h3k27me3  (Millipore)


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  • 99
    Name:
    Imprint Anti H3K27me3 antibody
    Description:

    Catalog Number:
    SAB4800015
    Molecular Weight:
    antigen mol wt ~17 kDa
    Price:
    None
    Host:
    rabbit
    Conjugate:
    unconjugated
    Category:
    Antibodies
    Source:
    rabbit
    Reactivity:
    human
    Score:
    85
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    Structured Review

    Millipore h3k27me3
    Imprint Anti H3K27me3 antibody

    https://www.bioz.com/result/h3k27me3/product/Millipore
    Average 99 stars, based on 210 article reviews
    Price from $9.99 to $1999.99
    h3k27me3 - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells"

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    Journal:

    doi: 10.1016/j.nepig.2016.04.001

    Correlation between genes in normal NSPCS enriched with H3K27me3 or H4K20me3 without detectable transcripts and genes altered in MRI-classified group I and group II GBM A heatmap for differential expression of genes of human GBM specimens. Genes used
    Figure Legend Snippet: Correlation between genes in normal NSPCS enriched with H3K27me3 or H4K20me3 without detectable transcripts and genes altered in MRI-classified group I and group II GBM A heatmap for differential expression of genes of human GBM specimens. Genes used

    Techniques Used: Magnetic Resonance Imaging, Expressing

    Comparison among genes in normal NSPCS enriched with H3K27me3 or H4K20me3, genes without detectable transcripts in normal NSPCs, and genes elevated in MRI-classified group I and group II GBM
    Figure Legend Snippet: Comparison among genes in normal NSPCS enriched with H3K27me3 or H4K20me3, genes without detectable transcripts in normal NSPCs, and genes elevated in MRI-classified group I and group II GBM

    Techniques Used: Magnetic Resonance Imaging

    Quantification of H3K27me3 or H4K20me3 co-localization with cell type markers in baboon SVZ by flow cytometry
    Figure Legend Snippet: Quantification of H3K27me3 or H4K20me3 co-localization with cell type markers in baboon SVZ by flow cytometry

    Techniques Used: Flow Cytometry, Cytometry

    H3K27me3 and H4K20me3 distributions across SVZ subpopulations. Co-immunostaining of H3K27me3 or H4K20me3 with cell-type specific markers GFAP, Nestin, and DCX. Left panel presents coronal section of baboon brain and right panel presents coronal section
    Figure Legend Snippet: H3K27me3 and H4K20me3 distributions across SVZ subpopulations. Co-immunostaining of H3K27me3 or H4K20me3 with cell-type specific markers GFAP, Nestin, and DCX. Left panel presents coronal section of baboon brain and right panel presents coronal section

    Techniques Used: Immunostaining

    H3K27me3 and H4K20me3 genome-wide enrichment patterns in baboon SVZ
    Figure Legend Snippet: H3K27me3 and H4K20me3 genome-wide enrichment patterns in baboon SVZ

    Techniques Used: Genome Wide

    Colocalization of H3K27me3 and H4K20me3 in NSPCs of baboon SVZ
    Figure Legend Snippet: Colocalization of H3K27me3 and H4K20me3 in NSPCs of baboon SVZ

    Techniques Used:

    Experimental Design for Correlation between genes in normal NSPCS enriched with H3K27me3 or H4K20me3 without detectable transcripts and genes altered in MRI-classified group I and group II GBM
    Figure Legend Snippet: Experimental Design for Correlation between genes in normal NSPCS enriched with H3K27me3 or H4K20me3 without detectable transcripts and genes altered in MRI-classified group I and group II GBM

    Techniques Used: Magnetic Resonance Imaging

    EZH2/H3K27me3 influence cell cycle in the SVZ cells
    Figure Legend Snippet: EZH2/H3K27me3 influence cell cycle in the SVZ cells

    Techniques Used:

    2) Product Images from "Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development"

    Article Title: Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    Journal: eLife

    doi: 10.7554/eLife.09571

    H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014
    Figure Legend Snippet: H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014

    Techniques Used: DNA Methylation Assay

    Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035
    Figure Legend Snippet: Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035

    Techniques Used:

    H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029
    Figure Legend Snippet: H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029

    Techniques Used:

    H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10
    Figure Legend Snippet: H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10

    Techniques Used: DNA Methylation Assay

    H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value
    Figure Legend Snippet: H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value

    Techniques Used: DNA Methylation Assay, Genome Wide

    Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)
    Figure Legend Snippet: Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)

    Techniques Used:

    H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022
    Figure Legend Snippet: H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022

    Techniques Used: Flow Cytometry, Expressing

    LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013
    Figure Legend Snippet: LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013

    Techniques Used: Chromatin Immunoprecipitation, Sequencing

    3) Product Images from "Epigenetic repression of the Igk locus by STAT5-mediated Ezh2 recruitment"

    Article Title: Epigenetic repression of the Igk locus by STAT5-mediated Ezh2 recruitment

    Journal: Nature Immunology

    doi: 10.1038/ni.2136

    H3K27 trimethylation correlates with STAT5 target genes that are repressed throughout B cell development. ( a ) Heat map of genes targeted by both STAT5 and H3K27me3 indentified in Fig. 6 representing fold changes in expression (log 2 ) as a function of B cell development and maturation relative to the pro-B cell stage (ImmGen Consortium). ( b ) EMBER analysis combining STAT5 and H3K27me3 ChIP-seq results with total mouse genome expression microarrays from the ImmGen Consortium. Predominant expression patterns of genes within 100 kb of STAT5 binding with or without coincidental H3K27me3 marks were assessed. Coincidence required a minimum of 2 bp overlap. In some instances more than one gene is within 100 kb of a peak. For example, 69 genes are within 100 kb of the 53 STAT5 and H3K27me3 coincident peaks. All indicated B cell developmental stages are compared to expression at pro-B cell stage. Change in mean expression behavior is categorized as: ≤−3s.d. (--); between −1 and −3s.d. (-); between −1 and 1s.d. (0); between 1 and 3s.d. (+); greater than 3s.d., with s.d. defined as the sum of the two standard deviations calculated for the experimental replicates at the two conditions.
    Figure Legend Snippet: H3K27 trimethylation correlates with STAT5 target genes that are repressed throughout B cell development. ( a ) Heat map of genes targeted by both STAT5 and H3K27me3 indentified in Fig. 6 representing fold changes in expression (log 2 ) as a function of B cell development and maturation relative to the pro-B cell stage (ImmGen Consortium). ( b ) EMBER analysis combining STAT5 and H3K27me3 ChIP-seq results with total mouse genome expression microarrays from the ImmGen Consortium. Predominant expression patterns of genes within 100 kb of STAT5 binding with or without coincidental H3K27me3 marks were assessed. Coincidence required a minimum of 2 bp overlap. In some instances more than one gene is within 100 kb of a peak. For example, 69 genes are within 100 kb of the 53 STAT5 and H3K27me3 coincident peaks. All indicated B cell developmental stages are compared to expression at pro-B cell stage. Change in mean expression behavior is categorized as: ≤−3s.d. (--); between −1 and −3s.d. (-); between −1 and 1s.d. (0); between 1 and 3s.d. (+); greater than 3s.d., with s.d. defined as the sum of the two standard deviations calculated for the experimental replicates at the two conditions.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Binding Assay

    Epigenetic regulation of Eκi during B lymphopoiesis. ( a ) Wild-type pro-B, large pre-B, small pre-B and immature B cells were isolated by flow cytometry and assayed by ChIP using antibodies specific for H4Ac, H3K4me1, H3K27me3 or control IgG1. qPCR was performed with primers specific for Eκi (average ± s.d, n=3). * P
    Figure Legend Snippet: Epigenetic regulation of Eκi during B lymphopoiesis. ( a ) Wild-type pro-B, large pre-B, small pre-B and immature B cells were isolated by flow cytometry and assayed by ChIP using antibodies specific for H4Ac, H3K4me1, H3K27me3 or control IgG1. qPCR was performed with primers specific for Eκi (average ± s.d, n=3). * P

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

    STAT5 and E2A mediate repressive and activation marks respectively from Jκ through Cκ. ( a ) ChIP assays of Irf4 -/- Irf8 -/- pre-B cells expressing CA-STAT5B or vector alone and cultured for 48 h in low IL-7. ChIP using antibodies specific for H4Ac, H3K4me1, H3K27me3 or control IgG1 followed by qPCR for Eκi (average ± s.d, n=3). * P
    Figure Legend Snippet: STAT5 and E2A mediate repressive and activation marks respectively from Jκ through Cκ. ( a ) ChIP assays of Irf4 -/- Irf8 -/- pre-B cells expressing CA-STAT5B or vector alone and cultured for 48 h in low IL-7. ChIP using antibodies specific for H4Ac, H3K4me1, H3K27me3 or control IgG1 followed by qPCR for Eκi (average ± s.d, n=3). * P

    Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Expressing, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction

    Tetrameric STAT5 binding mediates H3K27 trimethylation in vivo . ( a-b ) ChIP-seq analysis of Rag2 -/- pro-B cell populations expanded in vitro for 2 days in the presence of IL-7 (10 ng/ml). ChIPs were performed using anti-STAT5 and anti-H3K27me3. ( a ) Schematic of the Igk locus noting the location of Vκ, Jκ, Eκi and Cκ gene segments (mm9 chromosome 6: 70,653,572-70,676,748). The regions of detected H3K27me3 and STAT5 binding are provided. ( b ) Schematic of the Igh locus noting the location of V H , D H , J H , Eμ, and C H gene segments (mm9 chromosome 12: 114,496,979-117,248,165). The regions of detected H3K27me3 and STAT5 binding are provided. ( a,b ) Results are presented as a smoothed tag density where tag = sequence read. ( c ) Conserved de novo DNA sequence motifs identified among STAT5-bound regions. ( d ) Venn diagram showing the common peaks obtained from STAT5 and H3K27me3 ChIP-seq analysis. ( e ) Predicted tetrameric STAT5 binding motif determined from the gene regulatory regions demonstrating STAT5 and H3K27me3 common peaks ( Supplementary Tables 3,4 ). The size of the letter at each position represents the percentage of presence of that nucleotide in that position. The size of the number represents the number of the nucleotides present between two STAT5 binding motifs.
    Figure Legend Snippet: Tetrameric STAT5 binding mediates H3K27 trimethylation in vivo . ( a-b ) ChIP-seq analysis of Rag2 -/- pro-B cell populations expanded in vitro for 2 days in the presence of IL-7 (10 ng/ml). ChIPs were performed using anti-STAT5 and anti-H3K27me3. ( a ) Schematic of the Igk locus noting the location of Vκ, Jκ, Eκi and Cκ gene segments (mm9 chromosome 6: 70,653,572-70,676,748). The regions of detected H3K27me3 and STAT5 binding are provided. ( b ) Schematic of the Igh locus noting the location of V H , D H , J H , Eμ, and C H gene segments (mm9 chromosome 12: 114,496,979-117,248,165). The regions of detected H3K27me3 and STAT5 binding are provided. ( a,b ) Results are presented as a smoothed tag density where tag = sequence read. ( c ) Conserved de novo DNA sequence motifs identified among STAT5-bound regions. ( d ) Venn diagram showing the common peaks obtained from STAT5 and H3K27me3 ChIP-seq analysis. ( e ) Predicted tetrameric STAT5 binding motif determined from the gene regulatory regions demonstrating STAT5 and H3K27me3 common peaks ( Supplementary Tables 3,4 ). The size of the letter at each position represents the percentage of presence of that nucleotide in that position. The size of the number represents the number of the nucleotides present between two STAT5 binding motifs.

    Techniques Used: Binding Assay, In Vivo, Chromatin Immunoprecipitation, In Vitro, Sequencing

    4) Product Images from "MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency"

    Article Title: MacroH2A histone variants act as a barrier upon reprogramming towards pluripotency

    Journal: Nature communications

    doi: 10.1038/ncomms2582

    macroH2A occupancy inhibits activation of Utx target genes required early in iPS reprogramming (a) Pie chart of Utx target genes with aberrant H3K27me3 methylation (in Utx KO) 33 composed of the four classes of genes from Fig. 4c . (b) Comparison of genes marked by H3K27me3, H3K27ac and macroH2A-bound genes (combined macroH2A1 and macroH2A2 targets) in wt DFs and genes reactivated early in iPS reprogramming with normal H3K27me3 demethylation in Utx KO cells (top) versus genes that aberrantly retain H3K27me3 in Utx KO cells (bottom) 35 . MacroH2A is enriched in genes that are unable to demethylate H3K27me3 in the absence of Utx . (c) ChIP-seq profile (UCSC browser) of two genes bound by macroH2A1, macroH2A2 and H3K27me3 in DFs that are not properly demethylated in the absence of Utx during reprogramming (Sall1 and Sall4 - grey bar represents the gene body region). Chromatin ‘domains’ identified with Sicer annotated under each profile (black bar). MacroH2A1 ChIP-seq in dKO DFs and Input used as controls. (d) Time course analysis of mRNA expression of three Utx target genes (Fgf4, Sall1 and Sall4) during reprogramming shows delayed induction in wild type DFs as compared to macroH2A dKO DFs. Relative expression is plotted using ribosomal L7 as a house keeping gene, and compared to DFs at day 0, mean ± s.d. (n=3).
    Figure Legend Snippet: macroH2A occupancy inhibits activation of Utx target genes required early in iPS reprogramming (a) Pie chart of Utx target genes with aberrant H3K27me3 methylation (in Utx KO) 33 composed of the four classes of genes from Fig. 4c . (b) Comparison of genes marked by H3K27me3, H3K27ac and macroH2A-bound genes (combined macroH2A1 and macroH2A2 targets) in wt DFs and genes reactivated early in iPS reprogramming with normal H3K27me3 demethylation in Utx KO cells (top) versus genes that aberrantly retain H3K27me3 in Utx KO cells (bottom) 35 . MacroH2A is enriched in genes that are unable to demethylate H3K27me3 in the absence of Utx . (c) ChIP-seq profile (UCSC browser) of two genes bound by macroH2A1, macroH2A2 and H3K27me3 in DFs that are not properly demethylated in the absence of Utx during reprogramming (Sall1 and Sall4 - grey bar represents the gene body region). Chromatin ‘domains’ identified with Sicer annotated under each profile (black bar). MacroH2A1 ChIP-seq in dKO DFs and Input used as controls. (d) Time course analysis of mRNA expression of three Utx target genes (Fgf4, Sall1 and Sall4) during reprogramming shows delayed induction in wild type DFs as compared to macroH2A dKO DFs. Relative expression is plotted using ribosomal L7 as a house keeping gene, and compared to DFs at day 0, mean ± s.d. (n=3).

    Techniques Used: Activation Assay, Methylation, Gene Knockout, Chromatin Immunoprecipitation, Expressing

    The histone variant macroH2A acts as a barrier to somatic cell reprogramming macroH2A is enriched in somatic cells enabling pluripotency genes to be maintained in a repressed state in differentiated cells. This deposition is correlated with enrichment of the repressive mark H3K27me3. Upon over-expression of reprogramming factors in differentiated cells, macroH2A provides an extra layer of silencing at pluripotency genes, and therefore acts as a barrier to reprogramming. The histone demethylase Utx is required to remove H3K27me3 at a subset of genes, also bound by macroH2A, that need to be reactivated upon reprogramming. Differentiation of pluripotent stem cells is not impaired in the absence of macroH2A, as it likely acts as a final ‘lock’ upon other silencing mechanisms. However, differentiated cells (e.g. EBs) lacking macroH2A are more amenable to reactivation of the pluripotent state, further suggesting that its presence induces a barrier to reprogramming in differentiated cells.
    Figure Legend Snippet: The histone variant macroH2A acts as a barrier to somatic cell reprogramming macroH2A is enriched in somatic cells enabling pluripotency genes to be maintained in a repressed state in differentiated cells. This deposition is correlated with enrichment of the repressive mark H3K27me3. Upon over-expression of reprogramming factors in differentiated cells, macroH2A provides an extra layer of silencing at pluripotency genes, and therefore acts as a barrier to reprogramming. The histone demethylase Utx is required to remove H3K27me3 at a subset of genes, also bound by macroH2A, that need to be reactivated upon reprogramming. Differentiation of pluripotent stem cells is not impaired in the absence of macroH2A, as it likely acts as a final ‘lock’ upon other silencing mechanisms. However, differentiated cells (e.g. EBs) lacking macroH2A are more amenable to reactivation of the pluripotent state, further suggesting that its presence induces a barrier to reprogramming in differentiated cells.

    Techniques Used: Variant Assay, Over Expression

    macroH2A deficiency improves iPS reprogramming efficiency (a) Chromatin extracts from wt and dKO DFs probed for macroH2A1 and macroH2A2; Amido Black of core histones used for loading. (b) IF of macroH2A1 (top) and H3K27me3 (bottom) in wt and dKO male and female DFs. Note loss of macroH2A staining in dKO cells and enrichment of H3K27me3 at the Xi (white arrows) in female cells of both wt and dKO genotypes. DAPI used to stain DNA; Scale bar, 5μm. (c) Experimental scheme of iPS reprogramming from DF (wt, m1KO, m2KO and dKO) to iPS colonies, using a polycistronic lentiviral vector (Stemcca) encoding four factors (4F): Oct4, Sox2, Klf4, and Myc. Reprogramming efficiency was analyzed by FACS analysis of SSEA1 positive cells, alkaline phosphatase (AP) staining of iPS colonies, and IF for Nanog; time frame of experiments shown. (d) FACS plots for SSEA1-PE stained DFs showing increased percentage of SSEA1 positive cells in dKO (male and female) at 8 days post-infection; quantitation on the right. (e) Representative wells of AP positive iPS colonies, indicating increased reprogramming efficiency in dKO DFs at 14 days post-infection. (f) Number of colonies (AP and Nanog positive) obtained at day 14 post-infection; mean ± s.d. (n=3); unpaired Student's test (two tailed) p
    Figure Legend Snippet: macroH2A deficiency improves iPS reprogramming efficiency (a) Chromatin extracts from wt and dKO DFs probed for macroH2A1 and macroH2A2; Amido Black of core histones used for loading. (b) IF of macroH2A1 (top) and H3K27me3 (bottom) in wt and dKO male and female DFs. Note loss of macroH2A staining in dKO cells and enrichment of H3K27me3 at the Xi (white arrows) in female cells of both wt and dKO genotypes. DAPI used to stain DNA; Scale bar, 5μm. (c) Experimental scheme of iPS reprogramming from DF (wt, m1KO, m2KO and dKO) to iPS colonies, using a polycistronic lentiviral vector (Stemcca) encoding four factors (4F): Oct4, Sox2, Klf4, and Myc. Reprogramming efficiency was analyzed by FACS analysis of SSEA1 positive cells, alkaline phosphatase (AP) staining of iPS colonies, and IF for Nanog; time frame of experiments shown. (d) FACS plots for SSEA1-PE stained DFs showing increased percentage of SSEA1 positive cells in dKO (male and female) at 8 days post-infection; quantitation on the right. (e) Representative wells of AP positive iPS colonies, indicating increased reprogramming efficiency in dKO DFs at 14 days post-infection. (f) Number of colonies (AP and Nanog positive) obtained at day 14 post-infection; mean ± s.d. (n=3); unpaired Student's test (two tailed) p

    Techniques Used: Staining, Plasmid Preparation, FACS, Infection, Quantitation Assay, Two Tailed Test

    macroH2A isoforms and H3K27me3 occupy pluripotency genes in DFs (a) qMS heatmap with H3 PTMs comparing iPS cells with DFs, in wt and mH2A dKO cells. H3K27me3 and H3K27ac are amongst the most striking changes upon reprogramming. Two technical replicates run for each of two biological samples. (b) Average signal (100bp window) of the ChIP-seq read counts normalized to total number of reads (counts per million reads), plotted against the distance (-5Kb, +5Kb), from the nearest annotated (USCS mm9) Transcription Start Site (TSS) of all autosomal genes. c) Heatmap of all annotated TSS (-5Kb, +5Kb) based on macroH2A1, macroH2A2, H3K27me3 and H3K27ac enrichment in wt and mH2A dKO DFs, with k-means clustering of all samples (k=4) allowing for four distinct classes of genes (I, II, III, IV). (d) Correlation plot of macroH2A1 and macroH2A2 ChIP-seq reads at the TSS of autosomal genes in wt DFs. Pearson correlation value (R=0.92) indicates a strong correlation between macroH2A1 and macroH2A2. (e) Venn diagram representing genes enriched in H3K27me3, macroH2A-bound genes (combined macroH2A1 and macroH2A2 targets) and bound by at least one of the four (OSKM) factors 37 . (f) ChIP-seq profiles (UCSC browser) of two major pluripotency genes ( Pou5f1 (Oct4) and Nanog) for macroH2A1, macroH2A2, H3K27me3 and H3K27ac; grey bar represents the gene body region. Chromatin ‘domains’ identified with Sicer annotated under each profile (black bar); macroH2A1 ChIP-seq in dKO DFs and Input used as controls.
    Figure Legend Snippet: macroH2A isoforms and H3K27me3 occupy pluripotency genes in DFs (a) qMS heatmap with H3 PTMs comparing iPS cells with DFs, in wt and mH2A dKO cells. H3K27me3 and H3K27ac are amongst the most striking changes upon reprogramming. Two technical replicates run for each of two biological samples. (b) Average signal (100bp window) of the ChIP-seq read counts normalized to total number of reads (counts per million reads), plotted against the distance (-5Kb, +5Kb), from the nearest annotated (USCS mm9) Transcription Start Site (TSS) of all autosomal genes. c) Heatmap of all annotated TSS (-5Kb, +5Kb) based on macroH2A1, macroH2A2, H3K27me3 and H3K27ac enrichment in wt and mH2A dKO DFs, with k-means clustering of all samples (k=4) allowing for four distinct classes of genes (I, II, III, IV). (d) Correlation plot of macroH2A1 and macroH2A2 ChIP-seq reads at the TSS of autosomal genes in wt DFs. Pearson correlation value (R=0.92) indicates a strong correlation between macroH2A1 and macroH2A2. (e) Venn diagram representing genes enriched in H3K27me3, macroH2A-bound genes (combined macroH2A1 and macroH2A2 targets) and bound by at least one of the four (OSKM) factors 37 . (f) ChIP-seq profiles (UCSC browser) of two major pluripotency genes ( Pou5f1 (Oct4) and Nanog) for macroH2A1, macroH2A2, H3K27me3 and H3K27ac; grey bar represents the gene body region. Chromatin ‘domains’ identified with Sicer annotated under each profile (black bar); macroH2A1 ChIP-seq in dKO DFs and Input used as controls.

