H3K9ac on the Mmp2 promoter upon saline (Con) or AngII infusion. H3K9ac in the regions of −919 to −784, −669 to −485, −404 to −261, and −260 to −105 bp of the Mmp2 promoter in the aortas of Apoe −/− and SVKO; Apoe −/− mice is shown. n = 3 per group. (E) ChIP of H4K16ac on the Mmp2 promoter in aortic tissues of AL-Con, CR-Con, AL-AngII, and CR-AngII Apoe −/− mice. n = 3 per group. (F) Western blotting examination of aortic SIRT1 expression upon saline (Con) or AngII infusion for 4 wk in Apoe −/− mice. The quantification of Western blots is provided. n = 3 per group. (G and H) SIRT1 deacetylase activity (G) and NAD + /NADH ratio (H) in the aortas of Apoe −/− mice in the indicated groups were examined. n = 6 per group. (I) SIRT1 enrichment in the Mmp2 promoter was markedly higher than normal IgG in the aortas of AL-Con Apoe −/− mice. n = 3 per group. (J) SIRT1 enrichment in the regions of −919 to −784, −669 to −485, −404 to −261, and −260 to −105 bp of the Mmp2 promoter in the aortas of Apoe −/− mice in the indicated groups. n = 3 per group. Three independent experiments were performed for ChIP assays. All values are shown as the means ± SEM. *, P

Techniques Used: Expressing, Western Blot, Mouse Assay, Chromatin Immunoprecipitation, Histone Deacetylase Assay, Activity Assay

18) Product Images from "Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis"

Article Title: Histone deacetylase inhibitor SAHA mediates mast cell death and epigenetic silencing of constitutively active D816V KIT in systemic mastocytosis

Journal: Oncotarget

doi: 10.18632/oncotarget.14181

SAHA effects on ROSA KIT WT and ROSA KIT D816V cells A . SAHA induced a dose- and time-dependent decrease in viability, mainly due to apoptotic cell death, in ROSA cells. The effects were seen earlier and at lower SAHA concentrations in ROSA KIT D816V , where a significant decrease in viability was seen already at 24 h with 1.25 μM SAHA, compared to ROSA KIT WT . B . There was a significant decrease in percentage KIT positive cells in both cell lines in response to SAHA, and also here ROSA KIT D816V were more profoundly affected by SAHA. C . A significant increase in H3K9ac was seen for both ROSA KIT WT and ROSA KIT D816V cells. KIT was significantly decreased in ROSA KIT WT cells, however note that in the DMSO control, KIT is increasing significantly at 6 and 24 h compared to baseline, thus the results are difficult to interpret. For ROSA KIT D816V , there is a tendency to decrease in KIT at 24 h for both SAHA doses. *=p
Figure Legend Snippet: SAHA effects on ROSA KIT WT and ROSA KIT D816V cells A . SAHA induced a dose- and time-dependent decrease in viability, mainly due to apoptotic cell death, in ROSA cells. The effects were seen earlier and at lower SAHA concentrations in ROSA KIT D816V , where a significant decrease in viability was seen already at 24 h with 1.25 μM SAHA, compared to ROSA KIT WT . B . There was a significant decrease in percentage KIT positive cells in both cell lines in response to SAHA, and also here ROSA KIT D816V were more profoundly affected by SAHA. C . A significant increase in H3K9ac was seen for both ROSA KIT WT and ROSA KIT D816V cells. KIT was significantly decreased in ROSA KIT WT cells, however note that in the DMSO control, KIT is increasing significantly at 6 and 24 h compared to baseline, thus the results are difficult to interpret. For ROSA KIT D816V , there is a tendency to decrease in KIT at 24 h for both SAHA doses. *=p

Techniques Used:

19) Product Images from "The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation"

Article Title: The Carboxyl Terminus of Rtt109 Functions in Chaperone Control of Histone Acetylation

Journal:

doi: 10.1128/EC.00291-12

The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota
Figure Legend Snippet: The carboxyl terminus of fungal Rtt109s contain a Lys/Arg-rich sequence which is essential for H3K9ac in S. cerevisiae . (A) Using the ClustalW algorithm, S. cerevisiae Rtt109 was aligned with predicted Rtt109 sequences from fungi of the Ascomycota , Basidiomycota

Techniques Used: Sequencing

Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed
Figure Legend Snippet: Lysine 290 of Rtt109 is important for in vivo H3K56ac and H3K9ac. (A) 12MYC-RTT109K290Q rtt109 Δ and 12MYC-RTT109K290R rtt109 Δ strains show significantly decreased levels of H3K9ac but not H3K56ac in vivo . Western blotting was performed

Techniques Used: In Vivo, Western Blot

20) Product Images from "Absence of canonical active chromatin marks in developmentally regulated genes"

Article Title: Absence of canonical active chromatin marks in developmentally regulated genes

Journal: Nature genetics

doi: 10.1038/ng.3381

Gene expression and histone modifications in regulated broadly-expressed and stable tissue-specific genes at third instar larvae a, Diagrams of developmentally regulated genes broadly-expressed across multiple tissues at third instar-larvae L3 (left panel), and stable genes expressed in only one tissue at L3 (right panel). b, Gene expression levels at L3 measured by whole organism RNASeq (left panel). The number of genes in each category is given under the boxplots. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the IQR from the median. Outliers are plotted as dots. Validation by qPCR of the expression at L3 of regulated broadly-expressed genes compared to a stable gene ( Bmcp ) and a silent gene ( CG5367 ) (right panel). Error bars represent the Standard Error of the Mean (SEM) from three independent replicates. c, Levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac on whole L3 individuals. The seven regulated genes broadly-expressed at L3 are depicted as red dots within the boxplots. P-values were computed using the Wilcoxon test (two-sided). d, Validation by individual ChIPs and qPCR of H3K4me3 and H3K9ac in regulated genes broadly-expressed at L3. H3K4me3 and H3K9ac ChIPs are represented as enrichment of the marks over the silent gene ( CG5367 ). Error bars represent the SEM from three independent replicates.
Figure Legend Snippet: Gene expression and histone modifications in regulated broadly-expressed and stable tissue-specific genes at third instar larvae a, Diagrams of developmentally regulated genes broadly-expressed across multiple tissues at third instar-larvae L3 (left panel), and stable genes expressed in only one tissue at L3 (right panel). b, Gene expression levels at L3 measured by whole organism RNASeq (left panel). The number of genes in each category is given under the boxplots. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the IQR from the median. Outliers are plotted as dots. Validation by qPCR of the expression at L3 of regulated broadly-expressed genes compared to a stable gene ( Bmcp ) and a silent gene ( CG5367 ) (right panel). Error bars represent the Standard Error of the Mean (SEM) from three independent replicates. c, Levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac on whole L3 individuals. The seven regulated genes broadly-expressed at L3 are depicted as red dots within the boxplots. P-values were computed using the Wilcoxon test (two-sided). d, Validation by individual ChIPs and qPCR of H3K4me3 and H3K9ac in regulated genes broadly-expressed at L3. H3K4me3 and H3K9ac ChIPs are represented as enrichment of the marks over the silent gene ( CG5367 ). Error bars represent the SEM from three independent replicates.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Distribution of histone modification levels in stable, regulated and silent genes during fly development a , Expression of stable, regulated, and silent genes during fly development at the time point of maximum expression for each gene. Gene expression was computed as FPKMs by the modENCODE consortium. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the Inter Quartile Range (IQR) from the median. Outliers are plotted as dots. b, Normalized levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac at the time point of maximum expression during D. melanogaster development. These values represent the maximum height of the ChIPSeq peak within the gene body. P-values were computed using the Wilcoxon text (two-sided). c , Profiles of H3K4me3 during the 12 fly developmental time points in CG8636 , a gene stably expressed during fly development, and CG16733 , a pupa-specific gene. The expression (measured as FPKMs) along these points for the two genes is given on the left. d , Levels of H3K27me3 and H3K9me3 at the time point of maximum expression, computed as the average height of the ChIPSeq signal within the gene body, in stable, regulated and silent genes.
Figure Legend Snippet: Distribution of histone modification levels in stable, regulated and silent genes during fly development a , Expression of stable, regulated, and silent genes during fly development at the time point of maximum expression for each gene. Gene expression was computed as FPKMs by the modENCODE consortium. The bottom and top of the boxes are the first and third quartiles, and the line within, the median. The whiskers denote the interval within 1.5 times the Inter Quartile Range (IQR) from the median. Outliers are plotted as dots. b, Normalized levels of H3K4me3, H3K9ac, H3K4me1 and H3K27ac at the time point of maximum expression during D. melanogaster development. These values represent the maximum height of the ChIPSeq peak within the gene body. P-values were computed using the Wilcoxon text (two-sided). c , Profiles of H3K4me3 during the 12 fly developmental time points in CG8636 , a gene stably expressed during fly development, and CG16733 , a pupa-specific gene. The expression (measured as FPKMs) along these points for the two genes is given on the left. d , Levels of H3K27me3 and H3K9me3 at the time point of maximum expression, computed as the average height of the ChIPSeq signal within the gene body, in stable, regulated and silent genes.

Techniques Used: Modification, Expressing, Stable Transfection

Related Articles

Centrifugation:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: The cell nuclei were recovered by centrifugation and resuspended in lysate buffer (150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 0.1% SDS, 20 mM Tris, 1 mM DTT, 1X protease inhibitor cocktail). .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Positive Control:

Article Title: Hepcidin is regulated by promoter-associated histone acetylation and HDAC3
Article Snippet: Antibodies were H3k9ac Abcam ab10812 0.5 µg per IP; H3k4me3 Diagenode C15410003 1 µg per IP; RNA pol ii Diagenode C15200004 1 µg per IP; H3K27me3 Diagenode C15410195 1 µg per IP; HDAC3 Abcam ab7030 4 µL per IP. .. ChIP for HDAC3 was performed using the IDeal ChIP-Seq kit for transcription factors (Diagenode). qPCR using primers to amplify sections of genomic DNA of interest (Fig. ; Supplementary Fig. ) and Fast SYBR Green Master Mix (Applied Biosystems) to measure relative enrichment of sample relative to input was performed (%input).