    Techniques Used: Chromatin Immunoprecipitation

    5) Product Images from "Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development"

    Article Title: Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    Journal: eLife

    doi: 10.7554/eLife.09571

    H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014
    Figure Legend Snippet: H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014

    Techniques Used: DNA Methylation Assay

    Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035
    Figure Legend Snippet: Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035

    Techniques Used:

    H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029
    Figure Legend Snippet: H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029

    Techniques Used:

    H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10
    Figure Legend Snippet: H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10

    Techniques Used: DNA Methylation Assay

    H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value
    Figure Legend Snippet: H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value

    Techniques Used: DNA Methylation Assay, Genome Wide

    Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)
    Figure Legend Snippet: Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)

    Techniques Used:

    H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022
    Figure Legend Snippet: H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022

    Techniques Used: Flow Cytometry, Expressing

    LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013
    Figure Legend Snippet: LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013

    Techniques Used: Chromatin Immunoprecipitation, Sequencing

    6) Product Images from "Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13"

    Article Title: Distal Limb Patterning Requires Modulation of cis-Regulatory Activities by HOX13

    Journal: Cell reports

    doi: 10.1016/j.celrep.2016.11.039

    Hox13-Dependent Gene Regulation in Presumptive Digit Cells (A) UCSC genome browser views of Gsc , Odz4 and Sall1 genomic regions. (Top) ChIP-seq tracks for HOXA13 and HOXD13. (Left) Schematics of the tissue used for ChIP-seq. Below the H3K27ac and H3K27me3 profiles in Hox13 −/− limb buds, green and red boxes indicate gains and losses of histone marks between WT and Hox13 −/− late-distal limbs. The darkened regions spanning all tracks indicate genomic regions with no detectable change in H3K27ac or H3K27me3. Loss of spatial restriction in Hox13 −/− forelimb buds (bottom). Hox13 −/− fore- and hindlimbs are shown for Sall1 . WT buds at slightly different developmental stages (E11.25–E11.5) are shown in order to match the morphology of mutant buds. (B) UCSC genome browser views of Hes1 , Jag1 , and Sgk1 genomic regions. Track annotations are the same as in (A). Validated enhancer elements are highlighted as colored circles. VISTA identifiers ( Visel et al., 2007 ) are indicated along with the lacZ staining of the validated enhancer and ISH on WT and Hox13 −/−− limbs.
    Figure Legend Snippet: Hox13-Dependent Gene Regulation in Presumptive Digit Cells (A) UCSC genome browser views of Gsc , Odz4 and Sall1 genomic regions. (Top) ChIP-seq tracks for HOXA13 and HOXD13. (Left) Schematics of the tissue used for ChIP-seq. Below the H3K27ac and H3K27me3 profiles in Hox13 −/− limb buds, green and red boxes indicate gains and losses of histone marks between WT and Hox13 −/− late-distal limbs. The darkened regions spanning all tracks indicate genomic regions with no detectable change in H3K27ac or H3K27me3. Loss of spatial restriction in Hox13 −/− forelimb buds (bottom). Hox13 −/− fore- and hindlimbs are shown for Sall1 . WT buds at slightly different developmental stages (E11.25–E11.5) are shown in order to match the morphology of mutant buds. (B) UCSC genome browser views of Hes1 , Jag1 , and Sgk1 genomic regions. Track annotations are the same as in (A). Validated enhancer elements are highlighted as colored circles. VISTA identifiers ( Visel et al., 2007 ) are indicated along with the lacZ staining of the validated enhancer and ISH on WT and Hox13 −/−− limbs.

    Techniques Used: Chromatin Immunoprecipitation, Mutagenesis, Staining, In Situ Hybridization

    Genome-wide Mapping of HOXA13 and HOXD13 Binding in Distal Limbs (A) ISH on E11.5 WT limbs using Hoxa13-exon1 and Hoxd13-exon1 probes. Red dashed lines indicate the proximal edge of the tissue used for ChIP-seq. (B) Venn diagram showing the overlap between ChIP-seq peaks for HOXA13 and HOXD13. Boxplots showing enrichment (in terms of p value from MACS peak calling) for regions common to HOXA13 and HOXD13 (red) compared to regions unique for either TF (gray). (C) The logo shows the most enriched motif from de novo motif discovery for HOXA13 (left) and HOXD13 (right). (D) Fraction of VISTA elements ( Visel et al., 2007 ) either positive in limb (limb+), negative in limb but positive in another tissue (limb−), or negative in all tissues (VISTA−) overlapping one or more HOXA13 or HOXD13 binding site. p values from Fisher’s exact test are indicated. (E) Selection of ten VISTA elements ( Visel et al., 2007 ) active in the late-distal limbs, showing binding by both HOXA13 and HOXD13 (ChIP-seq, red tracks). (F) H3K27ac- and H3K27me3-enriched regions (intergenic and intronic peaks defined as farther than 2.5 kbp from the annotated TSS) in WT early and late-distal limbs. (G) Genomic annotations of H3K27ac and H3K27me3 profiles, classified according to the gain or loss observed in Hox13 −/− .
    Figure Legend Snippet: Genome-wide Mapping of HOXA13 and HOXD13 Binding in Distal Limbs (A) ISH on E11.5 WT limbs using Hoxa13-exon1 and Hoxd13-exon1 probes. Red dashed lines indicate the proximal edge of the tissue used for ChIP-seq. (B) Venn diagram showing the overlap between ChIP-seq peaks for HOXA13 and HOXD13. Boxplots showing enrichment (in terms of p value from MACS peak calling) for regions common to HOXA13 and HOXD13 (red) compared to regions unique for either TF (gray). (C) The logo shows the most enriched motif from de novo motif discovery for HOXA13 (left) and HOXD13 (right). (D) Fraction of VISTA elements ( Visel et al., 2007 ) either positive in limb (limb+), negative in limb but positive in another tissue (limb−), or negative in all tissues (VISTA−) overlapping one or more HOXA13 or HOXD13 binding site. p values from Fisher’s exact test are indicated. (E) Selection of ten VISTA elements ( Visel et al., 2007 ) active in the late-distal limbs, showing binding by both HOXA13 and HOXD13 (ChIP-seq, red tracks). (F) H3K27ac- and H3K27me3-enriched regions (intergenic and intronic peaks defined as farther than 2.5 kbp from the annotated TSS) in WT early and late-distal limbs. (G) Genomic annotations of H3K27ac and H3K27me3 profiles, classified according to the gain or loss observed in Hox13 −/− .

    Techniques Used: Genome Wide, Binding Assay, In Situ Hybridization, Chromatin Immunoprecipitation, Magnetic Cell Separation, Selection

    Changes in Histone Marks from Early to Late-Distal Limbs Largely Occur at HOX13-Bound Loci (A) Histograms showing the distribution of the distances between the indicated subsets of H3K27ac and H3K27me3 regions and the nearest HOX13 peak summit. (B) Same as (A) but considering H3K27ac or H3K27me3 loss in Hox13 −/− distal limbs. In the absence of HOX13, H3K27ac loss occurs within 10 bp and 1 kb of the nearest HOX13 peak (green and black arrows), while depletions of H3K27me3 primarily occur within 1 kb and 10 kb of a HOX13 peak (blue arrow). The numbers on each panel show the fraction of HOX13-bound and unbound sites in each subset. For example, 41% of the late-distal specific H3K27ac are HOX13-bound CRMs, while 76% of late-distal-specific CRMs showing loss of H3K27ac in Hox13 −/− limb buds are HOX13 bound. (C) Heatmaps showing the cumulative chromatin profile across 5 kb from the center of the CRMs undergoing loss or gain of H3K27ac (left) or H3K27me3 (right) in Hox13 −/− . HOX13-bound (defined according to A and B) regions are indicated by a red tag on the left side of both heatmaps.
    Figure Legend Snippet: Changes in Histone Marks from Early to Late-Distal Limbs Largely Occur at HOX13-Bound Loci (A) Histograms showing the distribution of the distances between the indicated subsets of H3K27ac and H3K27me3 regions and the nearest HOX13 peak summit. (B) Same as (A) but considering H3K27ac or H3K27me3 loss in Hox13 −/− distal limbs. In the absence of HOX13, H3K27ac loss occurs within 10 bp and 1 kb of the nearest HOX13 peak (green and black arrows), while depletions of H3K27me3 primarily occur within 1 kb and 10 kb of a HOX13 peak (blue arrow). The numbers on each panel show the fraction of HOX13-bound and unbound sites in each subset. For example, 41% of the late-distal specific H3K27ac are HOX13-bound CRMs, while 76% of late-distal-specific CRMs showing loss of H3K27ac in Hox13 −/− limb buds are HOX13 bound. (C) Heatmaps showing the cumulative chromatin profile across 5 kb from the center of the CRMs undergoing loss or gain of H3K27ac (left) or H3K27me3 (right) in Hox13 −/− . HOX13-bound (defined according to A and B) regions are indicated by a red tag on the left side of both heatmaps.

    Techniques Used:

    TAD-Centered Distribution of HOX13-Dependent CRMs and DEGs (A) TADs ( Dixon et al., 2012 ) classified based on the differential expression of genes in Hox13 −/− limbs. As compared to TADs showing no de-regulation in gene expression, those with downregulated gene(s) in Hox13 −/− late-distal buds show significantly more HOX13-dependent loss of acetylation (p = 1.3e-9, chi-square test). Similarly, TADs with upregulated gene(s) show HOX13-dependent loss of H3K27me3 in the mutant (p = 5.4e-6). (B) Boxplots showing the total number of non-limb ENCODE samples that show an enrichment for the indicated mark for different subsets of regions as defined throughout this study. The subset of regions with H3K27Ac or H3K27me3 loss in Hox13 −/− shows the highest tissue-specificity. The rightmost panel shows the subset of genes with HOX13-dependent H3K27me3 loss in the late-distal limbs but enriched for H3K27Ac in other tissues.
    Figure Legend Snippet: TAD-Centered Distribution of HOX13-Dependent CRMs and DEGs (A) TADs ( Dixon et al., 2012 ) classified based on the differential expression of genes in Hox13 −/− limbs. As compared to TADs showing no de-regulation in gene expression, those with downregulated gene(s) in Hox13 −/− late-distal buds show significantly more HOX13-dependent loss of acetylation (p = 1.3e-9, chi-square test). Similarly, TADs with upregulated gene(s) show HOX13-dependent loss of H3K27me3 in the mutant (p = 5.4e-6). (B) Boxplots showing the total number of non-limb ENCODE samples that show an enrichment for the indicated mark for different subsets of regions as defined throughout this study. The subset of regions with H3K27Ac or H3K27me3 loss in Hox13 −/− shows the highest tissue-specificity. The rightmost panel shows the subset of genes with HOX13-dependent H3K27me3 loss in the late-distal limbs but enriched for H3K27Ac in other tissues.

    Techniques Used: Expressing, Mutagenesis

    Impact of HOX13 Inactivation on the Regulation of the HoxA and HoxD Gene Clusters (A) UCSC genome browser view of the regulatory landscape surrounding the HoxD cluster (purple shadow). Black lines indicate the TAD boundaries in ES cells as defined by Dixon et al. ( Dixon et al., 2012 ). ChIP-seq tracks for HOXA13 and HOXD13 are shown on top. Schematics for the dissected tissues used for H3K27ac and H3K27me3 profiling are shown on the left. Dark green and red boxes indicating gains and losses of histone marks between WT and Hox13 −/− late-distal limb buds are shown below the Hox13 −/− H3K27ac and H3K27me3 profiles. Previously validated 3′ (early/zeugopod) and 5′ (digit) enhancers ( Gonzalez et al., 2007 ; Montavon et al., 2011 ; Andrey et al., 2013 ) are highlighted (black circles) at the top of the panel. (Bottom left) lacZ expression in the proximal limb triggered by a validated VISTA element. (Bottom right) Hoxd ISH in Hox13 −/− limbs. (Note: the HoxD locus is shown with Hoxd1 on the left and Hoxd13 on the right.) (B) UCSC genome browser view of the regulatory landscape surrounding the HoxA cluster ( Berlivet et al., 2013 ; Woltering et al., 2014 ). The HoxA cluster is highlighted in purple. All tracks and annotations are the same as in (A). Previously validated enhancers on the 5′ side ( Berlivet et al., 2013 ) are shown above the HOXA13 track as black circles. (Bottom left) LacZ expression driven by the indicated VISTA element ( Visel et al., 2007 ). (Bottom right) ISH for Hoxa5 and Hoxa11 in Hox13 −/− limbs.
    Figure Legend Snippet: Impact of HOX13 Inactivation on the Regulation of the HoxA and HoxD Gene Clusters (A) UCSC genome browser view of the regulatory landscape surrounding the HoxD cluster (purple shadow). Black lines indicate the TAD boundaries in ES cells as defined by Dixon et al. ( Dixon et al., 2012 ). ChIP-seq tracks for HOXA13 and HOXD13 are shown on top. Schematics for the dissected tissues used for H3K27ac and H3K27me3 profiling are shown on the left. Dark green and red boxes indicating gains and losses of histone marks between WT and Hox13 −/− late-distal limb buds are shown below the Hox13 −/− H3K27ac and H3K27me3 profiles. Previously validated 3′ (early/zeugopod) and 5′ (digit) enhancers ( Gonzalez et al., 2007 ; Montavon et al., 2011 ; Andrey et al., 2013 ) are highlighted (black circles) at the top of the panel. (Bottom left) lacZ expression in the proximal limb triggered by a validated VISTA element. (Bottom right) Hoxd ISH in Hox13 −/− limbs. (Note: the HoxD locus is shown with Hoxd1 on the left and Hoxd13 on the right.) (B) UCSC genome browser view of the regulatory landscape surrounding the HoxA cluster ( Berlivet et al., 2013 ; Woltering et al., 2014 ). The HoxA cluster is highlighted in purple. All tracks and annotations are the same as in (A). Previously validated enhancers on the 5′ side ( Berlivet et al., 2013 ) are shown above the HOXA13 track as black circles. (Bottom left) LacZ expression driven by the indicated VISTA element ( Visel et al., 2007 ). (Bottom right) ISH for Hoxa5 and Hoxa11 in Hox13 −/− limbs.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, In Situ Hybridization

    7) Product Images from "A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis"

    Article Title: A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis

    Journal: The FASEB Journal

    doi: 10.1096/fj.13-241760

    A hypothetical model depicting the central role of G9a- and EZH2-mediated histone methylation and the interdependent and mutually reinforcing crosstalk between histone methylation and DNA methylation in COX-2 epigenetic silencing in IPF. G9a- and EZH2-mediated H3K9me3 and H3K27me3 result in the recruitment of Dnmts and HDAC-containing complexes via HP1 and EZH2/EED, respectively, to the COX-2 promoter, which then leads to or reinforces DNA methylation and histone deacetylation. DNA methylation in turn causes the recruitment of G9a, EZH2, and HDAC-containing complexes through MeCP2 to strengthen H3K9me3, H3K27me3, and histone deacetylation, leading to reinforced epigenetic silencing of the COX-2 gene in IPF. Therefore, G9a- and EZH2-mediated H3K9me3 and H3K27me3 interact with DNA methylation in a bidirectional and mutually dependent manner to reinforce COX-2 epigenetic silencing in IPF. Disruption of any of these epigenetic modifications by inhibition or knockdown of G9a, EZH2, or Dnmt leads to the removal of the other repressive epigenetic modifications, resulting in an active chromatin state and reactivation of COX-2 in IPF.
    Figure Legend Snippet: A hypothetical model depicting the central role of G9a- and EZH2-mediated histone methylation and the interdependent and mutually reinforcing crosstalk between histone methylation and DNA methylation in COX-2 epigenetic silencing in IPF. G9a- and EZH2-mediated H3K9me3 and H3K27me3 result in the recruitment of Dnmts and HDAC-containing complexes via HP1 and EZH2/EED, respectively, to the COX-2 promoter, which then leads to or reinforces DNA methylation and histone deacetylation. DNA methylation in turn causes the recruitment of G9a, EZH2, and HDAC-containing complexes through MeCP2 to strengthen H3K9me3, H3K27me3, and histone deacetylation, leading to reinforced epigenetic silencing of the COX-2 gene in IPF. Therefore, G9a- and EZH2-mediated H3K9me3 and H3K27me3 interact with DNA methylation in a bidirectional and mutually dependent manner to reinforce COX-2 epigenetic silencing in IPF. Disruption of any of these epigenetic modifications by inhibition or knockdown of G9a, EZH2, or Dnmt leads to the removal of the other repressive epigenetic modifications, resulting in an active chromatin state and reactivation of COX-2 in IPF.

    Techniques Used: Methylation, DNA Methylation Assay, Inhibition

    Histone H3 is repressively hypermethylated and HMT recruitment is increased at the COX-2 promoter in F-IPFs. Confluent and serum-starved F-NLs and F-IPFs were incubated with IL-1β (1 ng/ml) for the times indicated. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K4me3 ( A ), H3K9me3 ( B ), H3K27me3 ( C ), G9a ( D ), SUV39H1 ( E ), EZH2 ( F ), and total histone H3 ( A–C ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A–C ) or to input control ( D–F ). Data are expressed as means ± sem from experiments with 6 separate F-NL and F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: Histone H3 is repressively hypermethylated and HMT recruitment is increased at the COX-2 promoter in F-IPFs. Confluent and serum-starved F-NLs and F-IPFs were incubated with IL-1β (1 ng/ml) for the times indicated. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K4me3 ( A ), H3K9me3 ( B ), H3K27me3 ( C ), G9a ( D ), SUV39H1 ( E ), EZH2 ( F ), and total histone H3 ( A–C ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A–C ) or to input control ( D–F ). Data are expressed as means ± sem from experiments with 6 separate F-NL and F-IPF cell lines performed in duplicate. * P

    Techniques Used: HMT Assay, Incubation, Sonication, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    Epigenetic inhibitors of G9a, EZH2, and Dnmt1 reduce H3K9me3 and H3K27me3 and increase histone H3 and H4 acetylation at the COX-2 promoter in F-IPFs. F-IPFs were incubated without or with BIX-01294 (100 nM), RG109 (5 μM), or DZNep (10 nM) in medium with serum for 2 d before they reached confluence and then were treated without or with the inhibitors in serum-free medium for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the inhibitors for a further 4 h. The protein-DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), HP1 ( B ), H3K27me3 ( C ), and acetylated histone H3 ( D ) and H4 ( E ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A , C , D ), total histone H4 ( E ), or input control ( B ). Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: Epigenetic inhibitors of G9a, EZH2, and Dnmt1 reduce H3K9me3 and H3K27me3 and increase histone H3 and H4 acetylation at the COX-2 promoter in F-IPFs. F-IPFs were incubated without or with BIX-01294 (100 nM), RG109 (5 μM), or DZNep (10 nM) in medium with serum for 2 d before they reached confluence and then were treated without or with the inhibitors in serum-free medium for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the inhibitors for a further 4 h. The protein-DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), HP1 ( B ), H3K27me3 ( C ), and acetylated histone H3 ( D ) and H4 ( E ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A , C , D ), total histone H4 ( E ), or input control ( B ). Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P

    Techniques Used: Incubation, Sonication, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    G9a and EZH2 siRNAs reduce H3K9me3 and H3K27me3 and increase histone H3 and H4 acetylation and H3K4me3 at the COX-2 promoter in F-IPFs. F-IPFs were transfected with control siRNA, G9a siRNA, or EZH2 siRNA in medium with serum for 2 d and serum starved for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the siRNAs for a further 4 h. The protein-DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), HP1 ( B ), H3K27me3 ( C ), acetylated histone H3 ( D ) and H4 ( E ), CBP, p300, PCAF ( F ), and H3K4me3 ( G ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A , C , D , G ), total histone H4 ( E ), or input control ( B , F ). Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P
    Figure Legend Snippet: G9a and EZH2 siRNAs reduce H3K9me3 and H3K27me3 and increase histone H3 and H4 acetylation and H3K4me3 at the COX-2 promoter in F-IPFs. F-IPFs were transfected with control siRNA, G9a siRNA, or EZH2 siRNA in medium with serum for 2 d and serum starved for 1 d before being incubated without or with IL-1β (1 ng/ml) in the presence or absence of the siRNAs for a further 4 h. The protein-DNA complexes were then cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), HP1 ( B ), H3K27me3 ( C ), acetylated histone H3 ( D ) and H4 ( E ), CBP, p300, PCAF ( F ), and H3K4me3 ( G ) were immunoprecipitated with specific antibodies. The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to total histone H3 ( A , C , D , G ), total histone H4 ( E ), or input control ( B , F ). Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate. * P

    Techniques Used: Transfection, Incubation, Sonication, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    HP1, PRC1, and repressive epigenetic enzymes are associated with the COX-2 promoter in F-IPFs. Confluent F-IPFs were serum starved for 24 h. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), H3K27me3 ( B ), HP1 ( C and D ), EZH2 ( E ), and EED ( F ) were immunoprecipitated with specific antibodies first, and then the IPs were immunoprecipitated again with antibodies against HP1 ( A ), PRC1 ( B ), G9a ( C ), Dnmt1, Dnmt3a ( C and E ), EED ( E ), and NCoR, CoREST, and mSin3a ( D , F ). The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to input control. Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate.
    Figure Legend Snippet: HP1, PRC1, and repressive epigenetic enzymes are associated with the COX-2 promoter in F-IPFs. Confluent F-IPFs were serum starved for 24 h. The protein-DNA complexes were cross-linked by formaldehyde treatment, and chromatin pellets were extracted and sonicated. H3K9me3 ( A ), H3K27me3 ( B ), HP1 ( C and D ), EZH2 ( E ), and EED ( F ) were immunoprecipitated with specific antibodies first, and then the IPs were immunoprecipitated again with antibodies against HP1 ( A ), PRC1 ( B ), G9a ( C ), Dnmt1, Dnmt3a ( C and E ), EED ( E ), and NCoR, CoREST, and mSin3a ( D , F ). The associated COX-2 promoter DNA was amplified by real-time PCR, and the amount was calculated and normalized to input control. Data are expressed as means ± sem from experiments with 6 separate F-IPF cell lines performed in duplicate.

    Techniques Used: Sonication, Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction

    8) Product Images from "Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity"

    Article Title: Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003698

    Validation of the temporal sequence of changes in gene expression and H3K27me3 level induced by Ras signaling. ( A ) Time course of changes in H3K27me3 level at the Itgb5 , Adcy7 , and Smad6 loci as determined by ChIP-seq analysis of Ras0 cells and cells infected with the retroviral vector for H-Ras(G12V) for 2, 4, 7, or 12 days. The regions for which the mean H3K27me3 level and corresponding t-half were calculated are highlighted in pink. ( B ) Changes in gene expression (FPKM) and mean H3K27me3 level for Itgb5 , Adcy7 , and Smad6 . The t-half values are indicated by the dashed lines. ( C ) RT-qPCR analysis of gene expression and ChIP-qPCR analysis of the ratio of H3K27me3 to total H3 for Itgb5 , Adcy7 , and Smad6 at the indicated times after introduction of the retroviral vector for H-Ras(G12V). Data are expressed relative to the corresponding value for time 0. The positions of PCR primers are indicated by arrowheads in (A), and correspond to positions e for Itgb5 and i for Adcy7 shown in Figure S4 . Data are representative of four independent experiments. ( D ) Gene expression (RT-qPCR) and the ratio of H3K27me3 to total H3 (ChIP-qPCR) at the indicated times after exposure of NIH 3T3 cells expressing Raf-ER to 4HT. Data are representative of four independent experiments.
    Figure Legend Snippet: Validation of the temporal sequence of changes in gene expression and H3K27me3 level induced by Ras signaling. ( A ) Time course of changes in H3K27me3 level at the Itgb5 , Adcy7 , and Smad6 loci as determined by ChIP-seq analysis of Ras0 cells and cells infected with the retroviral vector for H-Ras(G12V) for 2, 4, 7, or 12 days. The regions for which the mean H3K27me3 level and corresponding t-half were calculated are highlighted in pink. ( B ) Changes in gene expression (FPKM) and mean H3K27me3 level for Itgb5 , Adcy7 , and Smad6 . The t-half values are indicated by the dashed lines. ( C ) RT-qPCR analysis of gene expression and ChIP-qPCR analysis of the ratio of H3K27me3 to total H3 for Itgb5 , Adcy7 , and Smad6 at the indicated times after introduction of the retroviral vector for H-Ras(G12V). Data are expressed relative to the corresponding value for time 0. The positions of PCR primers are indicated by arrowheads in (A), and correspond to positions e for Itgb5 and i for Adcy7 shown in Figure S4 . Data are representative of four independent experiments. ( D ) Gene expression (RT-qPCR) and the ratio of H3K27me3 to total H3 (ChIP-qPCR) at the indicated times after exposure of NIH 3T3 cells expressing Raf-ER to 4HT. Data are representative of four independent experiments.

    Techniques Used: Sequencing, Expressing, Chromatin Immunoprecipitation, Infection, Plasmid Preparation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Comprehensive analysis of Ras-induced changes in gene transcription and H3K27me3 content in the gene body. ( A ) Venn diagram indicating the number of genes showing Ras-induced changes in expression and in the mean H3K27me3 level of the gene body. ( B ) Clustering of the temporal profiles of mean H3K27me3 level in the gene body. Each line represents one of 115 genes whose H3K27me3 level in the gene body and expression changed in NIH 3T3 cells during expression of H-Ras(G12V) for the indicated times. Results of hierarchical clustering are depicted on the left with colors of purple, gray, and brown. Changes in expression level (FPKM) of individual genes (as determined in Figure S1B ) are depicted on the right with colors of red (increase) or blue (decrease). ( C ) Averaged changes in expression and H3K27me3 level for the purple cluster (upper) and the gray cluster (lower) of genes shown in (B). Dashed lines represent “t-half,” the time corresponding to half of the difference between the values for Ras0 cells and cells expressing H-Ras(G12V) for 12 days. ( D ) The t-half values for expression and mean H3K27me3 level in the gene body for the purple and the gray clusters in (B).
    Figure Legend Snippet: Comprehensive analysis of Ras-induced changes in gene transcription and H3K27me3 content in the gene body. ( A ) Venn diagram indicating the number of genes showing Ras-induced changes in expression and in the mean H3K27me3 level of the gene body. ( B ) Clustering of the temporal profiles of mean H3K27me3 level in the gene body. Each line represents one of 115 genes whose H3K27me3 level in the gene body and expression changed in NIH 3T3 cells during expression of H-Ras(G12V) for the indicated times. Results of hierarchical clustering are depicted on the left with colors of purple, gray, and brown. Changes in expression level (FPKM) of individual genes (as determined in Figure S1B ) are depicted on the right with colors of red (increase) or blue (decrease). ( C ) Averaged changes in expression and H3K27me3 level for the purple cluster (upper) and the gray cluster (lower) of genes shown in (B). Dashed lines represent “t-half,” the time corresponding to half of the difference between the values for Ras0 cells and cells expressing H-Ras(G12V) for 12 days. ( D ) The t-half values for expression and mean H3K27me3 level in the gene body for the purple and the gray clusters in (B).

    Techniques Used: Expressing

    Activation of Ras signaling increases H3K27me3 abundance at the Fas locus in NIH 3T3 cells. ( A ) Immunoblot analysis of H-Ras, phosphorylated (p-) and total forms of Erk1/2, and α-tubulin (loading control) in the cytosolic fraction of NIH 3T3 cells expressing human H-Ras(G12V) (Ras cells) and control (Vec) cells. ( B ) Phase-contrast images of Ras and Vec cells. Scale bars, 100 µm. ( C ) RT-qPCR analysis of Fas , Acta2 , and Stambpl1 expression in Ras cells relative to that in Vec cells. Data are means ± SE from five independent experiments. ( D ) ChIP-qPCR analysis of H3K9me2, H3K9me3, and H3K27me3 at the Fas locus in Ras and Vec cells. The positions of genes on the chromosome and their transcriptional orientation are indicated at the bottom of the panel. Data are expressed as fold enrichment relative to the value for Vec cells at each position, and are means ± SE from at least two independent experiments.
    Figure Legend Snippet: Activation of Ras signaling increases H3K27me3 abundance at the Fas locus in NIH 3T3 cells. ( A ) Immunoblot analysis of H-Ras, phosphorylated (p-) and total forms of Erk1/2, and α-tubulin (loading control) in the cytosolic fraction of NIH 3T3 cells expressing human H-Ras(G12V) (Ras cells) and control (Vec) cells. ( B ) Phase-contrast images of Ras and Vec cells. Scale bars, 100 µm. ( C ) RT-qPCR analysis of Fas , Acta2 , and Stambpl1 expression in Ras cells relative to that in Vec cells. Data are means ± SE from five independent experiments. ( D ) ChIP-qPCR analysis of H3K9me2, H3K9me3, and H3K27me3 at the Fas locus in Ras and Vec cells. The positions of genes on the chromosome and their transcriptional orientation are indicated at the bottom of the panel. Data are expressed as fold enrichment relative to the value for Vec cells at each position, and are means ± SE from at least two independent experiments.

    Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Genome-wide identification of genomic regions at which H3K27me3 enrichment is associated with transcriptional repression. ( A ) Representative distribution of gene expression level and H3K27me3 abundance (as determined by RNA-seq and ChIP-seq, respectively) in control (Ras0) cells. Both parameters are normalized by total read counts. ( B ) Clustering of 2000 randomly selected genes of control cells based on H3K27me3 level. Each line represents an individual gene, including the upstream region, gene body, and downstream region. The length of the gene body is defined as 100% (consisting of 200 data points), and the flanking regions are ±100% of the gene body. Results of hierarchical clustering are depicted on the left with colors of brown, gray, and purple. The expression level (FPKM) for individual genes is depicted on the right with colors from blue (low FPKM) to red (high FPKM). ( C ) Definition of the gene body and the region around the TSS for the purposes of this study. ( D ) Relation between gene expression (FPKM) and H3K27me3 level both in and around the gene body (left) and in the region around the TSS (right) for all RefSeq genes. ( E ) Relation between gene expression (FPKM) and mean H3K27me3 level either in the gene body (left) or in the region around the TSS (right) for all RefSeq genes. The plots show the median, 25th and 75th percentiles, and range.
    Figure Legend Snippet: Genome-wide identification of genomic regions at which H3K27me3 enrichment is associated with transcriptional repression. ( A ) Representative distribution of gene expression level and H3K27me3 abundance (as determined by RNA-seq and ChIP-seq, respectively) in control (Ras0) cells. Both parameters are normalized by total read counts. ( B ) Clustering of 2000 randomly selected genes of control cells based on H3K27me3 level. Each line represents an individual gene, including the upstream region, gene body, and downstream region. The length of the gene body is defined as 100% (consisting of 200 data points), and the flanking regions are ±100% of the gene body. Results of hierarchical clustering are depicted on the left with colors of brown, gray, and purple. The expression level (FPKM) for individual genes is depicted on the right with colors from blue (low FPKM) to red (high FPKM). ( C ) Definition of the gene body and the region around the TSS for the purposes of this study. ( D ) Relation between gene expression (FPKM) and H3K27me3 level both in and around the gene body (left) and in the region around the TSS (right) for all RefSeq genes. ( E ) Relation between gene expression (FPKM) and mean H3K27me3 level either in the gene body (left) or in the region around the TSS (right) for all RefSeq genes. The plots show the median, 25th and 75th percentiles, and range.

    Techniques Used: Genome Wide, Expressing, RNA Sequencing Assay, Chromatin Immunoprecipitation

    Signaling-induced changes in H3K27me3 level are not required for those in transcriptional activity. ( A ) Time line for transfection with control (Ctrl) or Suz12 siRNAs, treatment with 4HT or ethanol (EtOH) vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (B) through (D). ( B ) Immunoblot analysis of Suz12 (arrow) and α-tubulin in the cytosolic fraction as well as of H3K27me3 and total H3 in the chromatin fraction. ( C ) ChIP-qPCR analysis of H3K27me3 normalized by total H3 for the regions of Itgb5 , Adcy7 , and Smad6 indicated in Figure 4A . Data are means ± SE from two independent experiments. ( D ) RT-qPCR analysis of relative Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from three independent experiments. ( E ) Time line for transfection with control or Suz12 siRNAs, treatment with 4HT or ethanol vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (F) and (G). ( F ) ChIP-qPCR analysis of H3K27me3 normalized by total H3 at Itgb5 , Adcy7 , and Smad6 . Data are means ± SE from two independent experiments. ( G ) RT-qPCR analysis of relative Suz12 , Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from two independent experiments.
    Figure Legend Snippet: Signaling-induced changes in H3K27me3 level are not required for those in transcriptional activity. ( A ) Time line for transfection with control (Ctrl) or Suz12 siRNAs, treatment with 4HT or ethanol (EtOH) vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (B) through (D). ( B ) Immunoblot analysis of Suz12 (arrow) and α-tubulin in the cytosolic fraction as well as of H3K27me3 and total H3 in the chromatin fraction. ( C ) ChIP-qPCR analysis of H3K27me3 normalized by total H3 for the regions of Itgb5 , Adcy7 , and Smad6 indicated in Figure 4A . Data are means ± SE from two independent experiments. ( D ) RT-qPCR analysis of relative Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from three independent experiments. ( E ) Time line for transfection with control or Suz12 siRNAs, treatment with 4HT or ethanol vehicle, and sample analysis (arrow) for NIH 3T3–Raf-ER cells studied in (F) and (G). ( F ) ChIP-qPCR analysis of H3K27me3 normalized by total H3 at Itgb5 , Adcy7 , and Smad6 . Data are means ± SE from two independent experiments. ( G ) RT-qPCR analysis of relative Suz12 , Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from two independent experiments.

    Techniques Used: Activity Assay, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    Ras-induced H3K27me3 accumulation and transcriptional changes are reversed by inactivation of Ras signaling. ( A ) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–ER-Ras cells studied in (B) through (D). ( B ) Immunoblot analysis of ER-Ras (arrow) and α-tubulin in the cytosolic fraction of the cells. ( C ) RT-qPCR analysis of relative Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from two independent experiments. ( D ) ChIP-qPCR analysis of H3K27me3 and total H3 levels for the regions of Itgb5 , Adcy7 , and Smad6 indicated in Figure 4A . Data are means ± SE from two independent experiments. ( E ) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–Raf-ER cells studied in (F) and (G). ( F ) RT-qPCR analysis of relative Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from two independent experiments. ( G ) ChIP-qPCR analysis of H3K27me3 and total H3 at Itgb5 , Adcy7 , and Smad6 . Data are means ± SE from two independent experiments. The position of PCR primers of Itgb5 correspond to positions c in Figure S4A .
    Figure Legend Snippet: Ras-induced H3K27me3 accumulation and transcriptional changes are reversed by inactivation of Ras signaling. ( A ) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–ER-Ras cells studied in (B) through (D). ( B ) Immunoblot analysis of ER-Ras (arrow) and α-tubulin in the cytosolic fraction of the cells. ( C ) RT-qPCR analysis of relative Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from two independent experiments. ( D ) ChIP-qPCR analysis of H3K27me3 and total H3 levels for the regions of Itgb5 , Adcy7 , and Smad6 indicated in Figure 4A . Data are means ± SE from two independent experiments. ( E ) Time line for exposure to and removal of 4HT as well as sample analysis (arrows) for NIH 3T3–Raf-ER cells studied in (F) and (G). ( F ) RT-qPCR analysis of relative Itgb5 , Adcy7 , and Smad6 expression. Data are means ± SE from two independent experiments. ( G ) ChIP-qPCR analysis of H3K27me3 and total H3 at Itgb5 , Adcy7 , and Smad6 . Data are means ± SE from two independent experiments. The position of PCR primers of Itgb5 correspond to positions c in Figure S4A .

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

    Signaling-induced changes in the production of novel transcripts from intergenic regions occur before changes in H3K27me3 level. ( A ) ChIP-seq analysis of H3K27me3 level as well as strand-specific assignment of sequencing reads from RNA-seq analysis by SOLiD sequencing for Col1a1 and Mink1 loci in Ras and Vec cells. Antisense transcription from the region upstream of each gene was detected predominantly in the vicinity of regions that showed changes in H3K27me3 level, with the predicted transcribed region being denoted schematically by the magenta box. ( B ) RT-qPCR analysis of transcripts derived from the regions upstream of Col1a1 ( uCol1a1 ) and Mink1 ( uMink1 ) in Ras and Vec cells. The analysis was performed with or without the RT reaction. Primers (red arrows) were targeted to the intergenic regions sensitive to Ras-induced modulation of H3K27me3 content. Data are means ± SE from four independent experiments. ( C ) RT-qPCR analysis of expression as well as ChIP-qPCR analysis of H3K27me3 normalized by total H3 for uCol1a1 and uMink1 at the indicated times after exposure of NIH 3T3 cells expressing Raf-ER to 4HT. Data are expressed relative to the values for time 0 and are representative of four independent experiments. ( D ) RT-qPCR analysis of the relative abundance of transcripts derived from uCol1a1 and uMink1 in NIH 3T3–Raf-ER cells transfected with Suz12 or control siRNAs and exposed to 4HT or ethanol as in Figure 5A . Data are means ± SE from two independent experiments.
    Figure Legend Snippet: Signaling-induced changes in the production of novel transcripts from intergenic regions occur before changes in H3K27me3 level. ( A ) ChIP-seq analysis of H3K27me3 level as well as strand-specific assignment of sequencing reads from RNA-seq analysis by SOLiD sequencing for Col1a1 and Mink1 loci in Ras and Vec cells. Antisense transcription from the region upstream of each gene was detected predominantly in the vicinity of regions that showed changes in H3K27me3 level, with the predicted transcribed region being denoted schematically by the magenta box. ( B ) RT-qPCR analysis of transcripts derived from the regions upstream of Col1a1 ( uCol1a1 ) and Mink1 ( uMink1 ) in Ras and Vec cells. The analysis was performed with or without the RT reaction. Primers (red arrows) were targeted to the intergenic regions sensitive to Ras-induced modulation of H3K27me3 content. Data are means ± SE from four independent experiments. ( C ) RT-qPCR analysis of expression as well as ChIP-qPCR analysis of H3K27me3 normalized by total H3 for uCol1a1 and uMink1 at the indicated times after exposure of NIH 3T3 cells expressing Raf-ER to 4HT. Data are expressed relative to the values for time 0 and are representative of four independent experiments. ( D ) RT-qPCR analysis of the relative abundance of transcripts derived from uCol1a1 and uMink1 in NIH 3T3–Raf-ER cells transfected with Suz12 or control siRNAs and exposed to 4HT or ethanol as in Figure 5A . Data are means ± SE from two independent experiments.