Construct:

Article Title: DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia
Article Snippet: The pLKO shRNA library and individual constructs were obtained from the RNAi Consortium at the Broad Institute. .. Antibodies for Sirt1 (Abcam; ab12193), Suv39h1 (Cell Signaling; #8729), H3K9me2 (Abcam; ab1220) , H3K9ac (Abcam; ab4441) , H3K27me3 (Upstate; 07-449) , H3K36me3 (Abcam; ab9050) , H3K79me2 (Abcam; ab3594) were used for immunoblot and immunoprecipitation experiments.

Real-time Polymerase Chain Reaction:

Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity
Article Snippet: Paragraph title: ChIP and quantitative PCR ... The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described .

Incubation:

Article Title: Hepcidin is regulated by promoter-associated histone acetylation and HDAC3
Article Snippet: Antibodies bound to chromatin were incubated with Protein A/G beads at 4 degrees for 5 h, and were then washed five times with RIPA buffer (50 mM Hepes-KOH, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate). .. Antibodies were H3k9ac Abcam ab10812 0.5 µg per IP; H3k4me3 Diagenode C15410003 1 µg per IP; RNA pol ii Diagenode C15200004 1 µg per IP; H3K27me3 Diagenode C15410195 1 µg per IP; HDAC3 Abcam ab7030 4 µL per IP.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Samples were incubated for 5 h at 4°C with 5–20 μg of antibodies and 20 μl of 50% slurry with protein A beads (Millipore; Billerica, MA, USA). .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Expressing:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: It is worth noting that the change in transcript levels associated with H3K9ac occurs at a lower largazole dose than the observed changes in H3K27ac spread (Figure ). .. To explore a possible synergy between H3K9ac and –K27ac with respect to transcriptional effects, we also looked at the mRNA expression levels of 3115 genes that developed enrichment for both histone marks (Figure ). .. Similar to mRNA expression patterns from genes hyperacetylated at H3K9 or –K27, mRNAs from genes whose coding regions exhibited spread of both acetylation marks showed both events of up- and down-regulation (Figure ).

Immunoprecipitation:

Article Title: DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia
Article Snippet: The sh-LUC (sequence D11), sh-Sirt1 (sequence A1) and sh-Suv39h1 (sequence C2) were used in all the shRNA experiments unless indicated otherwise. .. Antibodies for Sirt1 (Abcam; ab12193), Suv39h1 (Cell Signaling; #8729), H3K9me2 (Abcam; ab1220) , H3K9ac (Abcam; ab4441) , H3K27me3 (Upstate; 07-449) , H3K36me3 (Abcam; ab9050) , H3K79me2 (Abcam; ab3594) were used for immunoblot and immunoprecipitation experiments. .. EPZ4777 was obtained from Epizyme and J. Bradner.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Sequencing libraries were prepared using an Illumina ChIP-Seq DNA Sample Prep Kit (cat. # IP-102-1001), with a starting sample varying from 2 to 20 ng of DNA isolated from the immunoprecipitation step. .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity
Article Snippet: WT CD4+ or CD8+ , Tcf7 −/− Lef1 −/− CD8+ mature thymocytes or splenic CD8+ T cells were sorted and cross-linked with 1% formaldehyde in medium for 5 minutes, processed using truChIP Chromatin Shearing Reagent Kit (Covaris), and sonicated for 5 minutes on Covaris S2 ultrasonicator. .. The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described . .. For calculation of histone mark signals in a given cell type in ChIP-PCR experiments, each histone mark ChIP sample was normalized to corresponding IgG ChIP sample for a target region.

Protease Inhibitor:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: The cell nuclei were recovered by centrifugation and resuspended in lysate buffer (150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 0.1% SDS, 20 mM Tris, 1 mM DTT, 1X protease inhibitor cocktail). .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

other:

Article Title: Hepcidin is regulated by promoter-associated histone acetylation and HDAC3
Article Snippet: For H3K9ac and H3K4me3, results in regions of interest were normalized to positive controls to improve comparability between replicates (%input/ %input).

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Since H3K9ac and –K27ac are functionally associated with proximal promoters and the most drastic acetylation changes occurred at protein-coding regions, we further examined genes that were occupied by H3K9ac and –K27ac under basal conditions.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Although H3K9ac and H3K27ac undergo a broad dose-dependent increase in the gene body region, we observe dramatic differences between the two marks at enhancer regions.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Collectively, our data indicates that H3K9ac and H3K27ac undergo similar dose-dependent changes along gene body regions but opposite trends at enhancer locations in response to largazole.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: This approach allowed us to effectively assign each gene a largazole dosage sensitivity for both H3K9ac and –K27ac changes (Figure ).

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Overall, our data shows that dose-dependent changes in H3K9ac and H3K27ac by largazole have distinct dose-response behaviors.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: However, within the population of gene regions associated with H3K27ac, we observed a higher number of genes responsive to low concentrations of largazole as compared to H3K9ac.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: It is worth noting that the change in transcript levels associated with H3K9ac occurs at a lower largazole dose than the observed changes in H3K27ac spread (Figure ).

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Specifically, we observe loss of H3K27ac but not H3K9ac with higher doses of largazole (Figure and ).

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: As expected, both H3K9ac and H3K27ac signals elevated significantly over the range of largazole doses used but the EC50 of the two marks appear to be different (Figure ).

Sequencing:

Article Title: DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia
Article Snippet: The sh-LUC (sequence D11), sh-Sirt1 (sequence A1) and sh-Suv39h1 (sequence C2) were used in all the shRNA experiments unless indicated otherwise. .. Antibodies for Sirt1 (Abcam; ab12193), Suv39h1 (Cell Signaling; #8729), H3K9me2 (Abcam; ab1220) , H3K9ac (Abcam; ab4441) , H3K27me3 (Upstate; 07-449) , H3K36me3 (Abcam; ab9050) , H3K79me2 (Abcam; ab3594) were used for immunoblot and immunoprecipitation experiments.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Sequencing libraries were prepared using an Illumina ChIP-Seq DNA Sample Prep Kit (cat. # IP-102-1001), with a starting sample varying from 2 to 20 ng of DNA isolated from the immunoprecipitation step. .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Sonication:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Resuspended samples were sonicated for 25 cycles (30 s ‘on’ at high level and 30s ‘off’ per cycle) using a Bioruptor (Diagenode; Denville, NJ, USA) and spun for 10 min at 16 000 × g in a microcentrifuge. .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity
Article Snippet: WT CD4+ or CD8+ , Tcf7 −/− Lef1 −/− CD8+ mature thymocytes or splenic CD8+ T cells were sorted and cross-linked with 1% formaldehyde in medium for 5 minutes, processed using truChIP Chromatin Shearing Reagent Kit (Covaris), and sonicated for 5 minutes on Covaris S2 ultrasonicator. .. The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described .

Binding Assay:

Article Title: Hepcidin is regulated by promoter-associated histone acetylation and HDAC3
Article Snippet: Antibodies were H3k9ac Abcam ab10812 0.5 µg per IP; H3k4me3 Diagenode C15410003 1 µg per IP; RNA pol ii Diagenode C15200004 1 µg per IP; H3K27me3 Diagenode C15410195 1 µg per IP; HDAC3 Abcam ab7030 4 µL per IP. .. ChIP for HDAC3 was performed using the IDeal ChIP-Seq kit for transcription factors (Diagenode). qPCR using primers to amplify sections of genomic DNA of interest (Fig. ; Supplementary Fig. ) and Fast SYBR Green Master Mix (Applied Biosystems) to measure relative enrichment of sample relative to input was performed (%input).

Immunofluorescence:

Article Title: Live imaging of H3K9 acetylation in plant cells
Article Snippet: Paragraph title: Immunofluorescence staining of BY-2 cells ... To detect H3K9ac and microtubules, anti-H3K9ac (ab4441, Abcam, http://www.abcam.co.jp/ ) and anti-α-tubulin (DM1A, CP06, Merck Millipore, http://www.merckmillipore.com ) were used at 1:1,000 dilutions.

ChIP-sequencing:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Sequencing libraries were prepared using an Illumina ChIP-Seq DNA Sample Prep Kit (cat. # IP-102-1001), with a starting sample varying from 2 to 20 ng of DNA isolated from the immunoprecipitation step. .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Raw ChIP-seq data for p300, MLL4, as well as the GRO-seq data were processed in the same manner as mentioned above. .. With the exception of H3K9ac and –K27ac, signal analyses for all ChIP-seq datasets experiments were performed using MACS2 version 2.1.0.20150731 ( ) under default settings and a P -value cutoff of 1e–05. .. We used –broad -g hs –keep-dup = auto -p 1e-5 -m 10 200 –bw 200 and selected broad peak calls.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: To gain a better understanding of the signal distribution of H3K9/27ac, we divided the human genome into five territories based on the RefSeq hg18 gene alignment from the UCSC Genome Browser ( ): proximal promoter (±2 kb from the TSS), gene region (–2 kb from TSS to end of annotated gene), 3′ end (end of annotated gene to 2 kb downstream), enhancer elements (±2 kb from the determined center), and intergenic regions. .. In unstimulated cells, ∼5% of the genome was associated with H3K9ac and –K27ac ChIP-seq signal, with both marks heavily enriched at enhancer elements (54.0% for H3K27ac and 42.4% for H3K9ac) and transcription start sites (23.6% for H3K27ac and 26.1% for H3K9ac) (Figure and ). .. Increasing doses of largazole (nM) exposure shows a general trend of gradual H3K9ac enrichment for all genomic territories analyzed, with the gene body regions displaying the highest elevation (expanding purple region of the log2 ratio of largazole/vehicle) (Figure and ).