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, RNA Sequencing Assay, Quantitative RT-PCR, Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection

    9) Product Images from "Polycomb Repressive Complex 2 Controls the Embryo-to-Seedling Phase Transition"

    Article Title: Polycomb Repressive Complex 2 Controls the Embryo-to-Seedling Phase Transition

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002014

    The set of H3K4 trimethylated genes changes only marginally between wild type and fie and is under-represented among H3K27me3 targets. VENN Diagram representing the number of genes marked by H3K27me3 in wild type (green) and H3K4me3 in wild type (purple) and fie (pink). The mutual overlap of the H3K4me3 targets in wild type and fie is larger than expected for two independent sets, while the overlap of the H3K4me3 marked genes with the H3K27me3 marked genes is significantly smaller (wild type H3K4me3 and fie H3K4me3: rf = 1.9, p
    Figure Legend Snippet: The set of H3K4 trimethylated genes changes only marginally between wild type and fie and is under-represented among H3K27me3 targets. VENN Diagram representing the number of genes marked by H3K27me3 in wild type (green) and H3K4me3 in wild type (purple) and fie (pink). The mutual overlap of the H3K4me3 targets in wild type and fie is larger than expected for two independent sets, while the overlap of the H3K4me3 marked genes with the H3K27me3 marked genes is significantly smaller (wild type H3K4me3 and fie H3K4me3: rf = 1.9, p

    Techniques Used:

    Overrepresented gene ontology categories in the set of H3K27me3 targets that are up-regulated in fie . BiNGO (the Biological Network Gene Ontology tool) analysis representing over-represented categories of the ontology Biological Process among the genes that are marked by H3K27me3 in the wild type (20 DAS) and are significantly up-regulated in 7 DAS fie mutant seedlings. The size of the nodes is proportional to the number of genes in the test set which are annotated to that node. Colored nodes are significantly over-represented, with a color scale ranging from yellow (p-value = 0.05) to dark orange (p-value = 5.00 E -7). Statistical testing was as described by Maere et al. (2005) [31] .
    Figure Legend Snippet: Overrepresented gene ontology categories in the set of H3K27me3 targets that are up-regulated in fie . BiNGO (the Biological Network Gene Ontology tool) analysis representing over-represented categories of the ontology Biological Process among the genes that are marked by H3K27me3 in the wild type (20 DAS) and are significantly up-regulated in 7 DAS fie mutant seedlings. The size of the nodes is proportional to the number of genes in the test set which are annotated to that node. Colored nodes are significantly over-represented, with a color scale ranging from yellow (p-value = 0.05) to dark orange (p-value = 5.00 E -7). Statistical testing was as described by Maere et al. (2005) [31] .

    Techniques Used: Mutagenesis, Significance Assay

    PRC2 represses seed maturation and dormancy genes in the seedling. All genes provided in this model have been identified as H3K27me3 targets and were significantly up-regulated in fie mutants. They include master regulators of seed development such as AGL15 , LEC2 , ABI3 , FUS3 , further downstream regulators such as WRI , integrators of environmental signals such as FLC and finally genes involved in seed storage and desiccation tolerance. * For detailed information which members of the oleosins (oil body coat proteins) and LEAs (late embryogenesis abundant proteins) are affected see Table S5 . For LEC2 the up-regulation was only observed in qRT-PCR ( Table S4 ). We find here that the ABA and GA hormonal signaling pathways that play a pivotal role in the transition form seed to seedling are under PRC2 control since genes playing a positive role in ABA signaling as well as genes with a negative role in GA signaling are H3K27me3 marked in wild type and up-regulated in fie . Large letters stand for high and small for low ABA and GA levels, respectively. Abscisic acid (ABA), Gibberellic acid (GA), AGAMOUS-Like 15 (AGL15, AT5G13790), LEAFY COTYLEDON 2 (LEC2, AT1G28300), ABA INSENSITIVE 3 (ABI3, AT3G24650), FUSCA 3 (FUS3, AT3G26790), WRINKLED1 (WRI, AT3G54320), FLOWERING LOCUS C (FLC, AT5G10140), CRUCIFERIN 3 (CRU3, AT4G28520), CRUCIFERINA (CRA1, AT5G44120), SEED STORAGE ALBUMIN 1 (2S1, AT4G27140), SEED STORAGE ALBUMIN 2, (2S2, AT4G27150), HYDROXYSTEROID DEHYDROGENASE 1 (AtHSD1, AT5G50600), Peroxiredoxin 1 (PER1, AT1G48130), ABA INSENSITIVE 4 (ABI4, AT2G40220), DELAY OF GERMINATION 1 (DOG1, AT5G45830), CHOTTO 1/AINTEGUMENTA-LIKE 5 (CHO1/AIL5, AT5G5739), SOMNUS (SOM, AT1G03790), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8, AT1G02065), AT-hook protein of GA feedback 1 (AGF1, AT4G35390), GIBBERELLIC ACID METHYLTRANSFERASE 2 (GAMT2, AT5G56300).
    Figure Legend Snippet: PRC2 represses seed maturation and dormancy genes in the seedling. All genes provided in this model have been identified as H3K27me3 targets and were significantly up-regulated in fie mutants. They include master regulators of seed development such as AGL15 , LEC2 , ABI3 , FUS3 , further downstream regulators such as WRI , integrators of environmental signals such as FLC and finally genes involved in seed storage and desiccation tolerance. * For detailed information which members of the oleosins (oil body coat proteins) and LEAs (late embryogenesis abundant proteins) are affected see Table S5 . For LEC2 the up-regulation was only observed in qRT-PCR ( Table S4 ). We find here that the ABA and GA hormonal signaling pathways that play a pivotal role in the transition form seed to seedling are under PRC2 control since genes playing a positive role in ABA signaling as well as genes with a negative role in GA signaling are H3K27me3 marked in wild type and up-regulated in fie . Large letters stand for high and small for low ABA and GA levels, respectively. Abscisic acid (ABA), Gibberellic acid (GA), AGAMOUS-Like 15 (AGL15, AT5G13790), LEAFY COTYLEDON 2 (LEC2, AT1G28300), ABA INSENSITIVE 3 (ABI3, AT3G24650), FUSCA 3 (FUS3, AT3G26790), WRINKLED1 (WRI, AT3G54320), FLOWERING LOCUS C (FLC, AT5G10140), CRUCIFERIN 3 (CRU3, AT4G28520), CRUCIFERINA (CRA1, AT5G44120), SEED STORAGE ALBUMIN 1 (2S1, AT4G27140), SEED STORAGE ALBUMIN 2, (2S2, AT4G27150), HYDROXYSTEROID DEHYDROGENASE 1 (AtHSD1, AT5G50600), Peroxiredoxin 1 (PER1, AT1G48130), ABA INSENSITIVE 4 (ABI4, AT2G40220), DELAY OF GERMINATION 1 (DOG1, AT5G45830), CHOTTO 1/AINTEGUMENTA-LIKE 5 (CHO1/AIL5, AT5G5739), SOMNUS (SOM, AT1G03790), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8, AT1G02065), AT-hook protein of GA feedback 1 (AGF1, AT4G35390), GIBBERELLIC ACID METHYLTRANSFERASE 2 (GAMT2, AT5G56300).

    Techniques Used: Quantitative RT-PCR, Binding Assay

    Genome-wide distribution of H3K27me3 and H3K4me3 marks. Genome browser view of Chromosome 4 with the H3K27me3 profile in wild type (first panel) and fie (second panel), boxed annotation of genes in blue (coding region) and grey (introns) (third row), boxed annotation of transposable elements and other heterochromatic regions in brown and orange (transposable element genes) (forth row), the H3K4me3 profile in wild type (fifth panel) and in fie (sixth panel). Enrichment in H3K27me3 or H3K4me3 marks is shown in green bars. (B) and (C) are close-ups and show the major loss of H3K27me3 in fie whereas H3K4me3 distribution is basically unchanged. Raw data have been deposited at GEO database (GSE24163, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24163 ).
    Figure Legend Snippet: Genome-wide distribution of H3K27me3 and H3K4me3 marks. Genome browser view of Chromosome 4 with the H3K27me3 profile in wild type (first panel) and fie (second panel), boxed annotation of genes in blue (coding region) and grey (introns) (third row), boxed annotation of transposable elements and other heterochromatic regions in brown and orange (transposable element genes) (forth row), the H3K4me3 profile in wild type (fifth panel) and in fie (sixth panel). Enrichment in H3K27me3 or H3K4me3 marks is shown in green bars. (B) and (C) are close-ups and show the major loss of H3K27me3 in fie whereas H3K4me3 distribution is basically unchanged. Raw data have been deposited at GEO database (GSE24163, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24163 ).

    Techniques Used: Genome Wide

    Western blot detection of the H3K27me3 mark. Western blot analysis of H3K27me3 in wild type and fie mutants. (A) Comparison between wild type and fie nuclear extracts revealed major loss of H3K27me3-signal in the mutant; however, after over-exposure (OE) a faint signal becomes visible in fie mutants. Detection of histone H3 was used a loading control. (B) Under less stringent conditions a weak signal in fie is detectable by the H3K27me3 antibody. Pre-incubating the antibody with a surplus of H3K27me3 peptides, reduces the signal intensity to only of faint band in wild-type extracts that roughly matches the intensity of the remaining signal seen in fie mutants. Detection of histone H3 was used as loading control. (C) Peptide competition assay using H3K27me3 antibody with increasing concentrations of H3K27me1- and H3K27me2-peptide in fie mutant resulted in a strong reduction of the remaining signal detected by the H3K27me3 antibody. (D) Both peptides could not reduce the H3K27me3 signal in wild-type extract indicating that the antibody works properly given that sufficient antigen is provided.
    Figure Legend Snippet: Western blot detection of the H3K27me3 mark. Western blot analysis of H3K27me3 in wild type and fie mutants. (A) Comparison between wild type and fie nuclear extracts revealed major loss of H3K27me3-signal in the mutant; however, after over-exposure (OE) a faint signal becomes visible in fie mutants. Detection of histone H3 was used a loading control. (B) Under less stringent conditions a weak signal in fie is detectable by the H3K27me3 antibody. Pre-incubating the antibody with a surplus of H3K27me3 peptides, reduces the signal intensity to only of faint band in wild-type extracts that roughly matches the intensity of the remaining signal seen in fie mutants. Detection of histone H3 was used as loading control. (C) Peptide competition assay using H3K27me3 antibody with increasing concentrations of H3K27me1- and H3K27me2-peptide in fie mutant resulted in a strong reduction of the remaining signal detected by the H3K27me3 antibody. (D) Both peptides could not reduce the H3K27me3 signal in wild-type extract indicating that the antibody works properly given that sufficient antigen is provided.

    Techniques Used: Western Blot, Mutagenesis, Competitive Binding Assay

    10) Product Images from "Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development"

    Article Title: Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    Journal: eLife

    doi: 10.7554/eLife.09571

    H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014
    Figure Legend Snippet: H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014

    Techniques Used: DNA Methylation Assay

    Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035
    Figure Legend Snippet: Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035

    Techniques Used:

    H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029
    Figure Legend Snippet: H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029

    Techniques Used:

    H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10
    Figure Legend Snippet: H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10

    Techniques Used: DNA Methylation Assay

    H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value
    Figure Legend Snippet: H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value

    Techniques Used: DNA Methylation Assay, Genome Wide

    Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)
    Figure Legend Snippet: Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)

    Techniques Used:

    H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022
    Figure Legend Snippet: H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022

    Techniques Used: Flow Cytometry, Expressing

    LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013
    Figure Legend Snippet: LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013

    Techniques Used: Chromatin Immunoprecipitation, Sequencing

    11) Product Images from "Polycomb Repressive Complex 2 Controls the Embryo-to-Seedling Phase Transition"

    Article Title: Polycomb Repressive Complex 2 Controls the Embryo-to-Seedling Phase Transition

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002014

    The set of H3K4 trimethylated genes changes only marginally between wild type and fie and is under-represented among H3K27me3 targets. VENN Diagram representing the number of genes marked by H3K27me3 in wild type (green) and H3K4me3 in wild type (purple) and fie (pink). The mutual overlap of the H3K4me3 targets in wild type and fie is larger than expected for two independent sets, while the overlap of the H3K4me3 marked genes with the H3K27me3 marked genes is significantly smaller (wild type H3K4me3 and fie H3K4me3: rf = 1.9, p
    Figure Legend Snippet: The set of H3K4 trimethylated genes changes only marginally between wild type and fie and is under-represented among H3K27me3 targets. VENN Diagram representing the number of genes marked by H3K27me3 in wild type (green) and H3K4me3 in wild type (purple) and fie (pink). The mutual overlap of the H3K4me3 targets in wild type and fie is larger than expected for two independent sets, while the overlap of the H3K4me3 marked genes with the H3K27me3 marked genes is significantly smaller (wild type H3K4me3 and fie H3K4me3: rf = 1.9, p

    Techniques Used:

    Overrepresented gene ontology categories in the set of H3K27me3 targets that are up-regulated in fie . BiNGO (the Biological Network Gene Ontology tool) analysis representing over-represented categories of the ontology Biological Process among the genes that are marked by H3K27me3 in the wild type (20 DAS) and are significantly up-regulated in 7 DAS fie mutant seedlings. The size of the nodes is proportional to the number of genes in the test set which are annotated to that node. Colored nodes are significantly over-represented, with a color scale ranging from yellow (p-value = 0.05) to dark orange (p-value = 5.00 E -7). Statistical testing was as described by Maere et al. (2005) [31] .
    Figure Legend Snippet: Overrepresented gene ontology categories in the set of H3K27me3 targets that are up-regulated in fie . BiNGO (the Biological Network Gene Ontology tool) analysis representing over-represented categories of the ontology Biological Process among the genes that are marked by H3K27me3 in the wild type (20 DAS) and are significantly up-regulated in 7 DAS fie mutant seedlings. The size of the nodes is proportional to the number of genes in the test set which are annotated to that node. Colored nodes are significantly over-represented, with a color scale ranging from yellow (p-value = 0.05) to dark orange (p-value = 5.00 E -7). Statistical testing was as described by Maere et al. (2005) [31] .

    Techniques Used: Mutagenesis, Significance Assay

    PRC2 represses seed maturation and dormancy genes in the seedling. All genes provided in this model have been identified as H3K27me3 targets and were significantly up-regulated in fie mutants. They include master regulators of seed development such as AGL15 , LEC2 , ABI3 , FUS3 , further downstream regulators such as WRI , integrators of environmental signals such as FLC and finally genes involved in seed storage and desiccation tolerance. * For detailed information which members of the oleosins (oil body coat proteins) and LEAs (late embryogenesis abundant proteins) are affected see Table S5 . For LEC2 the up-regulation was only observed in qRT-PCR ( Table S4 ). We find here that the ABA and GA hormonal signaling pathways that play a pivotal role in the transition form seed to seedling are under PRC2 control since genes playing a positive role in ABA signaling as well as genes with a negative role in GA signaling are H3K27me3 marked in wild type and up-regulated in fie . Large letters stand for high and small for low ABA and GA levels, respectively. Abscisic acid (ABA), Gibberellic acid (GA), AGAMOUS-Like 15 (AGL15, AT5G13790), LEAFY COTYLEDON 2 (LEC2, AT1G28300), ABA INSENSITIVE 3 (ABI3, AT3G24650), FUSCA 3 (FUS3, AT3G26790), WRINKLED1 (WRI, AT3G54320), FLOWERING LOCUS C (FLC, AT5G10140), CRUCIFERIN 3 (CRU3, AT4G28520), CRUCIFERINA (CRA1, AT5G44120), SEED STORAGE ALBUMIN 1 (2S1, AT4G27140), SEED STORAGE ALBUMIN 2, (2S2, AT4G27150), HYDROXYSTEROID DEHYDROGENASE 1 (AtHSD1, AT5G50600), Peroxiredoxin 1 (PER1, AT1G48130), ABA INSENSITIVE 4 (ABI4, AT2G40220), DELAY OF GERMINATION 1 (DOG1, AT5G45830), CHOTTO 1/AINTEGUMENTA-LIKE 5 (CHO1/AIL5, AT5G5739), SOMNUS (SOM, AT1G03790), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8, AT1G02065), AT-hook protein of GA feedback 1 (AGF1, AT4G35390), GIBBERELLIC ACID METHYLTRANSFERASE 2 (GAMT2, AT5G56300).
    Figure Legend Snippet: PRC2 represses seed maturation and dormancy genes in the seedling. All genes provided in this model have been identified as H3K27me3 targets and were significantly up-regulated in fie mutants. They include master regulators of seed development such as AGL15 , LEC2 , ABI3 , FUS3 , further downstream regulators such as WRI , integrators of environmental signals such as FLC and finally genes involved in seed storage and desiccation tolerance. * For detailed information which members of the oleosins (oil body coat proteins) and LEAs (late embryogenesis abundant proteins) are affected see Table S5 . For LEC2 the up-regulation was only observed in qRT-PCR ( Table S4 ). We find here that the ABA and GA hormonal signaling pathways that play a pivotal role in the transition form seed to seedling are under PRC2 control since genes playing a positive role in ABA signaling as well as genes with a negative role in GA signaling are H3K27me3 marked in wild type and up-regulated in fie . Large letters stand for high and small for low ABA and GA levels, respectively. Abscisic acid (ABA), Gibberellic acid (GA), AGAMOUS-Like 15 (AGL15, AT5G13790), LEAFY COTYLEDON 2 (LEC2, AT1G28300), ABA INSENSITIVE 3 (ABI3, AT3G24650), FUSCA 3 (FUS3, AT3G26790), WRINKLED1 (WRI, AT3G54320), FLOWERING LOCUS C (FLC, AT5G10140), CRUCIFERIN 3 (CRU3, AT4G28520), CRUCIFERINA (CRA1, AT5G44120), SEED STORAGE ALBUMIN 1 (2S1, AT4G27140), SEED STORAGE ALBUMIN 2, (2S2, AT4G27150), HYDROXYSTEROID DEHYDROGENASE 1 (AtHSD1, AT5G50600), Peroxiredoxin 1 (PER1, AT1G48130), ABA INSENSITIVE 4 (ABI4, AT2G40220), DELAY OF GERMINATION 1 (DOG1, AT5G45830), CHOTTO 1/AINTEGUMENTA-LIKE 5 (CHO1/AIL5, AT5G5739), SOMNUS (SOM, AT1G03790), SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8, AT1G02065), AT-hook protein of GA feedback 1 (AGF1, AT4G35390), GIBBERELLIC ACID METHYLTRANSFERASE 2 (GAMT2, AT5G56300).

    Techniques Used: Quantitative RT-PCR, Binding Assay

    Genome-wide distribution of H3K27me3 and H3K4me3 marks. Genome browser view of Chromosome 4 with the H3K27me3 profile in wild type (first panel) and fie (second panel), boxed annotation of genes in blue (coding region) and grey (introns) (third row), boxed annotation of transposable elements and other heterochromatic regions in brown and orange (transposable element genes) (forth row), the H3K4me3 profile in wild type (fifth panel) and in fie (sixth panel). Enrichment in H3K27me3 or H3K4me3 marks is shown in green bars. (B) and (C) are close-ups and show the major loss of H3K27me3 in fie whereas H3K4me3 distribution is basically unchanged. Raw data have been deposited at GEO database (GSE24163, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24163 ).
    Figure Legend Snippet: Genome-wide distribution of H3K27me3 and H3K4me3 marks. Genome browser view of Chromosome 4 with the H3K27me3 profile in wild type (first panel) and fie (second panel), boxed annotation of genes in blue (coding region) and grey (introns) (third row), boxed annotation of transposable elements and other heterochromatic regions in brown and orange (transposable element genes) (forth row), the H3K4me3 profile in wild type (fifth panel) and in fie (sixth panel). Enrichment in H3K27me3 or H3K4me3 marks is shown in green bars. (B) and (C) are close-ups and show the major loss of H3K27me3 in fie whereas H3K4me3 distribution is basically unchanged. Raw data have been deposited at GEO database (GSE24163, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE24163 ).

    Techniques Used: Genome Wide

    Western blot detection of the H3K27me3 mark. Western blot analysis of H3K27me3 in wild type and fie mutants. (A) Comparison between wild type and fie nuclear extracts revealed major loss of H3K27me3-signal in the mutant; however, after over-exposure (OE) a faint signal becomes visible in fie mutants. Detection of histone H3 was used a loading control. (B) Under less stringent conditions a weak signal in fie is detectable by the H3K27me3 antibody. Pre-incubating the antibody with a surplus of H3K27me3 peptides, reduces the signal intensity to only of faint band in wild-type extracts that roughly matches the intensity of the remaining signal seen in fie mutants. Detection of histone H3 was used as loading control. (C) Peptide competition assay using H3K27me3 antibody with increasing concentrations of H3K27me1- and H3K27me2-peptide in fie mutant resulted in a strong reduction of the remaining signal detected by the H3K27me3 antibody. (D) Both peptides could not reduce the H3K27me3 signal in wild-type extract indicating that the antibody works properly given that sufficient antigen is provided.
    Figure Legend Snippet: Western blot detection of the H3K27me3 mark. Western blot analysis of H3K27me3 in wild type and fie mutants. (A) Comparison between wild type and fie nuclear extracts revealed major loss of H3K27me3-signal in the mutant; however, after over-exposure (OE) a faint signal becomes visible in fie mutants. Detection of histone H3 was used a loading control. (B) Under less stringent conditions a weak signal in fie is detectable by the H3K27me3 antibody. Pre-incubating the antibody with a surplus of H3K27me3 peptides, reduces the signal intensity to only of faint band in wild-type extracts that roughly matches the intensity of the remaining signal seen in fie mutants. Detection of histone H3 was used as loading control. (C) Peptide competition assay using H3K27me3 antibody with increasing concentrations of H3K27me1- and H3K27me2-peptide in fie mutant resulted in a strong reduction of the remaining signal detected by the H3K27me3 antibody. (D) Both peptides could not reduce the H3K27me3 signal in wild-type extract indicating that the antibody works properly given that sufficient antigen is provided.

    Techniques Used: Western Blot, Mutagenesis, Competitive Binding Assay

    12) Product Images from "Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development"

    Article Title: Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    Journal: eLife

    doi: 10.7554/eLife.09571

    H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014
    Figure Legend Snippet: H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014

    Techniques Used: DNA Methylation Assay

    Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035
    Figure Legend Snippet: Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035

    Techniques Used:

    H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029
    Figure Legend Snippet: H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029

    Techniques Used:

    H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10
    Figure Legend Snippet: H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10

    Techniques Used: DNA Methylation Assay

    H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value
    Figure Legend Snippet: H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value

    Techniques Used: DNA Methylation Assay, Genome Wide

    Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)
    Figure Legend Snippet: Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)

    Techniques Used:

    H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022
    Figure Legend Snippet: H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022

    Techniques Used: Flow Cytometry, Expressing

    LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013
    Figure Legend Snippet: LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013

    Techniques Used: Chromatin Immunoprecipitation, Sequencing

    13) Product Images from "Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development"

    Article Title: Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    Journal: eLife

    doi: 10.7554/eLife.09571

    H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014
    Figure Legend Snippet: H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014

    Techniques Used: DNA Methylation Assay

    Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035
    Figure Legend Snippet: Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035

    Techniques Used:

    H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029
    Figure Legend Snippet: H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029

    Techniques Used:

    H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10
    Figure Legend Snippet: H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10

    Techniques Used: DNA Methylation Assay

    H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value
    Figure Legend Snippet: H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value

    Techniques Used: DNA Methylation Assay, Genome Wide

    Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)
    Figure Legend Snippet: Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)

    Techniques Used:

    H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022
    Figure Legend Snippet: H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022

    Techniques Used: Flow Cytometry, Expressing

    LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013
    Figure Legend Snippet: LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013

    Techniques Used: Chromatin Immunoprecipitation, Sequencing

    14) Product Images from "Prognostic Value of EZH2 Expression and Activity in Renal Cell Carcinoma: A Prospective Study"

    Article Title: Prognostic Value of EZH2 Expression and Activity in Renal Cell Carcinoma: A Prospective Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081484

    Comparisons of the receiver operating characteristic (ROC) curves for prediction of survival by the EZH2 score, TNM stage, and H3K27me3 score. (A) (E) DFS, (B) (F) OS in the training set. (C) (G) DFS, (D) (H) OS in the validation set. (A-D) the area under the ROC curves (AUROC) of EZH2 score versus the AUROC of TNM stage, or H3K27me3 score. (E-H) the AUROC of the combined EZH2 and TNM stage model versus the AUROC of the TNM stage or EZH2 expression alone model.
    Figure Legend Snippet: Comparisons of the receiver operating characteristic (ROC) curves for prediction of survival by the EZH2 score, TNM stage, and H3K27me3 score. (A) (E) DFS, (B) (F) OS in the training set. (C) (G) DFS, (D) (H) OS in the validation set. (A-D) the area under the ROC curves (AUROC) of EZH2 score versus the AUROC of TNM stage, or H3K27me3 score. (E-H) the AUROC of the combined EZH2 and TNM stage model versus the AUROC of the TNM stage or EZH2 expression alone model.

    Techniques Used: Expressing

    The expression of EZH2 and H3K27me3 in human samples. Representative EZH2 and H3K27me3 immunohistochemical staining in renal cell carcinoma (200×magnification). (A-H) Positive EZH2 (black arrow) and H3K27me3 (white arrow) display a nuclear staining. (A) negative in RCC (E) negative in none tumor tissue (B-D) EZH2 positive (F-H) H3K27me3 positive (B) (F) staining intensity index-1socre (C) (G) staining intensity index-2 score (D) (H) staining intensity index-3 score (I) the expression of EZH2 and H3K27me3 was detected in all 10 cases of RCC tissues compared to adjacent non-RCC tissues. N, non-RCC tissue; T, RCC tissue.
    Figure Legend Snippet: The expression of EZH2 and H3K27me3 in human samples. Representative EZH2 and H3K27me3 immunohistochemical staining in renal cell carcinoma (200×magnification). (A-H) Positive EZH2 (black arrow) and H3K27me3 (white arrow) display a nuclear staining. (A) negative in RCC (E) negative in none tumor tissue (B-D) EZH2 positive (F-H) H3K27me3 positive (B) (F) staining intensity index-1socre (C) (G) staining intensity index-2 score (D) (H) staining intensity index-3 score (I) the expression of EZH2 and H3K27me3 was detected in all 10 cases of RCC tissues compared to adjacent non-RCC tissues. N, non-RCC tissue; T, RCC tissue.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    Kaplan-Meier analysis of disease-free survival (DFS) in renal cell carcinoma according to expression of the EZH2 or H3K27me3 score. (A), (D) all patients in the training set. (B), (E) patients with I+II stage disease in the training set. (C), (F) patients with III+IV stage disease in the training set. (G), (J) all patients in the validation set. (H), (K) patients with I+II stage disease in the validation set. (I), (L) patients with III+IV stage disease in the validation set.
    Figure Legend Snippet: Kaplan-Meier analysis of disease-free survival (DFS) in renal cell carcinoma according to expression of the EZH2 or H3K27me3 score. (A), (D) all patients in the training set. (B), (E) patients with I+II stage disease in the training set. (C), (F) patients with III+IV stage disease in the training set. (G), (J) all patients in the validation set. (H), (K) patients with I+II stage disease in the validation set. (I), (L) patients with III+IV stage disease in the validation set.

    Techniques Used: Expressing

    Kaplan-Meier analysis of overall survival (OS) in renal cell carcinoma according to expression of the EZH2 or H3K27me3 score. (A), (D) all patients in the training set. (B), (E) patients with I+II stage disease in the training set. (C), (F) patients with III+IV stage disease in the training set. (G), (J) all patients in the validation set. (H), (K) patients with I+II stage disease in the validation set. (I), (L) patients with III+IV stage disease in the validation set.
    Figure Legend Snippet: Kaplan-Meier analysis of overall survival (OS) in renal cell carcinoma according to expression of the EZH2 or H3K27me3 score. (A), (D) all patients in the training set. (B), (E) patients with I+II stage disease in the training set. (C), (F) patients with III+IV stage disease in the training set. (G), (J) all patients in the validation set. (H), (K) patients with I+II stage disease in the validation set. (I), (L) patients with III+IV stage disease in the validation set.

    Techniques Used: Expressing

    15) Product Images from "Prognostic Value of EZH2 Expression and Activity in Renal Cell Carcinoma: A Prospective Study"

    Article Title: Prognostic Value of EZH2 Expression and Activity in Renal Cell Carcinoma: A Prospective Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081484

    Comparisons of the receiver operating characteristic (ROC) curves for prediction of survival by the EZH2 score, TNM stage, and H3K27me3 score. (A) (E) DFS, (B) (F) OS in the training set. (C) (G) DFS, (D) (H) OS in the validation set. (A-D) the area under the ROC curves (AUROC) of EZH2 score versus the AUROC of TNM stage, or H3K27me3 score. (E-H) the AUROC of the combined EZH2 and TNM stage model versus the AUROC of the TNM stage or EZH2 expression alone model.
    Figure Legend Snippet: Comparisons of the receiver operating characteristic (ROC) curves for prediction of survival by the EZH2 score, TNM stage, and H3K27me3 score. (A) (E) DFS, (B) (F) OS in the training set. (C) (G) DFS, (D) (H) OS in the validation set. (A-D) the area under the ROC curves (AUROC) of EZH2 score versus the AUROC of TNM stage, or H3K27me3 score. (E-H) the AUROC of the combined EZH2 and TNM stage model versus the AUROC of the TNM stage or EZH2 expression alone model.

    Techniques Used: Expressing

    The expression of EZH2 and H3K27me3 in human samples. Representative EZH2 and H3K27me3 immunohistochemical staining in renal cell carcinoma (200×magnification). (A-H) Positive EZH2 (black arrow) and H3K27me3 (white arrow) display a nuclear staining. (A) negative in RCC (E) negative in none tumor tissue (B-D) EZH2 positive (F-H) H3K27me3 positive (B) (F) staining intensity index-1socre (C) (G) staining intensity index-2 score (D) (H) staining intensity index-3 score (I) the expression of EZH2 and H3K27me3 was detected in all 10 cases of RCC tissues compared to adjacent non-RCC tissues. N, non-RCC tissue; T, RCC tissue.
    Figure Legend Snippet: The expression of EZH2 and H3K27me3 in human samples. Representative EZH2 and H3K27me3 immunohistochemical staining in renal cell carcinoma (200×magnification). (A-H) Positive EZH2 (black arrow) and H3K27me3 (white arrow) display a nuclear staining. (A) negative in RCC (E) negative in none tumor tissue (B-D) EZH2 positive (F-H) H3K27me3 positive (B) (F) staining intensity index-1socre (C) (G) staining intensity index-2 score (D) (H) staining intensity index-3 score (I) the expression of EZH2 and H3K27me3 was detected in all 10 cases of RCC tissues compared to adjacent non-RCC tissues. N, non-RCC tissue; T, RCC tissue.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    Kaplan-Meier analysis of disease-free survival (DFS) in renal cell carcinoma according to expression of the EZH2 or H3K27me3 score. (A), (D) all patients in the training set. (B), (E) patients with I+II stage disease in the training set. (C), (F) patients with III+IV stage disease in the training set. (G), (J) all patients in the validation set. (H), (K) patients with I+II stage disease in the validation set. (I), (L) patients with III+IV stage disease in the validation set.
    Figure Legend Snippet: Kaplan-Meier analysis of disease-free survival (DFS) in renal cell carcinoma according to expression of the EZH2 or H3K27me3 score. (A), (D) all patients in the training set. (B), (E) patients with I+II stage disease in the training set. (C), (F) patients with III+IV stage disease in the training set. (G), (J) all patients in the validation set. (H), (K) patients with I+II stage disease in the validation set. (I), (L) patients with III+IV stage disease in the validation set.

    Techniques Used: Expressing

    Kaplan-Meier analysis of overall survival (OS) in renal cell carcinoma according to expression of the EZH2 or H3K27me3 score. (A), (D) all patients in the training set. (B), (E) patients with I+II stage disease in the training set. (C), (F) patients with III+IV stage disease in the training set. (G), (J) all patients in the validation set. (H), (K) patients with I+II stage disease in the validation set. (I), (L) patients with III+IV stage disease in the validation set.
    Figure Legend Snippet: Kaplan-Meier analysis of overall survival (OS) in renal cell carcinoma according to expression of the EZH2 or H3K27me3 score. (A), (D) all patients in the training set. (B), (E) patients with I+II stage disease in the training set. (C), (F) patients with III+IV stage disease in the training set. (G), (J) all patients in the validation set. (H), (K) patients with I+II stage disease in the validation set. (I), (L) patients with III+IV stage disease in the validation set.

    Techniques Used: Expressing

    16) Product Images from "Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development"

    Article Title: Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    Journal: eLife

    doi: 10.7554/eLife.09571

    H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014
    Figure Legend Snippet: H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014

    Techniques Used: DNA Methylation Assay

    Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035
    Figure Legend Snippet: Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035

    Techniques Used:

    H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029
    Figure Legend Snippet: H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029

    Techniques Used:

    H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10
    Figure Legend Snippet: H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10

    Techniques Used: DNA Methylation Assay

    H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value
    Figure Legend Snippet: H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value

    Techniques Used: DNA Methylation Assay, Genome Wide

    Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)
    Figure Legend Snippet: Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)

    Techniques Used:

    H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022
    Figure Legend Snippet: H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022

    Techniques Used: Flow Cytometry, Expressing

    LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013
    Figure Legend Snippet: LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013

    Techniques Used: Chromatin Immunoprecipitation, Sequencing

    17) Product Images from "Global analysis of H3K4me3 and H3K27me3 profiles in glioblastoma stem cells and identification of SLC17A7 as a bivalent tumor suppressor gene"

    Article Title: Global analysis of H3K4me3 and H3K27me3 profiles in glioblastoma stem cells and identification of SLC17A7 as a bivalent tumor suppressor gene

    Journal: Oncotarget

    doi:

    Properties of the H3K4 and H3K27 trimethylation patterns illustrated by the SN186 GSC line (A) The frequency distribution of peaks in bins of 200 bp is shown for H3K27me3 and H3K27me3 (X-axis, bins of 200 bp; Y-axis, the number of peaks in each bin). (B) The percentage of genes in the H3K4me3, H3K27me3, bivalent, and unmodified categories in GSC line SN186. (C) Number of genes identified in the H3K4me3, H3K27me3, bivalent, and unmodified categories in GSC line SN186. (D) The peak density profiles around TSSs (+/− 5kb) for H3K4me3-only genes in GSC line SN186. (E) The peak density profiles around TSSs (+/− 5kb) for H3K27me3-only genes in GSC line SN186. F, The peak density profiles around TSSs (+/− 5kb) for bivalent genes in GSC line SN186.
    Figure Legend Snippet: Properties of the H3K4 and H3K27 trimethylation patterns illustrated by the SN186 GSC line (A) The frequency distribution of peaks in bins of 200 bp is shown for H3K27me3 and H3K27me3 (X-axis, bins of 200 bp; Y-axis, the number of peaks in each bin). (B) The percentage of genes in the H3K4me3, H3K27me3, bivalent, and unmodified categories in GSC line SN186. (C) Number of genes identified in the H3K4me3, H3K27me3, bivalent, and unmodified categories in GSC line SN186. (D) The peak density profiles around TSSs (+/− 5kb) for H3K4me3-only genes in GSC line SN186. (E) The peak density profiles around TSSs (+/− 5kb) for H3K27me3-only genes in GSC line SN186. F, The peak density profiles around TSSs (+/− 5kb) for bivalent genes in GSC line SN186.

    Techniques Used:

    Correlation of H3K4 and H3K27 trimethylation with gene activity (A) The number of genes with H3K4me3 or H3K27me3 marks for the 8 GSC lines (X-axis, the number of genes). (B) A box plot of gene expression levels for the genes in the H3K4m3-only, H3K27me3-only, bivalent and unmodified categories in SN186 cells (Y-axis, normalized intensities from the array hybridization). (C) A box plot of gene expression levels for the genes in the H3K4m3-only, H3K27me3-only, bivalent and unmodified categories in more than four of the eight glioma stem cell lines (Y-axis, normalized intensities from array hybridization).
    Figure Legend Snippet: Correlation of H3K4 and H3K27 trimethylation with gene activity (A) The number of genes with H3K4me3 or H3K27me3 marks for the 8 GSC lines (X-axis, the number of genes). (B) A box plot of gene expression levels for the genes in the H3K4m3-only, H3K27me3-only, bivalent and unmodified categories in SN186 cells (Y-axis, normalized intensities from the array hybridization). (C) A box plot of gene expression levels for the genes in the H3K4m3-only, H3K27me3-only, bivalent and unmodified categories in more than four of the eight glioma stem cell lines (Y-axis, normalized intensities from array hybridization).

    Techniques Used: Activity Assay, Expressing, Hybridization

    Global identification of H3K4me3 and H3K27me3 binding profiles in astrocytes (A) The peak density heat map for H3K4me3 and H3K27me3 around the TSSs (transcription start sites) of the complete set of 39,865 RefSeq genes from UCSC. Tag densities within a window of +/− 5 kb on either side of the TSS coordinates were collected from each ChIP-seq dataset. (B) The peak density profiles around TSSs (+/− 5kb) for H3K27me3 and H3K27me3 in astrocytes. (C) A box plot of gene expression levels for the genes in the H3K4m3-only, H3K27me3-only, bivalent and unmodified categories in astrocytes (Y-axis, normalized intensities from array hybridization).
    Figure Legend Snippet: Global identification of H3K4me3 and H3K27me3 binding profiles in astrocytes (A) The peak density heat map for H3K4me3 and H3K27me3 around the TSSs (transcription start sites) of the complete set of 39,865 RefSeq genes from UCSC. Tag densities within a window of +/− 5 kb on either side of the TSS coordinates were collected from each ChIP-seq dataset. (B) The peak density profiles around TSSs (+/− 5kb) for H3K27me3 and H3K27me3 in astrocytes. (C) A box plot of gene expression levels for the genes in the H3K4m3-only, H3K27me3-only, bivalent and unmodified categories in astrocytes (Y-axis, normalized intensities from array hybridization).