Isolation:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Sequencing libraries were prepared using an Illumina ChIP-Seq DNA Sample Prep Kit (cat. # IP-102-1001), with a starting sample varying from 2 to 20 ng of DNA isolated from the immunoprecipitation step. .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Microscopy:

Article Title: Live imaging of H3K9 acetylation in plant cells
Article Snippet: To detect H3K9ac and microtubules, anti-H3K9ac (ab4441, Abcam, http://www.abcam.co.jp/ ) and anti-α-tubulin (DM1A, CP06, Merck Millipore, http://www.merckmillipore.com ) were used at 1:1,000 dilutions. .. To detect H3K9ac and microtubules, anti-H3K9ac (ab4441, Abcam, http://www.abcam.co.jp/ ) and anti-α-tubulin (DM1A, CP06, Merck Millipore, http://www.merckmillipore.com ) were used at 1:1,000 dilutions.

Purification:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: The immunoprecipitated chromatin was then recovered and DNA purified using phenol chloroform extraction. .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Polymerase Chain Reaction:

Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity
Article Snippet: The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described . .. The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described .

Chromatin Immunoprecipitation:

Article Title: Hepcidin is regulated by promoter-associated histone acetylation and HDAC3
Article Snippet: Paragraph title: Chromatin immunoprecipitation assays ... Antibodies were H3k9ac Abcam ab10812 0.5 µg per IP; H3k4me3 Diagenode C15410003 1 µg per IP; RNA pol ii Diagenode C15200004 1 µg per IP; H3K27me3 Diagenode C15410195 1 µg per IP; HDAC3 Abcam ab7030 4 µL per IP.

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Paragraph title: Chromatin immunoprecipitation ... Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Article Title: Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity
Article Snippet: Paragraph title: ChIP and quantitative PCR ... The resulting sheared chromatin fragments were in 200–500 bp range and were immunoprecipitated with anti-H3K4me3 (Millipore, 17–614), H3K9Ac (Abcam, ab4441), H3K27me3 (Millipore, 17–622), H3K27Ac (Abcam, ab4729), or control IgG and washed as previously described .

Negative Control:

Article Title: Hepcidin is regulated by promoter-associated histone acetylation and HDAC3
Article Snippet: Antibodies were H3k9ac Abcam ab10812 0.5 µg per IP; H3k4me3 Diagenode C15410003 1 µg per IP; RNA pol ii Diagenode C15200004 1 µg per IP; H3K27me3 Diagenode C15410195 1 µg per IP; HDAC3 Abcam ab7030 4 µL per IP. .. ChIP for HDAC3 was performed using the IDeal ChIP-Seq kit for transcription factors (Diagenode). qPCR using primers to amplify sections of genomic DNA of interest (Fig. ; Supplementary Fig. ) and Fast SYBR Green Master Mix (Applied Biosystems) to measure relative enrichment of sample relative to input was performed (%input).

shRNA:

Article Title: DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia
Article Snippet: The sh-LUC (sequence D11), sh-Sirt1 (sequence A1) and sh-Suv39h1 (sequence C2) were used in all the shRNA experiments unless indicated otherwise. .. Antibodies for Sirt1 (Abcam; ab12193), Suv39h1 (Cell Signaling; #8729), H3K9me2 (Abcam; ab1220) , H3K9ac (Abcam; ab4441) , H3K27me3 (Upstate; 07-449) , H3K36me3 (Abcam; ab9050) , H3K79me2 (Abcam; ab3594) were used for immunoblot and immunoprecipitation experiments.

Sample Prep:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: Sequencing libraries were prepared using an Illumina ChIP-Seq DNA Sample Prep Kit (cat. # IP-102-1001), with a starting sample varying from 2 to 20 ng of DNA isolated from the immunoprecipitation step. .. Antibodies used are as follows: RNAPII (Santa Cruz sc-899 lot # K0111); H3K9ac (abcam, cat. # ab4441); H3K27ac (abcam, cat. # ab4729); H3K4me1 (abcam, cat. # ab8895); H3K4me2 (abcam, cat. # ab7766).

Concentration Assay:

Article Title: Genome-wide dose-dependent inhibition of histone deacetylases studies reveal their roles in enhancer remodeling and suppression of oncogenic super-enhancers
Article Snippet: For example, in the EMC1 gene region newly acetylated histones are only detected in cells treated with largazole concentrations at or above 75 nM (Figure , bottom). .. To elucidate the sensitivity of each gene to largazole-induced acetylation changes we determined the largazole concentration necessary to induce a half-maximal acetylation response (EC50 ) in genes displaying 50% or greater H3K9ac or –K27ac signal coverage over the annotated gene lengths. .. Using FStitch calls, we calculated the total acetylation signal for both H3K9ac and –K27ac along the gene bodies for each of the nine corresponding ChIP-seq experiments and used the Sigmoidal Dose Response Search algorithm (SDRS) with a P = 0.05 cutoff ( ).

Staining:

Article Title: Live imaging of H3K9 acetylation in plant cells
Article Snippet: Paragraph title: Immunofluorescence staining of BY-2 cells ... To detect H3K9ac and microtubules, anti-H3K9ac (ab4441, Abcam, http://www.abcam.co.jp/ ) and anti-α-tubulin (DM1A, CP06, Merck Millipore, http://www.merckmillipore.com ) were used at 1:1,000 dilutions.

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    Abcam h3k9ac
    Methylation of H3K9 by Suv39h1 is involved in Sirt1-mediated silencing of the MLL-AF9 leukemic program upon suppression of Dot1L. ( a ) Scatterplots and boxplots showing changes in ChIP-seq signals for <t>H3K9ac</t> (x-axis; both panels), H3K9me2 (y-axis; left panel) and H3K27me3 (y-axis; right panel) at TSS ± 2 kb regions of genome (gray; 18,240 genes) and MLL-AF9 targets (129 genes) in sh- LUC (red) or sh- Sirt1 (blue) transduced mouse MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. ( b ) Venn diagram showing the overlap genes in “SIRT1-interacting proteins” and “candidate antagonists of Dot1L ”. ( c ) Effect of EPZ4777 on the proliferation of MLL-AF9 leukemic cells transduced with sh- LUC (green), sh- Sirt1 (red) or sh- Suv39h1 (blue). ( d ) Immunoblot of Suv39h1, Sirt1, H3K9me2 and histone H3 in MLL-AF9 leukemic cells transduced with sh- LUC or sh- Suv39h1 . ( e,g,h ) ChIP-qPCR of ( e ) H3K9me2, ( g ) Sirt1, and ( h ) Suv39h1 for Hoxa7 and Meis1 gene TSS regions in MLL-AF9 leukemic cells transduced with sh- LUC , sh- Sirt1 , or sh- Suv39h1 . ( f ) RT-qPCR of Hoxa7 and Meis1 in sh- LUC or sh- Suv39h1 transduced MLL-AF9 leukemic cells. ( i ) Bar-graph and boxplot showing changes in ATAC-seq signals at TSS ± 2 kb regions of MLL-AF9 targets (129 genes) in sh- LUC (green), sh- Sirt1 (red) and sh- Suv39h1 (blue) transduced MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. Individual MLL-AF9 target genes in bar-graph are ranked according to ATAC-seq (EPZ/DMSO) ratio in sh- LUC cells from high (left) to low (right). Cells were cultured in the presence of EPZ4777 or DMSO for ( a,e–i ) 6 days and ( c ) 9 days, respectively. Data represent the observed values and mean ± s.d. of ( c,e ) two and ( g,h ) four replicates, and ( f ) three independent experiments. NS, not significant; * P
    H3k9ac, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Validation of the ChIP-Seq analysis by ChIP-qPCR. a The results obtained by ChIP-qPCR. The DNA levels were normalized to input. H3K4me3: PAU023777.1, transcription factor MYC2; PAU023783.1, senescence-induced receptor-like serine/threonine-protein kinase; PAU017479.1, WRKY transcription factor 33; PAU019652.1, calcium-binding protein CML. H3K36me3: PAU003097.1, calmodulin; PAU019289.1, cyclic nucleotide gated channel; PAU007891.1, trans-cinnamate 4-monooxygenase; PAU019956.1, cinnamyl-alcohol dehydrogenase. <t>H3K9ac:</t> PAU012487.1, disease resistance protein RPM1; PAU023783.1, senescence-induced receptor-like serine/threonine-protein kinase; PAU023214.1, nitric-oxide synthase; PAU029105.1, trans-cinnamate 4-monooxygenase. b Changes of histone modifications derived from ChIP-Seq
    Anti H3k9ac Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 87/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Methylation of H3K9 by Suv39h1 is involved in Sirt1-mediated silencing of the MLL-AF9 leukemic program upon suppression of Dot1L. ( a ) Scatterplots and boxplots showing changes in ChIP-seq signals for H3K9ac (x-axis; both panels), H3K9me2 (y-axis; left panel) and H3K27me3 (y-axis; right panel) at TSS ± 2 kb regions of genome (gray; 18,240 genes) and MLL-AF9 targets (129 genes) in sh- LUC (red) or sh- Sirt1 (blue) transduced mouse MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. ( b ) Venn diagram showing the overlap genes in “SIRT1-interacting proteins” and “candidate antagonists of Dot1L ”. ( c ) Effect of EPZ4777 on the proliferation of MLL-AF9 leukemic cells transduced with sh- LUC (green), sh- Sirt1 (red) or sh- Suv39h1 (blue). ( d ) Immunoblot of Suv39h1, Sirt1, H3K9me2 and histone H3 in MLL-AF9 leukemic cells transduced with sh- LUC or sh- Suv39h1 . ( e,g,h ) ChIP-qPCR of ( e ) H3K9me2, ( g ) Sirt1, and ( h ) Suv39h1 for Hoxa7 and Meis1 gene TSS regions in MLL-AF9 leukemic cells transduced with sh- LUC , sh- Sirt1 , or sh- Suv39h1 . ( f ) RT-qPCR of Hoxa7 and Meis1 in sh- LUC or sh- Suv39h1 transduced MLL-AF9 leukemic cells. ( i ) Bar-graph and boxplot showing changes in ATAC-seq signals at TSS ± 2 kb regions of MLL-AF9 targets (129 genes) in sh- LUC (green), sh- Sirt1 (red) and sh- Suv39h1 (blue) transduced MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. Individual MLL-AF9 target genes in bar-graph are ranked according to ATAC-seq (EPZ/DMSO) ratio in sh- LUC cells from high (left) to low (right). Cells were cultured in the presence of EPZ4777 or DMSO for ( a,e–i ) 6 days and ( c ) 9 days, respectively. Data represent the observed values and mean ± s.d. of ( c,e ) two and ( g,h ) four replicates, and ( f ) three independent experiments. NS, not significant; * P