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Expressing, Hybridization

    Global identification of H3K4me3 and H3K27me3 binding profiles in glioma stem cells (GSCs) (A) Quantitative ChIP-PCR of the representative H3K4me3-marked gene GAPDH and the representative H3K27me3-marked gene SAT2. GAPDH, a house-keeping gene and positive control for H3K4me3 ChIP, was found to be enriched 118-fold, whereas SAT2 (satellite 2), a gene present in heterochromatin and a positive control for H3K27 methylation, was found to be enriched 528-fold (Y-axis, fold enrichment for the H3K4me3 or H3K27me3 ChIP DNAs versus the input DNA). (B) The peak density heat map for H3K4me3 around TSSs (transcription start sites). The whole set of TSSs from 39,865 RefSeq genes from UCSC is shown for the 8 GSC lines after clustering into 5 groups using the SeqMINER program. TSSs were used as reference coordinates. Tag densities within a window of +/−5 kb on either side of the TSS coordinates were collected from each ChIP-seq dataset. The right panel shows the H3K4me3 peak profiles in the 8 GSC lines for the genes in cluster 2, which is one of the clusters enriched for H2K4me3. The H3K4me3 profile exhibits a strong dual peak with a central notch. The plot was generated using the SeqMINER program. (C) The peak density heat map of H3K27me3 around the complete set of TSSs (transcription start sites) from 39,865 RefSeq genes from UCSC for the 8 GSC lines. Tag densities within a window of +/− 5 kb on either side of the TSS coordinates were collected from each ChIP-seq dataset. The peaks are much broader than those for H3K4me3.
    Figure Legend Snippet: Global identification of H3K4me3 and H3K27me3 binding profiles in glioma stem cells (GSCs) (A) Quantitative ChIP-PCR of the representative H3K4me3-marked gene GAPDH and the representative H3K27me3-marked gene SAT2. GAPDH, a house-keeping gene and positive control for H3K4me3 ChIP, was found to be enriched 118-fold, whereas SAT2 (satellite 2), a gene present in heterochromatin and a positive control for H3K27 methylation, was found to be enriched 528-fold (Y-axis, fold enrichment for the H3K4me3 or H3K27me3 ChIP DNAs versus the input DNA). (B) The peak density heat map for H3K4me3 around TSSs (transcription start sites). The whole set of TSSs from 39,865 RefSeq genes from UCSC is shown for the 8 GSC lines after clustering into 5 groups using the SeqMINER program. TSSs were used as reference coordinates. Tag densities within a window of +/−5 kb on either side of the TSS coordinates were collected from each ChIP-seq dataset. The right panel shows the H3K4me3 peak profiles in the 8 GSC lines for the genes in cluster 2, which is one of the clusters enriched for H2K4me3. The H3K4me3 profile exhibits a strong dual peak with a central notch. The plot was generated using the SeqMINER program. (C) The peak density heat map of H3K27me3 around the complete set of TSSs (transcription start sites) from 39,865 RefSeq genes from UCSC for the 8 GSC lines. Tag densities within a window of +/− 5 kb on either side of the TSS coordinates were collected from each ChIP-seq dataset. The peaks are much broader than those for H3K4me3.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Positive Control, Methylation, Generated

    18) Product Images from "Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development"

    Article Title: Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    Journal: eLife

    doi: 10.7554/eLife.09571

    H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014
    Figure Legend Snippet: H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014

    Techniques Used: DNA Methylation Assay

    Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035
    Figure Legend Snippet: Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035

    Techniques Used:

    H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029
    Figure Legend Snippet: H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029

    Techniques Used:

    H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10
    Figure Legend Snippet: H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10

    Techniques Used: DNA Methylation Assay

    H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value
    Figure Legend Snippet: H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value

    Techniques Used: DNA Methylation Assay, Genome Wide

    Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)
    Figure Legend Snippet: Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)

    Techniques Used:

    H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022
    Figure Legend Snippet: H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022

    Techniques Used: Flow Cytometry, Expressing

    LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013
    Figure Legend Snippet: LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013

    Techniques Used: Chromatin Immunoprecipitation, Sequencing

    19) Product Images from "Histone Methyltransferase MMSET/NSD2 Alters EZH2 Binding and Reprograms the Myeloma Epigenome through Global and Focal Changes in H3K36 and H3K27 Methylation"

    Article Title: Histone Methyltransferase MMSET/NSD2 Alters EZH2 Binding and Reprograms the Myeloma Epigenome through Global and Focal Changes in H3K36 and H3K27 Methylation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004566

    MMSET overexpression alters normal H3K36me2 distribution. (A) Loss of MMSET expression in t(4;14)+ cells depletes H3K36 methylation and increases H3K27 methylation. Immunoblot of nuclear extracts from t(4;14)+ KMS11 cells with inactivated translocated (TKO) or wild-type (NTKO) MMSET allele were probed using antibodies for MMSET, H3K36me2, H3K27me3 and total H4 as control. (B) Heatmaps of H3K36me2 distribution between NTKO (left) and TKO (right) cells. On the y-axis, genes were ranked based on their expression level from high (top) to low (bottom) and on the x-axis, normalized read density from ChIP-seq experiments was plotted in gene bodies and 10 kb upstream and downstream from the transcription start (TSS) and end (TES) sites. (C) MMSET alters H3K36me2 distribution in intragenic (top) and intergenic (bottom) regions. (Top) Average read density across 15,386 genes in NTKO (left, red) and TKO (right, green) cells. (Middle) UCSC genome browser view of the H3K36me2 distribution on a representative locus ( BTF3 ) in NTKO and TKO cells. (Bottom) Average normalized tag density of H3K36me2 in 6,172 intergenic regions in NTKO (left) and TKO (right) cells. (D) Representative UCSC genome browser view of the H3K36me2 distribution on chromosome 8 of NTKO (red) and TKO (green) cells. Top panel encompasses a gene-rich region of the chromosome and the bottom insert represents a 1MB region of the 8q24 gene desert. (E) ChIP-qPCR for H3K36me2 on the BTF3 locus. Methylation enrichment was tested 5 kb upstream of the TSS (5′) and at the TSS (right). Two independent biological replicates are shown.
    Figure Legend Snippet: MMSET overexpression alters normal H3K36me2 distribution. (A) Loss of MMSET expression in t(4;14)+ cells depletes H3K36 methylation and increases H3K27 methylation. Immunoblot of nuclear extracts from t(4;14)+ KMS11 cells with inactivated translocated (TKO) or wild-type (NTKO) MMSET allele were probed using antibodies for MMSET, H3K36me2, H3K27me3 and total H4 as control. (B) Heatmaps of H3K36me2 distribution between NTKO (left) and TKO (right) cells. On the y-axis, genes were ranked based on their expression level from high (top) to low (bottom) and on the x-axis, normalized read density from ChIP-seq experiments was plotted in gene bodies and 10 kb upstream and downstream from the transcription start (TSS) and end (TES) sites. (C) MMSET alters H3K36me2 distribution in intragenic (top) and intergenic (bottom) regions. (Top) Average read density across 15,386 genes in NTKO (left, red) and TKO (right, green) cells. (Middle) UCSC genome browser view of the H3K36me2 distribution on a representative locus ( BTF3 ) in NTKO and TKO cells. (Bottom) Average normalized tag density of H3K36me2 in 6,172 intergenic regions in NTKO (left) and TKO (right) cells. (D) Representative UCSC genome browser view of the H3K36me2 distribution on chromosome 8 of NTKO (red) and TKO (green) cells. Top panel encompasses a gene-rich region of the chromosome and the bottom insert represents a 1MB region of the 8q24 gene desert. (E) ChIP-qPCR for H3K36me2 on the BTF3 locus. Methylation enrichment was tested 5 kb upstream of the TSS (5′) and at the TSS (right). Two independent biological replicates are shown.

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

    Targeting MMSET in t(4;14)+ tumors prolongs survival. (A) Mouse xenograft model using t(4;14)+ KMS11 cells harboring a luciferase gene and a doxycycline-inducible MMSET-specific shRNA. Animals that were not administered doxycycline (−Dox) are shown on the top and animals that were given doxycycline are shown on the bottom (+Dox). Two representative animals are shown from each group (n = 5) at the following timepoints: 2 weeks after treatment; 4 weeks after treatment; 2 weeks after release from Dox; and 4 weeks after release from Dox. –Dox animals were sacrificed 26 days after treatment initiation due to tumor size. The same two animals are shown at each time points. (B) Kaplan-Meier curve of the xenograft mouse experiment from A. (C) Measurement of the luciferin signal in treated (black) and untreated (red) mice over time. (D) Immunoblot for H3K36me2 and H3K27me3 using nuclear extracts from tumors isolated from the untreated or doxycycline-treated mice (E) Proposed model for explaining how MMSET overexpression alters the epigenetic landscape of myeloma cells. Methylation of H3K36 by MMSET induces a global decrease of H3K27me3, leading to activation of gene expression (bottom). Additionally, an overabundance of K36 methylation alters genome-wide EZH2 binding, inducing focal increases in H3K27 methylation and gene repression.
    Figure Legend Snippet: Targeting MMSET in t(4;14)+ tumors prolongs survival. (A) Mouse xenograft model using t(4;14)+ KMS11 cells harboring a luciferase gene and a doxycycline-inducible MMSET-specific shRNA. Animals that were not administered doxycycline (−Dox) are shown on the top and animals that were given doxycycline are shown on the bottom (+Dox). Two representative animals are shown from each group (n = 5) at the following timepoints: 2 weeks after treatment; 4 weeks after treatment; 2 weeks after release from Dox; and 4 weeks after release from Dox. –Dox animals were sacrificed 26 days after treatment initiation due to tumor size. The same two animals are shown at each time points. (B) Kaplan-Meier curve of the xenograft mouse experiment from A. (C) Measurement of the luciferin signal in treated (black) and untreated (red) mice over time. (D) Immunoblot for H3K36me2 and H3K27me3 using nuclear extracts from tumors isolated from the untreated or doxycycline-treated mice (E) Proposed model for explaining how MMSET overexpression alters the epigenetic landscape of myeloma cells. Methylation of H3K36 by MMSET induces a global decrease of H3K27me3, leading to activation of gene expression (bottom). Additionally, an overabundance of K36 methylation alters genome-wide EZH2 binding, inducing focal increases in H3K27 methylation and gene repression.

    Techniques Used: Luciferase, shRNA, Mouse Assay, Isolation, Over Expression, Methylation, Activation Assay, Expressing, Genome Wide, Binding Assay

    MMSET alters EZH2 binding in t(4;14)+ myeloma cells. (A) Venn diagram showing overlap of genes bound at their promoters by EZH2 in NTKO (blue) and TKO (yellow) cells. (B) UCSC genome browser display of H3K27me3 (top, gray) and EZH2 binding (bottom, red) in NTKO cells. (C) UCSC genome browser display of EZH2 ChIP-seq tracks in NTKO (top, red) and TKO (bottom, green) cells associated with MMSET-repressed genes, CDCA7 (left) and DLL4 (right). (D) Heat map of over-represented gene categories among genes bound by EZH2 in either NTKO cells, TKO cells or both cell types. Enrichment was measured using iPAGE analysis [74] . (E) Motif analysis using HOMER [73] identified conserved sequences bound by EZH2 in NTKO cells, TKO cells or both cell types. (F) Relative cell number of MMSET-high and MMSET-low cells treated with indicated doses of the EZH2 small molecule inhibitor (GSK343). Inactive compound, GSK669, was used as a control. Graph represents four independent experiments +/− standard deviation. (G) Quantitative RT-PCR measurement of miR-126* expression in KMS11 cells treated with GSK669 or GSK 343. Graph represents average expression from three independent experiments +/− standard deviation (* p
    Figure Legend Snippet: MMSET alters EZH2 binding in t(4;14)+ myeloma cells. (A) Venn diagram showing overlap of genes bound at their promoters by EZH2 in NTKO (blue) and TKO (yellow) cells. (B) UCSC genome browser display of H3K27me3 (top, gray) and EZH2 binding (bottom, red) in NTKO cells. (C) UCSC genome browser display of EZH2 ChIP-seq tracks in NTKO (top, red) and TKO (bottom, green) cells associated with MMSET-repressed genes, CDCA7 (left) and DLL4 (right). (D) Heat map of over-represented gene categories among genes bound by EZH2 in either NTKO cells, TKO cells or both cell types. Enrichment was measured using iPAGE analysis [74] . (E) Motif analysis using HOMER [73] identified conserved sequences bound by EZH2 in NTKO cells, TKO cells or both cell types. (F) Relative cell number of MMSET-high and MMSET-low cells treated with indicated doses of the EZH2 small molecule inhibitor (GSK343). Inactive compound, GSK669, was used as a control. Graph represents four independent experiments +/− standard deviation. (G) Quantitative RT-PCR measurement of miR-126* expression in KMS11 cells treated with GSK669 or GSK 343. Graph represents average expression from three independent experiments +/− standard deviation (* p

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

    MMSET alters genome-wide patterns of H3K27me3 methylation. (A) Tag density profile of H3K27me3 distribution across different gene groups from Figure 2A . The ratio between read numbers in NTKO and TKO cells is presented on the y-axis. (B) UCSC genome browser display of H3K27me3 density tracks surrounding the transcription start site of two MMSET activated genes, CA2 (top) and CR2 (bottom). (C) GSEA analysis of genes upregulated by MMSET shows enrichment of previously identified EZH2 target genes. (D) UCSC genome browser display of H3K27me3 density tracks surrounding the transcription start site of two MMSET repressed genes, DLL4 (top) and CDCA7 (bottom). (E) ChIP-qPCR for H3K27me3 on CDCA7 locus. Methylation enrichment was tested on the promoter (TSS) and on the regions upstream (5′) and downstream (3′) from the TSS. Two independent biological replicates are shown. (F) UCSC genome browser of H3K27me3 enrichment on non-expressed genes of the HOXC cluster (left) and ChIP-qPCR for H3K27me3 on the HOXC10 locus (right). Two independent biological replicates are shown. (G) Tag density profile of H3K36me2 (left), H3K36me3 (middle) and H3K27me3 (right) distribution of differentially expressed genes in TKO cells.
    Figure Legend Snippet: MMSET alters genome-wide patterns of H3K27me3 methylation. (A) Tag density profile of H3K27me3 distribution across different gene groups from Figure 2A . The ratio between read numbers in NTKO and TKO cells is presented on the y-axis. (B) UCSC genome browser display of H3K27me3 density tracks surrounding the transcription start site of two MMSET activated genes, CA2 (top) and CR2 (bottom). (C) GSEA analysis of genes upregulated by MMSET shows enrichment of previously identified EZH2 target genes. (D) UCSC genome browser display of H3K27me3 density tracks surrounding the transcription start site of two MMSET repressed genes, DLL4 (top) and CDCA7 (bottom). (E) ChIP-qPCR for H3K27me3 on CDCA7 locus. Methylation enrichment was tested on the promoter (TSS) and on the regions upstream (5′) and downstream (3′) from the TSS. Two independent biological replicates are shown. (F) UCSC genome browser of H3K27me3 enrichment on non-expressed genes of the HOXC cluster (left) and ChIP-qPCR for H3K27me3 on the HOXC10 locus (right). Two independent biological replicates are shown. (G) Tag density profile of H3K36me2 (left), H3K36me3 (middle) and H3K27me3 (right) distribution of differentially expressed genes in TKO cells.

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

    20) Product Images from "Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development"

    Article Title: Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

    Journal: eLife

    doi: 10.7554/eLife.09571

    H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014
    Figure Legend Snippet: H3K9me2 and H3K27me3 correlate with distinct CpG and DNA methylation states. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. ( B ) Density contour plots showing correlation between levels of H3K9me2 (blue) or H3K27me3 (red) and DNA methylation at promoters in the epiblast. H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; HCP: high CpG density; ICP: intermediate CpG density; LCP: low CpG density. DOI: http://dx.doi.org/10.7554/eLife.09571.014

    Techniques Used: DNA Methylation Assay

    Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035
    Figure Legend Snippet: Density contour plots showing correlation between H3K27me3 and H3K9me2 enrichments at all promoters in E6.25 epiblast. DOI: http://dx.doi.org/10.7554/eLife.09571.035

    Techniques Used:

    H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029
    Figure Legend Snippet: H3K9me2 enriched enhancers are preferentially linked to the p53 pathway. ( A ) Selected enriched GO terms in H3K9me2-marked enhancers. ( B,C ) Bar plots showing top 20 enriched motifs in H3K9me2- ( B ) or H3K27me3- ( C ) marked enhancers. Source data file legends DOI: http://dx.doi.org/10.7554/eLife.09571.029

    Techniques Used:

    H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10
    Figure Legend Snippet: H3K9me2 marks a distinct set of enhancers. ( A ) Unbiased dynamics of ESC enhancers upon exit from naïve pluripotency. Classification was performed using self-organizing maps. Some enhancers become inactivated via acquisition of H3K27me3 or H3K9me2. Each of these modes is associated with distinct DNA methylation levels. ( B ) Box plots showing DNA methylation levels at enhancers in EpiLCs and EpiSCs. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. Effect size: *r≤0.10; **0.10

    Techniques Used: DNA Methylation Assay

    H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value
    Figure Legend Snippet: H3K9me2, H3K27me3 and DNA methylation dynamics between E6.25 epiblast and EpiSC. ( A ) Bar plots showing distribution of H3K9me2 (top) and H3K27me3 (bottom) genome-wide. 1 kB tiles were calculated for all chromosomes with a 500 bp offset, and each tile was intersected with annotated genomic regions. For each tile, enrichment was calculated. Shown are top and bottom 20% of enriched tiles. ( B ) Box plots showing gain of DNA methylation in EpiSC compared with E6.25 epiblast at promoters. Promoters were classified based on differential enrichment for H3K27me3 in EpiSC and E6.25 epiblast. Significance was calculated using unpaired Wilcoxon rank sum test with continuity correction. *p-value

    Techniques Used: DNA Methylation Assay, Genome Wide

    Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)
    Figure Legend Snippet: Accumulation of H3K9me2 or H3K27me3 at promoters of genes becoming repressed depends on CpG content. ( A ) Density contour plots showing H3K9me2 and H3K27me3 enrichment in E6.25 epiblast at promoters of HCP, ICP, and LCP. Shown are only promoters associated with genes becoming repressed in E6.25 epiblast when compared with E3.5 ICM (Log2(RPKM)

    Techniques Used:

    H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022
    Figure Legend Snippet: H3K9me2 accumulates at G9a-regulated repeat elements in the epiblast. ( A ) Box plots showing fractions of unique loci significantly marked by H3K9me2 or H3K27me3 within classes of repeat elements. Shown is data from lcChIP-seq of E6.25 epiblast. ( B ) Scatter plot showing correlation between the number of unique repeat loci in each subfamily with the number of loci upregulated in Ezh2 −/− E6.25 epiblast. Red points are subfamilies with significant H3K27me3 enrichment and increased proportion of upregulated loci. ( C,D ) Tables with subfamilies of repeat elements showing significantly increased proportion of unique loci marked by H3K9me2 ( C ) or H3K27me3 ( D ) and upregulated in Ehmt2 −/− ( C ) or Ezh2 −/− ( D ) E6.25 epiblast. ( E ) Density contour plots showing correlation between H3K9me2 and H3K27me3 enrichment at loci within two subclasses regulated by G9a. ( F ) Scatter plots FC in expression levels of unique loci in Ehmt2 −/− versus Ehmt2 +/+ E6.25 epiblast. Red triangles show loci that are significantly upregulated. DOI: http://dx.doi.org/10.7554/eLife.09571.022

    Techniques Used: Flow Cytometry, Expressing

    LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013
    Figure Legend Snippet: LcChIP-seq on E6.25 epiblast. ( A ) Validation of lcChIP. bar plots comparing enrichment of H3K27me3 and H3K9me2 in EpiSCs detected using large scale (5–10 × 10 6 cells) xChIP or lcChIP. Data are represented as mean (± SEM) fold enrichment relative to Gapdh from at least two independent biological replicates. ( B ) Validation of lcChIP-seq. Heatmap of unbiased Spearman’s rho correlation of H3K27me3 levels at promoter elements showing that all EpiSC ChIP-seq samples cluster together independently of the technique used. ( C ) Genome browser tracks showing H3K27me3 (red) and H3K9me2 (blue) enrichment in two biological replicates of E6.25 epiblast lcChIP-seq. Hoxc cluster and Pcsk5 are control regions for enrichment of H3K27me3 and H3K9me2, respectively. Otx2 is a highly expressed gene in the epiblast. Data is shown as a sliding window (1 kb and 300 bp for H3K9me2 and H3K27me3, respectively) of enrichment over input: Log2(RPM ChIP/RPM input). lcChIP-seq: low cell number chromatin immunoprecipitation with sequencing; H3K9me2: histone H3 lysine 9 dimethylation; H3K27me3: histone H3 lysine 27 trimethylation; EpiSCs: epiblast stem cells; xChIP: fixed ChIP; SEM: standard error of the mean, RPM: reads per million mapped. DOI: http://dx.doi.org/10.7554/eLife.09571.013

    Techniques Used: Chromatin Immunoprecipitation, Sequencing

    Related Articles

    Centrifugation:

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: The mean chromatin fragment size after sonication was ∼200–300 bp. .. After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C. .. Chromatin was immunoprecipitated overnight at 4°C with antibody-conjugated Dynabeads.