    Journal: Nature medicine

    Article Title: DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia

    doi: 10.1038/nm.3832

    Figure Lengend Snippet: Methylation of H3K9 by Suv39h1 is involved in Sirt1-mediated silencing of the MLL-AF9 leukemic program upon suppression of Dot1L. ( a ) Scatterplots and boxplots showing changes in ChIP-seq signals for H3K9ac (x-axis; both panels), H3K9me2 (y-axis; left panel) and H3K27me3 (y-axis; right panel) at TSS ± 2 kb regions of genome (gray; 18,240 genes) and MLL-AF9 targets (129 genes) in sh- LUC (red) or sh- Sirt1 (blue) transduced mouse MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. ( b ) Venn diagram showing the overlap genes in “SIRT1-interacting proteins” and “candidate antagonists of Dot1L ”. ( c ) Effect of EPZ4777 on the proliferation of MLL-AF9 leukemic cells transduced with sh- LUC (green), sh- Sirt1 (red) or sh- Suv39h1 (blue). ( d ) Immunoblot of Suv39h1, Sirt1, H3K9me2 and histone H3 in MLL-AF9 leukemic cells transduced with sh- LUC or sh- Suv39h1 . ( e,g,h ) ChIP-qPCR of ( e ) H3K9me2, ( g ) Sirt1, and ( h ) Suv39h1 for Hoxa7 and Meis1 gene TSS regions in MLL-AF9 leukemic cells transduced with sh- LUC , sh- Sirt1 , or sh- Suv39h1 . ( f ) RT-qPCR of Hoxa7 and Meis1 in sh- LUC or sh- Suv39h1 transduced MLL-AF9 leukemic cells. ( i ) Bar-graph and boxplot showing changes in ATAC-seq signals at TSS ± 2 kb regions of MLL-AF9 targets (129 genes) in sh- LUC (green), sh- Sirt1 (red) and sh- Suv39h1 (blue) transduced MLL-AF9 leukemic cells cultured in EPZ4777 versus DMSO. Individual MLL-AF9 target genes in bar-graph are ranked according to ATAC-seq (EPZ/DMSO) ratio in sh- LUC cells from high (left) to low (right). Cells were cultured in the presence of EPZ4777 or DMSO for ( a,e–i ) 6 days and ( c ) 9 days, respectively. Data represent the observed values and mean ± s.d. of ( c,e ) two and ( g,h ) four replicates, and ( f ) three independent experiments. NS, not significant; * P

    Article Snippet: Antibodies for Sirt1 (Abcam; ab12193), Suv39h1 (Cell Signaling; #8729), H3K9me2 (Abcam; ab1220) , H3K9ac (Abcam; ab4441) , H3K27me3 (Upstate; 07-449) , H3K36me3 (Abcam; ab9050) , H3K79me2 (Abcam; ab3594) were used for immunoblot and immunoprecipitation experiments.

    Techniques: Methylation, Chromatin Immunoprecipitation, Cell Culture, Transduction, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    Sirt1 localizes to active genes and mediates deacetylation of H3K9 in response to Dot1L inhibition. ( a–c ) Heatmaps showing ChIP-seq signal of ( a ) H3K79me2 and Sirt1, and ( b,c ) H3K9ac at TSS ± 5 kb regions for all genes in MLL-AF9 leukemic cells transduced with ( a,b ) sh- LUC or ( c ) sh- Sirt1 . Genes are ranked according to ChIP-seq signal of Sirt1 in EPZ4777 from high (top) to low (bottom). ( d,e ) Boxplots showing changes in ChIP-seq signal of ( d ) Sirt1 and ( e ) H3K9ac at TSS ± 2 kb regions of genome (gray; 18,420 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes) in mouse MLL-AF9 leukemic cells. Cells were cultured in DMSO or EPZ4777 for 6 days. Data represent mean ± s.d. NS, not significant; * P

    Journal: Nature medicine

    Article Title: DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia

    doi: 10.1038/nm.3832

    Figure Lengend Snippet: Sirt1 localizes to active genes and mediates deacetylation of H3K9 in response to Dot1L inhibition. ( a–c ) Heatmaps showing ChIP-seq signal of ( a ) H3K79me2 and Sirt1, and ( b,c ) H3K9ac at TSS ± 5 kb regions for all genes in MLL-AF9 leukemic cells transduced with ( a,b ) sh- LUC or ( c ) sh- Sirt1 . Genes are ranked according to ChIP-seq signal of Sirt1 in EPZ4777 from high (top) to low (bottom). ( d,e ) Boxplots showing changes in ChIP-seq signal of ( d ) Sirt1 and ( e ) H3K9ac at TSS ± 2 kb regions of genome (gray; 18,420 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes) in mouse MLL-AF9 leukemic cells. Cells were cultured in DMSO or EPZ4777 for 6 days. Data represent mean ± s.d. NS, not significant; * P

    Article Snippet: Antibodies for Sirt1 (Abcam; ab12193), Suv39h1 (Cell Signaling; #8729), H3K9me2 (Abcam; ab1220) , H3K9ac (Abcam; ab4441) , H3K27me3 (Upstate; 07-449) , H3K36me3 (Abcam; ab9050) , H3K79me2 (Abcam; ab3594) were used for immunoblot and immunoprecipitation experiments.

    Techniques: Inhibition, Chromatin Immunoprecipitation, Transduction, Cell Culture

    Unique H3K9 epigenomic signature at MLL-AF9 bound gene loci in MLL -fusion leukemia. ( a,b ) Scatterplots showing ChIP-seq signals for H3K79me2 (x-axis) and H3K9ac (y-axis) in mouse ( a ) MLL-AF9 leukemic cells, and ( b ) LSK cells sorted from normal mouse bone marrow. Hoxa cluster genes and Meis1 are highlighted in black circles. ( c,h ) Screen shots showing ChIP-seq profiles of MLL-AF9 fusion protein (black), H3K79me2 (blue) and H3K9ac (red) at select MLL-AF9 bound target ( HOXA cluster and MEIS1 ), active gene ( GAPDH ) and silent gene ( HBB/OLFR ) loci in ( c ) mouse and ( h ) human MLL-AF9 leukemic cell lines. The core occupied regions for MLL-AF9 fusion protein in mouse MLL-AF9 leukemia are highlighted ( c ; green-dashed box). ( d–g ) Boxplots showing ChIP-seq signal of H3K9ac in ( d ) mouse and ( e–g ) human MLL-AF9 leukemic cells including ( e ) Molm13, ( f ) Nomo1 and ( g ) MonoMac6 cells. ( a,b,d–g ) Data showing ChIP-seq signals of H3K79me2 or H3K9ac at TSS ± 2 kb regions of genome (gray; 18,240 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes). ( d–g ) Data represent mean ± s.d. * P

    Journal: Nature medicine

    Article Title: DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia

    doi: 10.1038/nm.3832

    Figure Lengend Snippet: Unique H3K9 epigenomic signature at MLL-AF9 bound gene loci in MLL -fusion leukemia. ( a,b ) Scatterplots showing ChIP-seq signals for H3K79me2 (x-axis) and H3K9ac (y-axis) in mouse ( a ) MLL-AF9 leukemic cells, and ( b ) LSK cells sorted from normal mouse bone marrow. Hoxa cluster genes and Meis1 are highlighted in black circles. ( c,h ) Screen shots showing ChIP-seq profiles of MLL-AF9 fusion protein (black), H3K79me2 (blue) and H3K9ac (red) at select MLL-AF9 bound target ( HOXA cluster and MEIS1 ), active gene ( GAPDH ) and silent gene ( HBB/OLFR ) loci in ( c ) mouse and ( h ) human MLL-AF9 leukemic cell lines. The core occupied regions for MLL-AF9 fusion protein in mouse MLL-AF9 leukemia are highlighted ( c ; green-dashed box). ( d–g ) Boxplots showing ChIP-seq signal of H3K9ac in ( d ) mouse and ( e–g ) human MLL-AF9 leukemic cells including ( e ) Molm13, ( f ) Nomo1 and ( g ) MonoMac6 cells. ( a,b,d–g ) Data showing ChIP-seq signals of H3K79me2 or H3K9ac at TSS ± 2 kb regions of genome (gray; 18,240 genes), active genes (red; 4,560 genes), MLL-AF9 targets (green; 129 genes) and silent genes (blue; 4,560 genes). ( d–g ) Data represent mean ± s.d. * P

    Article Snippet: Antibodies for Sirt1 (Abcam; ab12193), Suv39h1 (Cell Signaling; #8729), H3K9me2 (Abcam; ab1220) , H3K9ac (Abcam; ab4441) , H3K27me3 (Upstate; 07-449) , H3K36me3 (Abcam; ab9050) , H3K79me2 (Abcam; ab3594) were used for immunoblot and immunoprecipitation experiments.

    Techniques: Chromatin Immunoprecipitation

    SIRT1 activator SRT1720 sensitizes MLL -r leukemia to DOT1L inhibitor EPZ4777. ( a–e ) Mouse MLL-AF9 leukemic cells were treated with DMSO (blue), SRT1720 alone (1 μM; red), EPZ4777 alone (10 μM; orange) or the combination of SRT1720 plus EPZ4777 (green) for 6 days in tissue culture. ( a ) RT-qPCR and ( b ) H3K9ac-ChIP-qPCR for Hoxa7 and Meis1 genes. ( c ) Kaplan-Meier survival curves of mice transplanted with pretreated cells. ( d ) Percentage of GFP-positive MLL-AF9 leukemic blasts in the peripheral blood of the mice described in ( c ) on day 15 post-transplantation. ( e ) Relative blast colony count of pretreated cells further cultured in methylcellulose without the small molecules. ( f,g ) Effect of EPZ4777 on the proliferation of ( f ) mouse MLL-AF9 leukemic cells, and ( g ) human Molm13 ( MLL-AF9 ), MV4-11 ( MLL-AF4 ), SEMK2 ( MLL-AF4 ), Kasumi-1 ( AML1-ETO ) and HL-60 leukemic cell lines. Cells were co-treated with the indicated concentration of EPZ4777 and either DMSO (red) or SRT1720 (1 μM; green) for ( f ) 3, 6 and 10 days, and ( g ) 17 days, respectively. Data represent the observed values and mean ± s.d. of ( a,b,f,g ) two and ( e ) six replicates, and ( d ) ten mice per group. * P

    Journal: Nature medicine

    Article Title: DOT1L inhibits SIRT1-mediated epigenetic silencing to maintain leukemic gene expression in MLL-rearranged leukemia

    doi: 10.1038/nm.3832

    Figure Lengend Snippet: SIRT1 activator SRT1720 sensitizes MLL -r leukemia to DOT1L inhibitor EPZ4777. ( a–e ) Mouse MLL-AF9 leukemic cells were treated with DMSO (blue), SRT1720 alone (1 μM; red), EPZ4777 alone (10 μM; orange) or the combination of SRT1720 plus EPZ4777 (green) for 6 days in tissue culture. ( a ) RT-qPCR and ( b ) H3K9ac-ChIP-qPCR for Hoxa7 and Meis1 genes. ( c ) Kaplan-Meier survival curves of mice transplanted with pretreated cells. ( d ) Percentage of GFP-positive MLL-AF9 leukemic blasts in the peripheral blood of the mice described in ( c ) on day 15 post-transplantation. ( e ) Relative blast colony count of pretreated cells further cultured in methylcellulose without the small molecules. ( f,g ) Effect of EPZ4777 on the proliferation of ( f ) mouse MLL-AF9 leukemic cells, and ( g ) human Molm13 ( MLL-AF9 ), MV4-11 ( MLL-AF4 ), SEMK2 ( MLL-AF4 ), Kasumi-1 ( AML1-ETO ) and HL-60 leukemic cell lines. Cells were co-treated with the indicated concentration of EPZ4777 and either DMSO (red) or SRT1720 (1 μM; green) for ( f ) 3, 6 and 10 days, and ( g ) 17 days, respectively. Data represent the observed values and mean ± s.d. of ( a,b,f,g ) two and ( e ) six replicates, and ( d ) ten mice per group. * P

    Article Snippet: Antibodies for Sirt1 (Abcam; ab12193), Suv39h1 (Cell Signaling; #8729), H3K9me2 (Abcam; ab1220) , H3K9ac (Abcam; ab4441) , H3K27me3 (Upstate; 07-449) , H3K36me3 (Abcam; ab9050) , H3K79me2 (Abcam; ab3594) were used for immunoblot and immunoprecipitation experiments.

    Techniques: Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Mouse Assay, Transplantation Assay, Cell Culture, Concentration Assay

    Validation of the ChIP-Seq analysis by ChIP-qPCR. a The results obtained by ChIP-qPCR. The DNA levels were normalized to input. H3K4me3: PAU023777.1, transcription factor MYC2; PAU023783.1, senescence-induced receptor-like serine/threonine-protein kinase; PAU017479.1, WRKY transcription factor 33; PAU019652.1, calcium-binding protein CML. H3K36me3: PAU003097.1, calmodulin; PAU019289.1, cyclic nucleotide gated channel; PAU007891.1, trans-cinnamate 4-monooxygenase; PAU019956.1, cinnamyl-alcohol dehydrogenase. H3K9ac: PAU012487.1, disease resistance protein RPM1; PAU023783.1, senescence-induced receptor-like serine/threonine-protein kinase; PAU023214.1, nitric-oxide synthase; PAU029105.1, trans-cinnamate 4-monooxygenase. b Changes of histone modifications derived from ChIP-Seq

    Journal: BMC Genomics

    Article Title: Genome-wide analysis of three histone marks and gene expression in Paulownia fortunei with phytoplasma infection

    doi: 10.1186/s12864-019-5609-1

    Figure Lengend Snippet: Validation of the ChIP-Seq analysis by ChIP-qPCR. a The results obtained by ChIP-qPCR. The DNA levels were normalized to input. H3K4me3: PAU023777.1, transcription factor MYC2; PAU023783.1, senescence-induced receptor-like serine/threonine-protein kinase; PAU017479.1, WRKY transcription factor 33; PAU019652.1, calcium-binding protein CML. H3K36me3: PAU003097.1, calmodulin; PAU019289.1, cyclic nucleotide gated channel; PAU007891.1, trans-cinnamate 4-monooxygenase; PAU019956.1, cinnamyl-alcohol dehydrogenase. H3K9ac: PAU012487.1, disease resistance protein RPM1; PAU023783.1, senescence-induced receptor-like serine/threonine-protein kinase; PAU023214.1, nitric-oxide synthase; PAU029105.1, trans-cinnamate 4-monooxygenase. b Changes of histone modifications derived from ChIP-Seq

    Article Snippet: Subsequently, the chromatin was isolated, sonicated, and immunoprecipitated against anti-H3K4me3 antibody (abcam), anti-H3K36me3 antibody (abcam) and anti-H3K9ac antibody (abcam), respectively.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Derivative Assay

    Pathway analysis of differentially modified genes by H3K4me3 ( a ), H3K36me3 ( b ) and H3K9ac ( c ) marks under phytoplasma stress. Top 20 pathways are shown in the figure

    Journal: BMC Genomics

    Article Title: Genome-wide analysis of three histone marks and gene expression in Paulownia fortunei with phytoplasma infection

    doi: 10.1186/s12864-019-5609-1

    Figure Lengend Snippet: Pathway analysis of differentially modified genes by H3K4me3 ( a ), H3K36me3 ( b ) and H3K9ac ( c ) marks under phytoplasma stress. Top 20 pathways are shown in the figure

    Article Snippet: Subsequently, the chromatin was isolated, sonicated, and immunoprecipitated against anti-H3K4me3 antibody (abcam), anti-H3K36me3 antibody (abcam) and anti-H3K9ac antibody (abcam), respectively.

    Techniques: Modification

    GO analysis of genes differentially modified by H3K4me3, H3K36me3 and H3K9ac marks under phytoplasma stress

    Journal: BMC Genomics

    Article Title: Genome-wide analysis of three histone marks and gene expression in Paulownia fortunei with phytoplasma infection

    doi: 10.1186/s12864-019-5609-1

    Figure Lengend Snippet: GO analysis of genes differentially modified by H3K4me3, H3K36me3 and H3K9ac marks under phytoplasma stress

    Article Snippet: Subsequently, the chromatin was isolated, sonicated, and immunoprecipitated against anti-H3K4me3 antibody (abcam), anti-H3K36me3 antibody (abcam) and anti-H3K9ac antibody (abcam), respectively.

    Techniques: Modification

    Numbers of differentially modified regions in P. fortunei with phytoplasma infection compared to P. fortunei for H3K4me3, H3K36me3 and H3K9ac marks

    Journal: BMC Genomics

    Article Title: Genome-wide analysis of three histone marks and gene expression in Paulownia fortunei with phytoplasma infection

    doi: 10.1186/s12864-019-5609-1

    Figure Lengend Snippet: Numbers of differentially modified regions in P. fortunei with phytoplasma infection compared to P. fortunei for H3K4me3, H3K36me3 and H3K9ac marks

    Article Snippet: Subsequently, the chromatin was isolated, sonicated, and immunoprecipitated against anti-H3K4me3 antibody (abcam), anti-H3K36me3 antibody (abcam) and anti-H3K9ac antibody (abcam), respectively.

    Techniques: Modification, Infection

    Venn diagram of differentially modified genes by H3K4me3, H3K36me3 and H3K9ac marks in P. fortunei with phytoplasma infection compared to P. fortunei

    Journal: BMC Genomics

    Article Title: Genome-wide analysis of three histone marks and gene expression in Paulownia fortunei with phytoplasma infection

    doi: 10.1186/s12864-019-5609-1

    Figure Lengend Snippet: Venn diagram of differentially modified genes by H3K4me3, H3K36me3 and H3K9ac marks in P. fortunei with phytoplasma infection compared to P. fortunei

    Article Snippet: Subsequently, the chromatin was isolated, sonicated, and immunoprecipitated against anti-H3K4me3 antibody (abcam), anti-H3K36me3 antibody (abcam) and anti-H3K9ac antibody (abcam), respectively.