    Article Title: Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma
    Article Snippet: Cell pellet was resuspended in Cell Lysis Buffer and after 6000rpm centrifugation RIPA buffer were added to perform nuclei lysis. .. Antibodies used in this study were as follows: MYCN (B8.4.B, Santa Cruz Biotechnology), LSD1 (ab17721, ABCAM), H3K4me2 (07-030, Millipore), H3K27me3 (07-449, Millipore), H3Ac (06-599, Millipore).

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: After centrifugation, lysates were incubated with 2 μg antibody for 4 h (or overnight); 20 μl protein A/G PLUS agarose (Santa Cruz) was then added and incubated for another 4 h (or overnight) on a rotator at 4 °C. .. Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    Blocking Assay:

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: Human samples were obtained with informed consent, and the analysis was approved by the institutional review boards. .. For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. Cells and sections were washed in PBS three times for 5 min and incubated in Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, CA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) for 1 h. After washing with PBS three times for 5 min, coverslips were mounted using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

    Article Title:
    Article Snippet: After being stored at −20 °C for 1 h, embryos were rehydrated with 2% PTX (2% Triton X-100 in PBS) and treated in 2 m HCl for 1 h at room temperature followed by neutralizing in 100 mm Tris-HCl (pH 8.5) for 15 min. Embryos were washed with 2% PTX three times, 5 min each, incubated in the block solution (1% BSA, 10% goat serum, 0.3 m glycine in 2% PTX) for 1 h, and transferred to the block solution containing a primary antibody for incubation overnight at 4 °C, followed by washing with 2% PTX six times, 5 min for the first two times and 30 min for the last four times, at room temperature. .. The following primary antibodies were used: mouse anti-5mC (Abcam ab10805, 1:500), mouse anti-5mC (Abcam ab51552, 1:100), rabbit anti-H3 (Abcam ab1791, 1:500), mouse anti-H3 (EASYBIO BE3015, 1:200), mouse anti-BrdUTP (Santa Cruz sc-32323, 1:100), anti-ssDNA (IBL JP18731, 1:100), rabbit anti-Ung (GeneTex GTX103236, 1:200), rabbit anti-H3K4me3 (Abcam ab8580, 1:100), and rabbit anti-H3K27me3 (Millipore 07-449, 1:100).

    Real-time Polymerase Chain Reaction:

    Article Title: Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters
    Article Snippet: ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303). .. ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303).

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore). .. Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Polycomb Target Genes Are Silenced in Multiple Myeloma
    Article Snippet: Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode). .. Chromatin immunoprecipitation was performed using OneDay ChIP Kit™ (Diagenode, Liège, Belgium) according to the manufacturer's protocol.

    Article Title: Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice
    Article Snippet: Antibodies used were IgG (Santa Cruz #sc-2027), H3K4me2 (Millipore #07-030), H3K27me3 (Millipore, #07-449) and H3K9ac (Millipore #07-352). .. The immunoprecipitation, washes and DNA purification was done with Magna ChIP™ A Chromatin Immunoprecipitation Kit (Millipore #17-610) following manufacturer's protocol.

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: Genomic DNA was purified with a DNA purification kit (QIAGEN) and sent for real-time PCR or sequencing. .. Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176). .. DNA was purified from eluates using phenol-chloroform isoamyl alcohol extraction.

    Microarray:

    Article Title: Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression
    Article Snippet: Chromatin immunoprecipitations (ChIPs) were carried out in triplicate as described ( ) with antibodies against acetylated H3 (Millipore, 06–599) and H3K27me3 (Millipore, 07–449). .. ChIP and input DNA were amplified using WGA kit (Sigma), labelled with Cy-dyes and hybridized to H18-RefSeq promoter microarrays (Roche NimbleGen).

    Article Title: Integrative epigenomic mapping defines four main chromatin states in Arabidopsis
    Article Snippet: Paragraph title: Immunoprecipitation of chromatin and methylated DNA, labelling and microarray hybridization ... Specificity of the H3K27me2 and H3K9me3 antibodies was tested by peptide competition and western blotting analysis on nuclear extracts ( ) as described in using H3K27me3, H3K27me2, H3K27me1, H3K9me3 and H3K36me3 peptides (Millipore, 12-565, 12-566, 12-567, 12-568 and Diagenode sp-058-050, respectively).

    Incubation:

    Article Title: Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters
    Article Snippet: Following 15 min incubation on ice, the sample was sonicated using Bioruptor (Diagnode) to obtain fragments of average size 200 bp to 600 bp. .. ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303).

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: Proteins were cross-linked to DNA by addition of formaldehyde to the tissue suspension to a final concentration of 1% and incubation for 10 min at room temperature. .. Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: Human samples were obtained with informed consent, and the analysis was approved by the institutional review boards. .. For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. Cells and sections were washed in PBS three times for 5 min and incubated in Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, CA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) for 1 h. After washing with PBS three times for 5 min, coverslips were mounted using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

    Article Title: Efficient Reprogramming of Na?ve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System
    Article Snippet: AP staining was performed according to the manufacturer's (Sidansai, China) instructions. .. For the immunofluorescence staining, cells were fixed with 4% formaldehyde in DPBS for 15 min, permeabilized with 1% Triton X-100 in DPBS for 15 min, and blocked with 2% bovine serum albumin in DPBS for 1 h. Thereafter, cells were incubated with primary antibodies for 1 h, including those antibodies Oct4 (1∶200, Abcam), Sox2 (1∶200, Cell Signaling), Nanog (1∶200, Abcam), TRA-1-60 (Millipore, 1∶200), TRA-1-81 (Millipore, 1∶200), SSEA1 (1∶50, Developmental Studies Hybridoma Bank), SSEA3 (1∶50, Developmental Studies Hybridoma Bank), SSEA4 (1∶50, Developmental Studies Hybridoma Bank), 5-methyl cytidine (5-mC, 1∶200, Abcam), 5-hydroxymethyl cytidine (5-hmC, 1∶200, Active Motif), H3K27me3 (1∶250, Millipore). .. Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1∶500, Molecular Probes) and Alexa Fluor 594 (1∶500, Molecular Probes).

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: The mean chromatin fragment size after sonication was ∼200–300 bp. .. After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C. .. Chromatin was immunoprecipitated overnight at 4°C with antibody-conjugated Dynabeads.

    Article Title: Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice
    Article Snippet: Digestion mixes were incubated at 28°C heatblock for 6 min shaking at 800 rpm. .. Antibodies used were IgG (Santa Cruz #sc-2027), H3K4me2 (Millipore #07-030), H3K27me3 (Millipore, #07-449) and H3K9ac (Millipore #07-352).

    Article Title:
    Article Snippet: Next, embryos were incubated in fluorescence-conjugated secondary antibody overnight at 4 °C. .. The following primary antibodies were used: mouse anti-5mC (Abcam ab10805, 1:500), mouse anti-5mC (Abcam ab51552, 1:100), rabbit anti-H3 (Abcam ab1791, 1:500), mouse anti-H3 (EASYBIO BE3015, 1:200), mouse anti-BrdUTP (Santa Cruz sc-32323, 1:100), anti-ssDNA (IBL JP18731, 1:100), rabbit anti-Ung (GeneTex GTX103236, 1:200), rabbit anti-H3K4me3 (Abcam ab8580, 1:100), and rabbit anti-H3K27me3 (Millipore 07-449, 1:100).

    Article Title: Accelerated hepatocellular carcinoma development in CUL4B transgenic mice
    Article Snippet: Briefly, fresh liver tissue was cut into small pieces, then cross-linked with 1% formaldehyde, sonicated, pre-cleaned, and incubated with 5-10 μg of antibody per reaction. .. Antibodies used in ChIP assay were anti-CUL4B (Sigma), anti-EZH2 (BD, Franklin Lakes, NJ, USA), anti-H3K27me3 (Millipore) and anti-H2AK119ub1 (CST).

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: Genomic DNA was eluted with elution buffer (1% SDS and 100 mM NaHCO3 ) at 65 °C for 30 min. 5 M NaCl was added to a final concentration of 200 mM for further incubation at 65 °C for 4 h or overnight. .. Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: Pre-cleared lysates were then incubated overnight with 10 μg antibody at 4 °C. .. Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176).

    Mass Spectrometry:

    Article Title: Integrative epigenomic mapping defines four main chromatin states in Arabidopsis
    Article Snippet: All experiments were performed using wild-type Arabidopsis thaliana accession Columbia seedlings grown for 10 days either in liquid MS (whole seedlings) or on MS agar plates (roots and aerial parts) supplemented with 1% sucrose under long day conditions. .. Specificity of the H3K27me2 and H3K9me3 antibodies was tested by peptide competition and western blotting analysis on nuclear extracts ( ) as described in using H3K27me3, H3K27me2, H3K27me1, H3K9me3 and H3K36me3 peptides (Millipore, 12-565, 12-566, 12-567, 12-568 and Diagenode sp-058-050, respectively).

    Modification:

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. Cells and sections were washed in PBS three times for 5 min and incubated in Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, CA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) for 1 h. After washing with PBS three times for 5 min, coverslips were mounted using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

    Western Blot:

    Article Title: Prognostic Value of EZH2 Expression and Activity in Renal Cell Carcinoma: A Prospective Study
    Article Snippet: Western blot was carried out as previously described [ ]. .. Primary antibodies used in Western blot included those against EZH2 (Cell Signaling Technology), H3K27me3 (Millipore), H3 (Abcam, Cambridge, MA) and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). .. DFS is defined as the interval between curative surgery and recurrence or metastasis.

    Article Title: Integrative epigenomic mapping defines four main chromatin states in Arabidopsis
    Article Snippet: ChIP and Me-DIP assays were carried out essentially as described ( ) using commercially available antibodies ( ; ). .. Specificity of the H3K27me2 and H3K9me3 antibodies was tested by peptide competition and western blotting analysis on nuclear extracts ( ) as described in using H3K27me3, H3K27me2, H3K27me1, H3K9me3 and H3K36me3 peptides (Millipore, 12-565, 12-566, 12-567, 12-568 and Diagenode sp-058-050, respectively). .. Immunoprecipitated DNA (IP) and input DNA (INPUT) were amplified, differentially labelled and co-hybridized in dye-swap experiments as described ( ; ) for the chromosome 4 tiling array or according to the manufacturer's instructions for the Roche NimbleGen whole-genome tiling array.

    Article Title: Epigenetic silencing of Th1 type chemokines shapes tumor immunity and immunotherapy
    Article Snippet: Lentiviral shRNAs ( ) were provided by the Vector Core at the University of Michigan or kindly provided by Dr. Arul Chinnaiyan (University of Michigan). .. Antibodies including monoclonal anti-EZH2 (1:2000, BD Biosciences, 612667), anti-H3K27me3 (1:1000, Millipore, 07-449), H3K9me2 (1:2000, ab1220, Abcam), H3K9me3 (1:500, ab8898, Abcam), H3K4me1 (ab8895, Abcam), H3K4me2 (ab194678, Abcam), H3K4me3 (ab1012, Abcam), anti-Histone H3 (1:2000, Cell Signaling, 9715), anti-HA (1:200, Santa Cruz Biotechnology, sc-805), anti-DNMT1 (1:250, Abcam, ab13537) were used for Western blotting. .. Anti-human CXCR3 (1C6) blocking antibody was prepared from mouse hybridoma (ATCC, #HB-12330) by Hybridoma Core at the University of Michigan.

    Hybridization:

    Article Title: Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters
    Article Snippet: ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303). .. ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303).

    Article Title: Integrative epigenomic mapping defines four main chromatin states in Arabidopsis
    Article Snippet: Paragraph title: Immunoprecipitation of chromatin and methylated DNA, labelling and microarray hybridization ... Specificity of the H3K27me2 and H3K9me3 antibodies was tested by peptide competition and western blotting analysis on nuclear extracts ( ) as described in using H3K27me3, H3K27me2, H3K27me1, H3K9me3 and H3K36me3 peptides (Millipore, 12-565, 12-566, 12-567, 12-568 and Diagenode sp-058-050, respectively).

    Immunoprecipitation:

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: The cross-linking reaction was quenched and chromatin was sheared to an average length of 0.2–1.0 kb by sonication. .. Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore). .. In addition, 1/20 of the solution was collected before adding antibody to determine the amount of input DNA.

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C. .. After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Article Title: Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice
    Article Snippet: Antibodies used were IgG (Santa Cruz #sc-2027), H3K4me2 (Millipore #07-030), H3K27me3 (Millipore, #07-449) and H3K9ac (Millipore #07-352). .. Antibodies used were IgG (Santa Cruz #sc-2027), H3K4me2 (Millipore #07-030), H3K27me3 (Millipore, #07-449) and H3K9ac (Millipore #07-352).

    Article Title: Integrative epigenomic mapping defines four main chromatin states in Arabidopsis
    Article Snippet: Paragraph title: Immunoprecipitation of chromatin and methylated DNA, labelling and microarray hybridization ... Specificity of the H3K27me2 and H3K9me3 antibodies was tested by peptide competition and western blotting analysis on nuclear extracts ( ) as described in using H3K27me3, H3K27me2, H3K27me1, H3K9me3 and H3K36me3 peptides (Millipore, 12-565, 12-566, 12-567, 12-568 and Diagenode sp-058-050, respectively).

    Protease Inhibitor:

    Article Title: Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters
    Article Snippet: Fixed cells were washed well with 1 × PBS containing protease inhibitors at 4°C and resuspended in 2 ml lysis buffer supplemented with PMSF, DTT, and protease inhibitor cocktail (Roche). .. ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303).

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: GMPs from the BM of recipient mice were cross-linked with 0.5% formaldehyde for 5 min at room temperature, washed three times with PBS, and lysed with RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate [DOC], and protease inhibitor cocktail) and sonicated for 30 min using a Microson XL2000 Ultrasonic cell disruptor (Misonix). .. After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Magnetic Beads:

    Article Title: Accelerated hepatocellular carcinoma development in CUL4B transgenic mice
    Article Snippet: After being mixed with magnetic beads for 2 hours, complexes were washed with low and high salt buffers, and the DNA was extracted and precipitated. .. Antibodies used in ChIP assay were anti-CUL4B (Sigma), anti-EZH2 (BD, Franklin Lakes, NJ, USA), anti-H3K27me3 (Millipore) and anti-H2AK119ub1 (CST).

    Cell Culture:

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. Cells and sections were washed in PBS three times for 5 min and incubated in Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, CA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) for 1 h. After washing with PBS three times for 5 min, coverslips were mounted using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

    other:

    Article Title: Histone Methylation Regulator PTIP Is Required for PPARγ and C/EBPα Expression and Adipogenesis
    Article Snippet: Anti-H3K27me3 (07−449) was from Millipore.

    Article Title: Synthetic CpG islands reveal DNA sequence determinants of chromatin structure
    Article Snippet: α-H3K4me3 (Abcam[UK]-8580), α-H3K4me1(Abcam-8895), α-H3K27me3 (Millipore[UK]-07-449), α-H3K9/K14ac (Abcam 12,179), α-H3 (Abcam 1791), α-SUZ12 (Abcam 12,073-100), α-RNA Pol II N20 (Santa-Cruz[UK] 899), α-RNA Pol II S5P (Abcam 5131), α-RNA Pol II unphosphorylated CTD (Abcam 817), α-GFP (Chromotec [Germany] GFP-TRAP-A gta-20), α-IgG (Invitrogen 10500C).

    Polymerase Chain Reaction:

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore). .. Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C. .. After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Article Title: Polycomb Target Genes Are Silenced in Multiple Myeloma
    Article Snippet: Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode). .. Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode).

    Article Title: Accelerated hepatocellular carcinoma development in CUL4B transgenic mice
    Article Snippet: The enrichment of the DNA template was analyzed by conventional PCR using primers specific for each target gene promoter. .. Antibodies used in ChIP assay were anti-CUL4B (Sigma), anti-EZH2 (BD, Franklin Lakes, NJ, USA), anti-H3K27me3 (Millipore) and anti-H2AK119ub1 (CST).

    Sonication:

    Article Title: Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters
    Article Snippet: The sonicated chromatin was divided into 200 μl aliquots for chromatin immunoprecipitation (ChIP) using the ChIP assay kit (Upstate, #17–295) according to the manufacturer’s protocol. .. ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303).

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: The cross-linking reaction was quenched and chromatin was sheared to an average length of 0.2–1.0 kb by sonication. .. Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: The mean chromatin fragment size after sonication was ∼200–300 bp. .. After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Article Title: Polycomb Target Genes Are Silenced in Multiple Myeloma
    Article Snippet: Nuclei were lysed in RIPA buffer (MOPS (free acid), 0.5 M EDTA pH 8.0, 5N NaCl (to adjust pH to 7.0–7.2), 1% SDS; 10% NP-40, 10% DOC and protease inhibitors) for 10 min on ice and sonicated 3×15 min (30 sec ON/30 sec OFF) at ultrasonic wave output power 320 W in Bioruptor® (Diagenode, Liège, Belgium). .. Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode).

    Article Title: Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma
    Article Snippet: A small aliquot of sonicated material was put aside and remaining sample immunoprecipitated using 5 micrograms of ChIP-grade antibodies. .. Antibodies used in this study were as follows: MYCN (B8.4.B, Santa Cruz Biotechnology), LSD1 (ab17721, ABCAM), H3K4me2 (07-030, Millipore), H3K27me3 (07-449, Millipore), H3Ac (06-599, Millipore).

    Article Title: Accelerated hepatocellular carcinoma development in CUL4B transgenic mice
    Article Snippet: Briefly, fresh liver tissue was cut into small pieces, then cross-linked with 1% formaldehyde, sonicated, pre-cleaned, and incubated with 5-10 μg of antibody per reaction. .. Antibodies used in ChIP assay were anti-CUL4B (Sigma), anti-EZH2 (BD, Franklin Lakes, NJ, USA), anti-H3K27me3 (Millipore) and anti-H2AK119ub1 (CST).

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: Primary cells were washed, filtered through a 70-μm filter, lysed in buffer containing 1% SDS at a density of 0.1 g tissue per ml and sonicated for eight cycles of 30 s at 40% power followed by 30 s of rest. .. Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176).

    Affinity Purification:

    Article Title: PPAR? Interprets a Chromatin Signature of Pluripotency to Promote Embryonic Differentiation at Gastrulation
    Article Snippet: An affinity-purified polyclonal antibody against xenopus PPARβ was produced by immunizing rabbits with the peptide KLH-VQAPVSDSAAPDSPV (Eurogentec). .. We used anti-H3K4me3 from Abcam (ab8580), anti-H3K27me3 from Millipore (07-449), and anti–β-actin from Sigma (AC-40).