    Techniques: Modification, Infection

    The correlation between each biological replicates of P. fortunei and P. fortunei with phytoplasma infection for H3K4me3 ( a ), H3K36me3 ( b ) and H3K9ac ( c ) marks

    Journal: BMC Genomics

    Article Title: Genome-wide analysis of three histone marks and gene expression in Paulownia fortunei with phytoplasma infection

    doi: 10.1186/s12864-019-5609-1

    Figure Lengend Snippet: The correlation between each biological replicates of P. fortunei and P. fortunei with phytoplasma infection for H3K4me3 ( a ), H3K36me3 ( b ) and H3K9ac ( c ) marks

    Article Snippet: Subsequently, the chromatin was isolated, sonicated, and immunoprecipitated against anti-H3K4me3 antibody (abcam), anti-H3K36me3 antibody (abcam) and anti-H3K9ac antibody (abcam), respectively.

    Techniques: Infection

    The distribution patterns of H3K4me3, H3K36me3 and H3K9ac within different regions in Paulownia genome

    Journal: BMC Genomics

    Article Title: Genome-wide analysis of three histone marks and gene expression in Paulownia fortunei with phytoplasma infection

    doi: 10.1186/s12864-019-5609-1

    Figure Lengend Snippet: The distribution patterns of H3K4me3, H3K36me3 and H3K9ac within different regions in Paulownia genome

    Article Snippet: Subsequently, the chromatin was isolated, sonicated, and immunoprecipitated against anti-H3K4me3 antibody (abcam), anti-H3K36me3 antibody (abcam) and anti-H3K9ac antibody (abcam), respectively.

    Techniques:

    Genome-wide patterns of H3K4me3, H3K36me3 and H3K9ac in P. fortunei and P. fortunei with phytoplasma infection. a Numbers of histone modified regions detected by MACS software; b Average lengths of histone modified regions

    Journal: BMC Genomics

    Article Title: Genome-wide analysis of three histone marks and gene expression in Paulownia fortunei with phytoplasma infection

    doi: 10.1186/s12864-019-5609-1

    Figure Lengend Snippet: Genome-wide patterns of H3K4me3, H3K36me3 and H3K9ac in P. fortunei and P. fortunei with phytoplasma infection. a Numbers of histone modified regions detected by MACS software; b Average lengths of histone modified regions

    Article Snippet: Subsequently, the chromatin was isolated, sonicated, and immunoprecipitated against anti-H3K4me3 antibody (abcam), anti-H3K36me3 antibody (abcam) and anti-H3K9ac antibody (abcam), respectively.

    Techniques: Genome Wide, Infection, Modification, Magnetic Cell Separation, Software

    Western Blot analysis of FOXO1 and H3K9ac expression in BeWo cells after 48 h stimulation with human calcitriol. ( A ) Bar graph diagram showing a significant ( p = 0.011 corresponding to 0.01 µM, p = 0.021 corresponding to 0.1 µM, p = 0.021 corresponding to 1.0 µM), dose-dependent upregulation of FOXO1 after 48 h stimulation with human calcitriol. ( B ) Bar graph diagram showing a significant ( p = 0.008) downregulation of H3K9ac after 48 h stimulation with human calcitriol at 1.0 µM. No significance was shown for stimulation with 0.01 nor 0.1 µM. ( C ) Representative photographs of the western blot membrane with detected bands for H3K9, FOXO1 and endogenous control (β-Actin).

    Journal: International Journal of Molecular Sciences

    Article Title: Histone H3 Lysine 9 Acetylation is Downregulated in GDM Placentas and Calcitriol Supplementation Enhanced This Effect

    doi: 10.3390/ijms19124061

    Figure Lengend Snippet: Western Blot analysis of FOXO1 and H3K9ac expression in BeWo cells after 48 h stimulation with human calcitriol. ( A ) Bar graph diagram showing a significant ( p = 0.011 corresponding to 0.01 µM, p = 0.021 corresponding to 0.1 µM, p = 0.021 corresponding to 1.0 µM), dose-dependent upregulation of FOXO1 after 48 h stimulation with human calcitriol. ( B ) Bar graph diagram showing a significant ( p = 0.008) downregulation of H3K9ac after 48 h stimulation with human calcitriol at 1.0 µM. No significance was shown for stimulation with 0.01 nor 0.1 µM. ( C ) Representative photographs of the western blot membrane with detected bands for H3K9, FOXO1 and endogenous control (β-Actin).

    Article Snippet: Blocking of unspecific background staining was performed by incubating the membrane in 1× Casein solution (VECTASTAIN ABC-AmP Kit for rabbit IgG, Vector Laboratories, Burlingame, CA, USA) for 1 h. The primary antibodies, anti-H3K9ac (monoclonal rabbit IgG, Abcam, Cambridge, UK), anti-FOXO1 (monoclonal mouse IgG, Novus Biologicals Europe, Abingdon, UK) diluted at 1:500 in Casein and anti-β-Actin (monoclonal mouse IgG, Sigma-Aldrich, St. Louise, MO, USA) diluted at 1:1000 in Casein, were added for 16 h at 4 °C, plus an additional 2 × 15 min incubation at room temperature for anti-H3K9ac and anti-FOXO1.

    Techniques: Western Blot, Expressing

    Double immunofluorescence phenotyping of placenta cells. H3K9ac, marked with Cy-3-labled secondary antibody, is stained red in both rows. CK7 is stained green in the first row, marking EVT cells. CD31 is stained green in the second row, marking endothelial cells. Pictures were taken with a 400× lens.

    Journal: International Journal of Molecular Sciences

    Article Title: Histone H3 Lysine 9 Acetylation is Downregulated in GDM Placentas and Calcitriol Supplementation Enhanced This Effect

    doi: 10.3390/ijms19124061

    Figure Lengend Snippet: Double immunofluorescence phenotyping of placenta cells. H3K9ac, marked with Cy-3-labled secondary antibody, is stained red in both rows. CK7 is stained green in the first row, marking EVT cells. CD31 is stained green in the second row, marking endothelial cells. Pictures were taken with a 400× lens.

    Article Snippet: Blocking of unspecific background staining was performed by incubating the membrane in 1× Casein solution (VECTASTAIN ABC-AmP Kit for rabbit IgG, Vector Laboratories, Burlingame, CA, USA) for 1 h. The primary antibodies, anti-H3K9ac (monoclonal rabbit IgG, Abcam, Cambridge, UK), anti-FOXO1 (monoclonal mouse IgG, Novus Biologicals Europe, Abingdon, UK) diluted at 1:500 in Casein and anti-β-Actin (monoclonal mouse IgG, Sigma-Aldrich, St. Louise, MO, USA) diluted at 1:1000 in Casein, were added for 16 h at 4 °C, plus an additional 2 × 15 min incubation at room temperature for anti-H3K9ac and anti-FOXO1.

    Techniques: Immunofluorescence, Staining

    H3K9ac expression in syncytiotrophoblast, decidua and foetal endothelia cells of control and GDM placentas. Boxplots ( A , D , G ) showing the IRS for H3K9ac expression in syncytiotrophoblast (SCT), decidua and foetal endothelial to be highly significantly lower in GDM placentas ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Histone H3 Lysine 9 Acetylation is Downregulated in GDM Placentas and Calcitriol Supplementation Enhanced This Effect

    doi: 10.3390/ijms19124061

    Figure Lengend Snippet: H3K9ac expression in syncytiotrophoblast, decidua and foetal endothelia cells of control and GDM placentas. Boxplots ( A , D , G ) showing the IRS for H3K9ac expression in syncytiotrophoblast (SCT), decidua and foetal endothelial to be highly significantly lower in GDM placentas ( p

    Article Snippet: Blocking of unspecific background staining was performed by incubating the membrane in 1× Casein solution (VECTASTAIN ABC-AmP Kit for rabbit IgG, Vector Laboratories, Burlingame, CA, USA) for 1 h. The primary antibodies, anti-H3K9ac (monoclonal rabbit IgG, Abcam, Cambridge, UK), anti-FOXO1 (monoclonal mouse IgG, Novus Biologicals Europe, Abingdon, UK) diluted at 1:500 in Casein and anti-β-Actin (monoclonal mouse IgG, Sigma-Aldrich, St. Louise, MO, USA) diluted at 1:1000 in Casein, were added for 16 h at 4 °C, plus an additional 2 × 15 min incubation at room temperature for anti-H3K9ac and anti-FOXO1.

    Techniques: Expressing

    Positive and negative immunohistochemistry staining controls. Colon tissue was used as positive ( A , a ) as well as negative ( B , b ) control for H3K9ac antibody. For the H3K4me3 antibody, colon tissue was used as positive ( C , c ) as well as negative ( D , d ) control, as well. Negative control serum was added to the tissue for negative control staining. Brown staining indicates primary and secondary antibody binding, blue staining is due to haemalaun counter staining. Pictures were taken at a 100× (capital letters) and 250× (lower-case letters) magnification, respectively.

    Journal: International Journal of Molecular Sciences

    Article Title: Histone H3 Lysine 9 Acetylation is Downregulated in GDM Placentas and Calcitriol Supplementation Enhanced This Effect

    doi: 10.3390/ijms19124061

    Figure Lengend Snippet: Positive and negative immunohistochemistry staining controls. Colon tissue was used as positive ( A , a ) as well as negative ( B , b ) control for H3K9ac antibody. For the H3K4me3 antibody, colon tissue was used as positive ( C , c ) as well as negative ( D , d ) control, as well. Negative control serum was added to the tissue for negative control staining. Brown staining indicates primary and secondary antibody binding, blue staining is due to haemalaun counter staining. Pictures were taken at a 100× (capital letters) and 250× (lower-case letters) magnification, respectively.

    Article Snippet: Blocking of unspecific background staining was performed by incubating the membrane in 1× Casein solution (VECTASTAIN ABC-AmP Kit for rabbit IgG, Vector Laboratories, Burlingame, CA, USA) for 1 h. The primary antibodies, anti-H3K9ac (monoclonal rabbit IgG, Abcam, Cambridge, UK), anti-FOXO1 (monoclonal mouse IgG, Novus Biologicals Europe, Abingdon, UK) diluted at 1:500 in Casein and anti-β-Actin (monoclonal mouse IgG, Sigma-Aldrich, St. Louise, MO, USA) diluted at 1:1000 in Casein, were added for 16 h at 4 °C, plus an additional 2 × 15 min incubation at room temperature for anti-H3K9ac and anti-FOXO1.

    Techniques: Immunohistochemistry, Staining, Negative Control, Binding Assay

    Western Blot analysis of H3K9ac expression in HVT cells after 48 h stimulation with human calcitriol. ( A ) Bar graph diagram showing a significant ( p = 0.03) downregulation of H3K9ac after 48 h stimulation with human calcitriol at 1.0 µM. ( B ) Representative photograph of the western blot membrane with detected bands for H3K9 and endogenous control (β-Actin).

    Journal: International Journal of Molecular Sciences

    Article Title: Histone H3 Lysine 9 Acetylation is Downregulated in GDM Placentas and Calcitriol Supplementation Enhanced This Effect

    doi: 10.3390/ijms19124061

    Figure Lengend Snippet: Western Blot analysis of H3K9ac expression in HVT cells after 48 h stimulation with human calcitriol. ( A ) Bar graph diagram showing a significant ( p = 0.03) downregulation of H3K9ac after 48 h stimulation with human calcitriol at 1.0 µM. ( B ) Representative photograph of the western blot membrane with detected bands for H3K9 and endogenous control (β-Actin).

    Article Snippet: Blocking of unspecific background staining was performed by incubating the membrane in 1× Casein solution (VECTASTAIN ABC-AmP Kit for rabbit IgG, Vector Laboratories, Burlingame, CA, USA) for 1 h. The primary antibodies, anti-H3K9ac (monoclonal rabbit IgG, Abcam, Cambridge, UK), anti-FOXO1 (monoclonal mouse IgG, Novus Biologicals Europe, Abingdon, UK) diluted at 1:500 in Casein and anti-β-Actin (monoclonal mouse IgG, Sigma-Aldrich, St. Louise, MO, USA) diluted at 1:1000 in Casein, were added for 16 h at 4 °C, plus an additional 2 × 15 min incubation at room temperature for anti-H3K9ac and anti-FOXO1.

    Techniques: Western Blot, Expressing

    Two potential mechanisms leading to a reduction of H3K9ac in trophoblast cells. Immunohistochemical analysis showed a downregulation of H3K9ac (red triangle) in GDM placentas, while H3K4me3 (purple hexagons) showed no significant differences (solid pink arrow tail). The H3K9ac reduction may also be part of the aetiology of GDM (pink arrow broken line). Stimulation of trophoblast cells with calcitriol also lead to a reduction of H3K9ac, as well as an increase in FOXO1 (solid orange arrows). This may in turn be linked to the reduction of H3K9ac (yellow arrow broken line), ultimately leading to a downregulation of transcription (green arrows).

    Journal: International Journal of Molecular Sciences

    Article Title: Histone H3 Lysine 9 Acetylation is Downregulated in GDM Placentas and Calcitriol Supplementation Enhanced This Effect

    doi: 10.3390/ijms19124061

    Figure Lengend Snippet: Two potential mechanisms leading to a reduction of H3K9ac in trophoblast cells. Immunohistochemical analysis showed a downregulation of H3K9ac (red triangle) in GDM placentas, while H3K4me3 (purple hexagons) showed no significant differences (solid pink arrow tail). The H3K9ac reduction may also be part of the aetiology of GDM (pink arrow broken line). Stimulation of trophoblast cells with calcitriol also lead to a reduction of H3K9ac, as well as an increase in FOXO1 (solid orange arrows). This may in turn be linked to the reduction of H3K9ac (yellow arrow broken line), ultimately leading to a downregulation of transcription (green arrows).

    Article Snippet: Blocking of unspecific background staining was performed by incubating the membrane in 1× Casein solution (VECTASTAIN ABC-AmP Kit for rabbit IgG, Vector Laboratories, Burlingame, CA, USA) for 1 h. The primary antibodies, anti-H3K9ac (monoclonal rabbit IgG, Abcam, Cambridge, UK), anti-FOXO1 (monoclonal mouse IgG, Novus Biologicals Europe, Abingdon, UK) diluted at 1:500 in Casein and anti-β-Actin (monoclonal mouse IgG, Sigma-Aldrich, St. Louise, MO, USA) diluted at 1:1000 in Casein, were added for 16 h at 4 °C, plus an additional 2 × 15 min incubation at room temperature for anti-H3K9ac and anti-FOXO1.

    Techniques: Immunohistochemistry

    ACSS2 is chromatin-bound in differentiated CAD neurons (a) ChIP-seq in differentiated CAD neurons was performed in replicate with two different antibodies to ACSS2. Correlation plot displays relative enrichment over corresponding MACS peaks (default parameters with Input as control, 1598 peaks). (b) Correlation plot displays relative ChIP-seq enrichment genome-wide. (c) UCSC Genome Browser views of ChIP-seq tracks show that, upon CAD neuron differentiation, increases in H4K5, H4K12, and H3K9 acetylation over the NUDT1 gene locus co-occur with ACSS2 enrichment (U–undiff, D–diff; chr5: 140,326,845–140,339,655). (d) UCSC Genome Browser view of indicated ChIP-seq tracks in undifferentiated CAD cells (u) and differentiated CAD neurons (d) over Tab2 locus (chr10: 7,875,000–8,004,000). (e) GO enrichment analysis of the genes most proximate to ACSS2 peaks demonstrates that neuron-specific genes are enriched. (f) Frequency of ACSS2 peaks (T antibody) located upstream of their target gene associated with histone acetylation. (g) Frequency of ACSS2 peaks (CS antibody) located upstream of their target gene associated with histone acetylation. (h) Table shows % direct overlap of ACSS2 peaks with H3K9ac, H4K5ac, and H4K12ac broad MACS peaks. (i–k) Decile plots depict enrichment of H3K9ac (i), H4K5ac (j), and H4K12ac (k) over ranked deciles of ACSS2 peak enrichment (zeroes removed). (l–m) Differentiation-induced co-enrichment of ACSS2 and acetyl broad peaks (MACS). Peak enrichment correlation indicated for H3K9ac (i; spearman r = 0.28, p = 1.4e-07), H4K5ac (m; spearman r = 0.52, p = 1.1e-22), and H4K12ac (n; spearman r = 0.63, p = 6.3e–17). (o) Discovered de novo motifs for transcription factor binding sites predicted by HOMER from all ACSS2 ChIP-seq peaks called by MACS in differentiated CAD neurons. (p) ChIP-seq enrichment of differentiation-induced genes as a group show correlation with histone acetylation in differentiated CAD neurons.

    Journal: Nature

    Article Title: ACETYL-COA SYNTHETASE REGULATES HISTONE ACETYLATION AND HIPPOCAMPAL MEMORY

    doi: 10.1038/nature22405

    Figure Lengend Snippet: ACSS2 is chromatin-bound in differentiated CAD neurons (a) ChIP-seq in differentiated CAD neurons was performed in replicate with two different antibodies to ACSS2. Correlation plot displays relative enrichment over corresponding MACS peaks (default parameters with Input as control, 1598 peaks). (b) Correlation plot displays relative ChIP-seq enrichment genome-wide. (c) UCSC Genome Browser views of ChIP-seq tracks show that, upon CAD neuron differentiation, increases in H4K5, H4K12, and H3K9 acetylation over the NUDT1 gene locus co-occur with ACSS2 enrichment (U–undiff, D–diff; chr5: 140,326,845–140,339,655). (d) UCSC Genome Browser view of indicated ChIP-seq tracks in undifferentiated CAD cells (u) and differentiated CAD neurons (d) over Tab2 locus (chr10: 7,875,000–8,004,000). (e) GO enrichment analysis of the genes most proximate to ACSS2 peaks demonstrates that neuron-specific genes are enriched. (f) Frequency of ACSS2 peaks (T antibody) located upstream of their target gene associated with histone acetylation. (g) Frequency of ACSS2 peaks (CS antibody) located upstream of their target gene associated with histone acetylation. (h) Table shows % direct overlap of ACSS2 peaks with H3K9ac, H4K5ac, and H4K12ac broad MACS peaks. (i–k) Decile plots depict enrichment of H3K9ac (i), H4K5ac (j), and H4K12ac (k) over ranked deciles of ACSS2 peak enrichment (zeroes removed). (l–m) Differentiation-induced co-enrichment of ACSS2 and acetyl broad peaks (MACS). Peak enrichment correlation indicated for H3K9ac (i; spearman r = 0.28, p = 1.4e-07), H4K5ac (m; spearman r = 0.52, p = 1.1e-22), and H4K12ac (n; spearman r = 0.63, p = 6.3e–17). (o) Discovered de novo motifs for transcription factor binding sites predicted by HOMER from all ACSS2 ChIP-seq peaks called by MACS in differentiated CAD neurons. (p) ChIP-seq enrichment of differentiation-induced genes as a group show correlation with histone acetylation in differentiated CAD neurons.

    Article Snippet: Antibodies included Anti-H3 (Abcam ab1791), Anti-H3K9ac (Abcam ab4441), Anti-H3K27ac (Abcam ab4729), Anti-H3K122ac (Abcam ab33308), Anti-H4 (Millipore 05-858), Anti-H4K5ac (Millipore 39-584), Anti-H4K12ac (Abcam ab1761), Anti-ACSS2 (T) (Thermo MA5-145810), Anti-ACSS2 (CS) (Cell Signaling 3658), Anti-ACL (Proteintech 15421-1-AP), Anti-α-Tubulin (Sigma T8328), Anti-GAPDH (Fitzgerald Industries 10R-G109A), Anti-KAT3A/CBP (Abcam ab2832), Anti-SNAP25 (Abcam ab5666), Anti-Synaptophysin (Millipore MAB368), Anti-MAP2 C/D (Cell Signaling #8707), Anti-NR4A2 (Santa Cruz sc-991), Anti-NeuN (Millipore ABN78)

    Techniques: Chromatin Immunoprecipitation, Magnetic Cell Separation, Genome Wide, Binding Assay

    ACSS2 ChIP-seq localization is linked to histone acetylation in vivo in mouse hippocampus (HPC). (a) ChIP-seq for ACSS2 and H3K9ac in mouse HPC. Track-views show ACSS2 and H3K9ac for three canonical neuronal genes involved in memory: Arc, Egr2, and Nr2f2 (chr15:74496025–74506488, chr10:66991018–67006804, and chr7:77488549–77516626, respectively). (b) Venn diagram shows in vivo HPC ACSS2 and H3K9ac peaks co-localize with the nearest gene TSS (

    Journal: Nature

    Article Title: ACETYL-COA SYNTHETASE REGULATES HISTONE ACETYLATION AND HIPPOCAMPAL MEMORY

    doi: 10.1038/nature22405

    Figure Lengend Snippet: ACSS2 ChIP-seq localization is linked to histone acetylation in vivo in mouse hippocampus (HPC). (a) ChIP-seq for ACSS2 and H3K9ac in mouse HPC. Track-views show ACSS2 and H3K9ac for three canonical neuronal genes involved in memory: Arc, Egr2, and Nr2f2 (chr15:74496025–74506488, chr10:66991018–67006804, and chr7:77488549–77516626, respectively). (b) Venn diagram shows in vivo HPC ACSS2 and H3K9ac peaks co-localize with the nearest gene TSS (

    Article Snippet: Antibodies included Anti-H3 (Abcam ab1791), Anti-H3K9ac (Abcam ab4441), Anti-H3K27ac (Abcam ab4729), Anti-H3K122ac (Abcam ab33308), Anti-H4 (Millipore 05-858), Anti-H4K5ac (Millipore 39-584), Anti-H4K12ac (Abcam ab1761), Anti-ACSS2 (T) (Thermo MA5-145810), Anti-ACSS2 (CS) (Cell Signaling 3658), Anti-ACL (Proteintech 15421-1-AP), Anti-α-Tubulin (Sigma T8328), Anti-GAPDH (Fitzgerald Industries 10R-G109A), Anti-KAT3A/CBP (Abcam ab2832), Anti-SNAP25 (Abcam ab5666), Anti-Synaptophysin (Millipore MAB368), Anti-MAP2 C/D (Cell Signaling #8707), Anti-NR4A2 (Santa Cruz sc-991), Anti-NeuN (Millipore ABN78)

    Techniques: Chromatin Immunoprecipitation, In Vivo

    (a) Genome-wide compartment analysis of the in vivo hippocampal ChIP-seq of H3K9ac, and the mouse forebrain H3K9ac ChIP-seq from ENCODE, showing a similar peak distribution genome-wide: originating in different brain regions, the in vivo H3K9ac ChIP data are in strong agreement (Spearman R = 0.67) (b) Shown is a 4-way Venn diagram depicting overlap of RefSeq transcripts targeted by the indicated enzyme or modification (peaks for CBP (GSM1629373) and H3K27ac (GSM1629397) in mouse cortical neurons were called using MACS2 (narrow peaks, FDR 0.1%) with an input sonication efficiency control (GSM1629381); peaks were associated to the nearest TSS among all RefSeq transcripts) (c) Gene Ontology enrichment analysis has been performed on common CBP:ACSS2 targets, indicating that these enzymes co-target genes that modulate synapse biology and synaptic membrane potential.

    Journal: Nature

    Article Title: ACETYL-COA SYNTHETASE REGULATES HISTONE ACETYLATION AND HIPPOCAMPAL MEMORY

    doi: 10.1038/nature22405

    Figure Lengend Snippet: (a) Genome-wide compartment analysis of the in vivo hippocampal ChIP-seq of H3K9ac, and the mouse forebrain H3K9ac ChIP-seq from ENCODE, showing a similar peak distribution genome-wide: originating in different brain regions, the in vivo H3K9ac ChIP data are in strong agreement (Spearman R = 0.67) (b) Shown is a 4-way Venn diagram depicting overlap of RefSeq transcripts targeted by the indicated enzyme or modification (peaks for CBP (GSM1629373) and H3K27ac (GSM1629397) in mouse cortical neurons were called using MACS2 (narrow peaks, FDR 0.1%) with an input sonication efficiency control (GSM1629381); peaks were associated to the nearest TSS among all RefSeq transcripts) (c) Gene Ontology enrichment analysis has been performed on common CBP:ACSS2 targets, indicating that these enzymes co-target genes that modulate synapse biology and synaptic membrane potential.

    Article Snippet: Antibodies included Anti-H3 (Abcam ab1791), Anti-H3K9ac (Abcam ab4441), Anti-H3K27ac (Abcam ab4729), Anti-H3K122ac (Abcam ab33308), Anti-H4 (Millipore 05-858), Anti-H4K5ac (Millipore 39-584), Anti-H4K12ac (Abcam ab1761), Anti-ACSS2 (T) (Thermo MA5-145810), Anti-ACSS2 (CS) (Cell Signaling 3658), Anti-ACL (Proteintech 15421-1-AP), Anti-α-Tubulin (Sigma T8328), Anti-GAPDH (Fitzgerald Industries 10R-G109A), Anti-KAT3A/CBP (Abcam ab2832), Anti-SNAP25 (Abcam ab5666), Anti-Synaptophysin (Millipore MAB368), Anti-MAP2 C/D (Cell Signaling #8707), Anti-NR4A2 (Santa Cruz sc-991), Anti-NeuN (Millipore ABN78)

    Techniques: Genome Wide, In Vivo, Chromatin Immunoprecipitation, Modification, Sonication

    (a–d) Meta-gene enrichment analysis shows ChIP occupancy for ACSS2 (a), H3K9ac (b), H4K5ac (c), and for H4K12ac (d) across the top 5% of genes enriched for ACSS2 in differentiated CAD neurons (Top 5% DE; red). The bottom 80% of genes (Bot 80% DE) is shown in blue, and the average signal across all genes (All genes DE) is shown in green. (e–h) Meta-gene enrichment analysis shows ChIP occupancy for ACSS2 (e), H3K9ac (f), H4K5ac (g), and for H4K12ac (h) at the top 5% of genes that become dynamically bound by ACSS2 upon neuronal differentiation (Top 5% DE; red). The bottom 80% of genes (Bot 80% DE) is shown in blue, and the average signal across all genes (All genes DE) is shown in green. (i) Multiple linear regression analysis was implemented to model the interaction between genic ACSS2 enrichment and WT gene expression changes, and to visualize the interaction between differentiation-linked gene expression changes and ACSS2 recruitment to chromatin. The contour plot of this fitted regression model displays high levels of ACSS2 enrichment in red and low levels in blue, and is overlaid with the scatter plot of the independent gene expression variables. The visualized model demonstrates that high ACSS2 enrichment (red) corresponds to increased gene expression in the CAD neuronal differentiation.

    Journal: Nature

    Article Title: ACETYL-COA SYNTHETASE REGULATES HISTONE ACETYLATION AND HIPPOCAMPAL MEMORY

    doi: 10.1038/nature22405

    Figure Lengend Snippet: (a–d) Meta-gene enrichment analysis shows ChIP occupancy for ACSS2 (a), H3K9ac (b), H4K5ac (c), and for H4K12ac (d) across the top 5% of genes enriched for ACSS2 in differentiated CAD neurons (Top 5% DE; red). The bottom 80% of genes (Bot 80% DE) is shown in blue, and the average signal across all genes (All genes DE) is shown in green. (e–h) Meta-gene enrichment analysis shows ChIP occupancy for ACSS2 (e), H3K9ac (f), H4K5ac (g), and for H4K12ac (h) at the top 5% of genes that become dynamically bound by ACSS2 upon neuronal differentiation (Top 5% DE; red). The bottom 80% of genes (Bot 80% DE) is shown in blue, and the average signal across all genes (All genes DE) is shown in green. (i) Multiple linear regression analysis was implemented to model the interaction between genic ACSS2 enrichment and WT gene expression changes, and to visualize the interaction between differentiation-linked gene expression changes and ACSS2 recruitment to chromatin. The contour plot of this fitted regression model displays high levels of ACSS2 enrichment in red and low levels in blue, and is overlaid with the scatter plot of the independent gene expression variables. The visualized model demonstrates that high ACSS2 enrichment (red) corresponds to increased gene expression in the CAD neuronal differentiation.

    Article Snippet: Antibodies included Anti-H3 (Abcam ab1791), Anti-H3K9ac (Abcam ab4441), Anti-H3K27ac (Abcam ab4729), Anti-H3K122ac (Abcam ab33308), Anti-H4 (Millipore 05-858), Anti-H4K5ac (Millipore 39-584), Anti-H4K12ac (Abcam ab1761), Anti-ACSS2 (T) (Thermo MA5-145810), Anti-ACSS2 (CS) (Cell Signaling 3658), Anti-ACL (Proteintech 15421-1-AP), Anti-α-Tubulin (Sigma T8328), Anti-GAPDH (Fitzgerald Industries 10R-G109A), Anti-KAT3A/CBP (Abcam ab2832), Anti-SNAP25 (Abcam ab5666), Anti-Synaptophysin (Millipore MAB368), Anti-MAP2 C/D (Cell Signaling #8707), Anti-NR4A2 (Santa Cruz sc-991), Anti-NeuN (Millipore ABN78)

    Techniques: Chromatin Immunoprecipitation, Expressing