    Immunofluorescence:

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: Human samples were obtained with informed consent, and the analysis was approved by the institutional review boards. .. For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. Cells and sections were washed in PBS three times for 5 min and incubated in Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, CA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) for 1 h. After washing with PBS three times for 5 min, coverslips were mounted using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

    Article Title: Efficient Reprogramming of Na?ve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System
    Article Snippet: AP staining was performed according to the manufacturer's (Sidansai, China) instructions. .. For the immunofluorescence staining, cells were fixed with 4% formaldehyde in DPBS for 15 min, permeabilized with 1% Triton X-100 in DPBS for 15 min, and blocked with 2% bovine serum albumin in DPBS for 1 h. Thereafter, cells were incubated with primary antibodies for 1 h, including those antibodies Oct4 (1∶200, Abcam), Sox2 (1∶200, Cell Signaling), Nanog (1∶200, Abcam), TRA-1-60 (Millipore, 1∶200), TRA-1-81 (Millipore, 1∶200), SSEA1 (1∶50, Developmental Studies Hybridoma Bank), SSEA3 (1∶50, Developmental Studies Hybridoma Bank), SSEA4 (1∶50, Developmental Studies Hybridoma Bank), 5-methyl cytidine (5-mC, 1∶200, Abcam), 5-hydroxymethyl cytidine (5-hmC, 1∶200, Active Motif), H3K27me3 (1∶250, Millipore). .. Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1∶500, Molecular Probes) and Alexa Fluor 594 (1∶500, Molecular Probes).

    ChIP-sequencing:

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: Paragraph title: ChIP assay and ChIP assay coupled with massive parallel sequencing (ChIP-Seq). ... After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: Paragraph title: ChIP-qPCR and ChIP-seq ... Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: Paragraph title: ChIP assay and ChIP-Seq ... Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176).

    Fluorescence:

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. Cells and sections were washed in PBS three times for 5 min and incubated in Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, CA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) for 1 h. After washing with PBS three times for 5 min, coverslips were mounted using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

    Article Title:
    Article Snippet: Next, embryos were incubated in fluorescence-conjugated secondary antibody overnight at 4 °C. .. The following primary antibodies were used: mouse anti-5mC (Abcam ab10805, 1:500), mouse anti-5mC (Abcam ab51552, 1:100), rabbit anti-H3 (Abcam ab1791, 1:500), mouse anti-H3 (EASYBIO BE3015, 1:200), mouse anti-BrdUTP (Santa Cruz sc-32323, 1:100), anti-ssDNA (IBL JP18731, 1:100), rabbit anti-Ung (GeneTex GTX103236, 1:200), rabbit anti-H3K4me3 (Abcam ab8580, 1:100), and rabbit anti-H3K27me3 (Millipore 07-449, 1:100).

    Methylation:

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: Paragraph title: Chromatin immunoprecipitation assay for the Rassf1a-, p16INK4A -, Socs1-, Cx26- and Cdh1-associated histone lysine methylation ... Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Integrative epigenomic mapping defines four main chromatin states in Arabidopsis
    Article Snippet: Paragraph title: Immunoprecipitation of chromatin and methylated DNA, labelling and microarray hybridization ... Specificity of the H3K27me2 and H3K9me3 antibodies was tested by peptide competition and western blotting analysis on nuclear extracts ( ) as described in using H3K27me3, H3K27me2, H3K27me1, H3K9me3 and H3K36me3 peptides (Millipore, 12-565, 12-566, 12-567, 12-568 and Diagenode sp-058-050, respectively).

    Size-exclusion Chromatography:

    Article Title: Polycomb Target Genes Are Silenced in Multiple Myeloma
    Article Snippet: Nuclei were lysed in RIPA buffer (MOPS (free acid), 0.5 M EDTA pH 8.0, 5N NaCl (to adjust pH to 7.0–7.2), 1% SDS; 10% NP-40, 10% DOC and protease inhibitors) for 10 min on ice and sonicated 3×15 min (30 sec ON/30 sec OFF) at ultrasonic wave output power 320 W in Bioruptor® (Diagenode, Liège, Belgium). .. Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode).

    Microscopy:

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ).

    Purification:

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore). .. Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C. .. After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: Genomic DNA was purified with a DNA purification kit (QIAGEN) and sent for real-time PCR or sequencing. .. Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176). .. Eluates were treated with RNase and Proteinase K for 30 min at 37 °C then treated with NaCl and incubated overnight at 65 °C to reverse crosslinks.

    Sequencing:

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: Paragraph title: ChIP assay and ChIP assay coupled with massive parallel sequencing (ChIP-Seq). ... After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: Genomic DNA was purified with a DNA purification kit (QIAGEN) and sent for real-time PCR or sequencing. .. Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    Immunostaining:

    Article Title:
    Article Snippet: Paragraph title: Immunostaining ... The following primary antibodies were used: mouse anti-5mC (Abcam ab10805, 1:500), mouse anti-5mC (Abcam ab51552, 1:100), rabbit anti-H3 (Abcam ab1791, 1:500), mouse anti-H3 (EASYBIO BE3015, 1:200), mouse anti-BrdUTP (Santa Cruz sc-32323, 1:100), anti-ssDNA (IBL JP18731, 1:100), rabbit anti-Ung (GeneTex GTX103236, 1:200), rabbit anti-H3K4me3 (Abcam ab8580, 1:100), and rabbit anti-H3K27me3 (Millipore 07-449, 1:100).

    Confocal Microscopy:

    Article Title:
    Article Snippet: After being washed with 2% PTX six times, embryos were mounted and observed by confocal microscopy. .. The following primary antibodies were used: mouse anti-5mC (Abcam ab10805, 1:500), mouse anti-5mC (Abcam ab51552, 1:100), rabbit anti-H3 (Abcam ab1791, 1:500), mouse anti-H3 (EASYBIO BE3015, 1:200), mouse anti-BrdUTP (Santa Cruz sc-32323, 1:100), anti-ssDNA (IBL JP18731, 1:100), rabbit anti-Ung (GeneTex GTX103236, 1:200), rabbit anti-H3K4me3 (Abcam ab8580, 1:100), and rabbit anti-H3K27me3 (Millipore 07-449, 1:100).

    Staining:

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: Human samples were obtained with informed consent, and the analysis was approved by the institutional review boards. .. For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. Cells and sections were washed in PBS three times for 5 min and incubated in Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, CA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) for 1 h. After washing with PBS three times for 5 min, coverslips were mounted using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

    Article Title: Efficient Reprogramming of Na?ve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System
    Article Snippet: AP staining was performed according to the manufacturer's (Sidansai, China) instructions. .. For the immunofluorescence staining, cells were fixed with 4% formaldehyde in DPBS for 15 min, permeabilized with 1% Triton X-100 in DPBS for 15 min, and blocked with 2% bovine serum albumin in DPBS for 1 h. Thereafter, cells were incubated with primary antibodies for 1 h, including those antibodies Oct4 (1∶200, Abcam), Sox2 (1∶200, Cell Signaling), Nanog (1∶200, Abcam), TRA-1-60 (Millipore, 1∶200), TRA-1-81 (Millipore, 1∶200), SSEA1 (1∶50, Developmental Studies Hybridoma Bank), SSEA3 (1∶50, Developmental Studies Hybridoma Bank), SSEA4 (1∶50, Developmental Studies Hybridoma Bank), 5-methyl cytidine (5-mC, 1∶200, Abcam), 5-hydroxymethyl cytidine (5-hmC, 1∶200, Active Motif), H3K27me3 (1∶250, Millipore). .. Primary antibodies were detected using secondary antibodies conjugated to Alexa Fluor 488 (1∶500, Molecular Probes) and Alexa Fluor 594 (1∶500, Molecular Probes).

    Article Title:
    Article Snippet: Staining intensity measurement was done by ImageJ software. .. The following primary antibodies were used: mouse anti-5mC (Abcam ab10805, 1:500), mouse anti-5mC (Abcam ab51552, 1:100), rabbit anti-H3 (Abcam ab1791, 1:500), mouse anti-H3 (EASYBIO BE3015, 1:200), mouse anti-BrdUTP (Santa Cruz sc-32323, 1:100), anti-ssDNA (IBL JP18731, 1:100), rabbit anti-Ung (GeneTex GTX103236, 1:200), rabbit anti-H3K4me3 (Abcam ab8580, 1:100), and rabbit anti-H3K27me3 (Millipore 07-449, 1:100).

    Mouse Assay:

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: GMPs from the BM of recipient mice were cross-linked with 0.5% formaldehyde for 5 min at room temperature, washed three times with PBS, and lysed with RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate [DOC], and protease inhibitor cocktail) and sonicated for 30 min using a Microson XL2000 Ultrasonic cell disruptor (Misonix). .. After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Chromatin Immunoprecipitation:

    Article Title: Deacetylase activity-independent transcriptional activation by HDAC2 during TPA-induced HL-60 cell differentiation
    Article Snippet: These oligonucleotides were inserted into the Age I/ EcoR I site of the pLKO.1 TRC vector (Addgene). .. The antibodies used for the ChIP assays were directed against RNA Pol II (Santa Cruz [sc-899 X]), HDAC2 (Abcam [ab12169]), AcH3 (Abcam [ab47915]), PAX5 (Santa Cruz [sc-13146 X]), and H3K27me3 (Millipore [07–449]. .. Antibodies against PAX5 (Santa Cruz [sc-13146 X]) and Flag (Sigma [F3165]) were used in the immunoprecipitation (IP) and PAX5 (Santa Cruz [sc-1974]) and HDAC2 (Abcam [ab12169]) were used in the immunoblot assays.

    Article Title: Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters
    Article Snippet: The sonicated chromatin was divided into 200 μl aliquots for chromatin immunoprecipitation (ChIP) using the ChIP assay kit (Upstate, #17–295) according to the manufacturer’s protocol. .. ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303). .. Unenriched input fraction was retained as control in each case.

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: Paragraph title: Chromatin immunoprecipitation assay for the Rassf1a-, p16INK4A -, Socs1-, Cx26- and Cdh1-associated histone lysine methylation ... Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression
    Article Snippet: Paragraph title: Chromatin immunoprecipitation ... Chromatin immunoprecipitations (ChIPs) were carried out in triplicate as described ( ) with antibodies against acetylated H3 (Millipore, 06–599) and H3K27me3 (Millipore, 07–449).

    Article Title: Concurrent loss of Ezh2 and Tet2 cooperates in the pathogenesis of myelodysplastic disorders
    Article Snippet: Paragraph title: ChIP assay and ChIP assay coupled with massive parallel sequencing (ChIP-Seq). ... After centrifugation, the soluble chromatin fraction was recovered, precleared for 1 h at 4°C with a mixture of Protein A– and G–conjugated Dynabeads (Invitrogen) blocked with BSA and salmon (Invitrogen), and then incubated with an anti-H3K27me3 antibody (07–449; Millipore) for 2 h at 4°C.

    Article Title: Polycomb Target Genes Are Silenced in Multiple Myeloma
    Article Snippet: Chromatin was collected at 14 000 rpm for 15 min and divided in several fractions. .. Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode). .. Chromatin immunoprecipitation was performed using OneDay ChIP Kit™ (Diagenode, Liège, Belgium) according to the manufacturer's protocol.

    Article Title: Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice
    Article Snippet: Paragraph title: Native Chromatin Immunoprecipitation ... Antibodies used were IgG (Santa Cruz #sc-2027), H3K4me2 (Millipore #07-030), H3K27me3 (Millipore, #07-449) and H3K9ac (Millipore #07-352).

    Article Title: Integrative epigenomic mapping defines four main chromatin states in Arabidopsis
    Article Snippet: ChIP and Me-DIP assays were carried out essentially as described ( ) using commercially available antibodies ( ; ). .. Specificity of the H3K27me2 and H3K9me3 antibodies was tested by peptide competition and western blotting analysis on nuclear extracts ( ) as described in using H3K27me3, H3K27me2, H3K27me1, H3K9me3 and H3K36me3 peptides (Millipore, 12-565, 12-566, 12-567, 12-568 and Diagenode sp-058-050, respectively).

    Article Title: Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma
    Article Snippet: Paragraph title: Chromatin immunoprecipitation ... Antibodies used in this study were as follows: MYCN (B8.4.B, Santa Cruz Biotechnology), LSD1 (ab17721, ABCAM), H3K4me2 (07-030, Millipore), H3K27me3 (07-449, Millipore), H3Ac (06-599, Millipore).

    Article Title: Accelerated hepatocellular carcinoma development in CUL4B transgenic mice
    Article Snippet: The sequences of the primers were for p16 -a, 5′-GCAACAGGGAATGGAACT-3′ and 5′-AGGTATCTGGGCAGAAGG-3′ (−1800bp to −1400bp upstream of the TSS), for p16 -b, 5′-CAAAGTCACATACTAGAGGGAA-3′ and 5′-GGGTCTTATAGAGCGGATT-3′ (−1400bp to −1000bp), and for p16 -c, 5′-CTTCCCGCTTTCTCAATCTCC-3′ and 5′-CCCGGCTCTTCCTCTTTCC-3′ (−1000bp to −600bp). .. Antibodies used in ChIP assay were anti-CUL4B (Sigma), anti-EZH2 (BD, Franklin Lakes, NJ, USA), anti-H3K27me3 (Millipore) and anti-H2AK119ub1 (CST). .. Immunohistochemistry was performed as described previously [ ].

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: Paragraph title: ChIP-qPCR and ChIP-seq ... Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: Paragraph title: ChIP assay and ChIP-Seq ... Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176).

    Plasmid Preparation:

    Article Title: Epigenetic silencing of Th1 type chemokines shapes tumor immunity and immunotherapy
    Article Snippet: Lentiviral shRNAs ( ) were provided by the Vector Core at the University of Michigan or kindly provided by Dr. Arul Chinnaiyan (University of Michigan). .. Antibodies including monoclonal anti-EZH2 (1:2000, BD Biosciences, 612667), anti-H3K27me3 (1:1000, Millipore, 07-449), H3K9me2 (1:2000, ab1220, Abcam), H3K9me3 (1:500, ab8898, Abcam), H3K4me1 (ab8895, Abcam), H3K4me2 (ab194678, Abcam), H3K4me3 (ab1012, Abcam), anti-Histone H3 (1:2000, Cell Signaling, 9715), anti-HA (1:200, Santa Cruz Biotechnology, sc-805), anti-DNMT1 (1:250, Abcam, ab13537) were used for Western blotting.

    Software:

    Article Title: Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells
    Article Snippet: For immunofluorescence staining, cells and tissue sections were fixed with 4% formaldehyde for 15 min, washed three times in PBS and permeabilized by 1% Triton X-100 in PBS for 20 min. After washing five times in PBS, cells and sections were incubated in blocking buffer (1% BSA in PBS) for 30 min and then with mouse monoclonal antibodies directed against H3K4me3 (1:1000; Wako, Tokyo, Japan; 307-34813), H4K20me3 (1:1000; Abcam, Cambridge, MA; ab78517), Oct-3/4 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA; sc-5279) or ßIII-tubulin (1:500; Covance, Berkeley, CA; MMS-435P), or with rabbit polyclonal antibodies directed against acetylated H3K9 (H3K9ac) (1:500, Abcam; ab10812), H3K9me3 (1:1000, Millipore: 07-442), H3K27me3 (1:1000, Millipore; 07-449) or RNA polymerase II (RNAPII) (1:500, Abcam; ab5095) in the same buffer for 1 h. The specificity of antibodies against histone modifications was confirmed previously ( , ). .. Cells and sections were washed in PBS three times for 5 min and incubated in Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:1000, Invitrogen, Carlsbad, CA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000, Invitrogen) for 1 h. After washing with PBS three times for 5 min, coverslips were mounted using ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen).

    Article Title:
    Article Snippet: Staining intensity measurement was done by ImageJ software. .. The following primary antibodies were used: mouse anti-5mC (Abcam ab10805, 1:500), mouse anti-5mC (Abcam ab51552, 1:100), rabbit anti-H3 (Abcam ab1791, 1:500), mouse anti-H3 (EASYBIO BE3015, 1:200), mouse anti-BrdUTP (Santa Cruz sc-32323, 1:100), anti-ssDNA (IBL JP18731, 1:100), rabbit anti-Ung (GeneTex GTX103236, 1:200), rabbit anti-H3K4me3 (Abcam ab8580, 1:100), and rabbit anti-H3K27me3 (Millipore 07-449, 1:100).

    SYBR Green Assay:

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore). .. Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Polycomb Target Genes Are Silenced in Multiple Myeloma
    Article Snippet: Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode). .. Chromatin immunoprecipitation was performed using OneDay ChIP Kit™ (Diagenode, Liège, Belgium) according to the manufacturer's protocol.

    Article Title: Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice
    Article Snippet: Antibodies used were IgG (Santa Cruz #sc-2027), H3K4me2 (Millipore #07-030), H3K27me3 (Millipore, #07-449) and H3K9ac (Millipore #07-352). .. The immunoprecipitation, washes and DNA purification was done with Magna ChIP™ A Chromatin Immunoprecipitation Kit (Millipore #17-610) following manufacturer's protocol.

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176). .. DNA was purified from eluates using phenol-chloroform isoamyl alcohol extraction.

    Negative Control:

    Article Title: Polycomb Target Genes Are Silenced in Multiple Myeloma
    Article Snippet: Chromatin was collected at 14 000 rpm for 15 min and divided in several fractions. .. Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode). .. Chromatin immunoprecipitation was performed using OneDay ChIP Kit™ (Diagenode, Liège, Belgium) according to the manufacturer's protocol.

    shRNA:

    Article Title: Epigenetic silencing of Th1 type chemokines shapes tumor immunity and immunotherapy
    Article Snippet: Paragraph title: Plasmids, shRNA and antibodies ... Antibodies including monoclonal anti-EZH2 (1:2000, BD Biosciences, 612667), anti-H3K27me3 (1:1000, Millipore, 07-449), H3K9me2 (1:2000, ab1220, Abcam), H3K9me3 (1:500, ab8898, Abcam), H3K4me1 (ab8895, Abcam), H3K4me2 (ab194678, Abcam), H3K4me3 (ab1012, Abcam), anti-Histone H3 (1:2000, Cell Signaling, 9715), anti-HA (1:200, Santa Cruz Biotechnology, sc-805), anti-DNMT1 (1:250, Abcam, ab13537) were used for Western blotting.

    In Situ:

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: For ChIP experiments using primary liver tissue, livers were perfused in situ via the inferior vena cava with 30 ml of 2% formaldehyde over 5 min. Livers were removed, weighed, minced and homogenized in a Potter-Elvehjem hand-powered homogenizer. .. Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176).

    Ethanol Precipitation:

    Article Title: Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma
    Article Snippet: Immunoprecipitated DNA was purified using Phenol/Chloroform and Ethanol precipitation techniques. .. Antibodies used in this study were as follows: MYCN (B8.4.B, Santa Cruz Biotechnology), LSD1 (ab17721, ABCAM), H3K4me2 (07-030, Millipore), H3K27me3 (07-449, Millipore), H3Ac (06-599, Millipore).

    Produced:

    Article Title: PPAR? Interprets a Chromatin Signature of Pluripotency to Promote Embryonic Differentiation at Gastrulation
    Article Snippet: An affinity-purified polyclonal antibody against xenopus PPARβ was produced by immunizing rabbits with the peptide KLH-VQAPVSDSAAPDSPV (Eurogentec). .. We used anti-H3K4me3 from Abcam (ab8580), anti-H3K27me3 from Millipore (07-449), and anti–β-actin from Sigma (AC-40).

    Concentration Assay:

    Article Title: Mechanisms of epigenetic silencing of the Rassf1a gene during estrogen-induced breast carcinogenesis in ACI rats
    Article Snippet: Proteins were cross-linked to DNA by addition of formaldehyde to the tissue suspension to a final concentration of 1% and incubation for 10 min at room temperature. .. Chromatin was immunoprecipitated with primary anti-H3K9me3 and anti-H3K27me3 antibodies (Millipore).

    Article Title: Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice
    Article Snippet: Digestion was stopped by adding EDTA to final concentration of 10 µM and tubes were left on ice for at least 5 min. Non-soluble fractions were removed by centrifuging at 10,000 rpm (+4°C) for 10 min and collecting the supernatant. .. Antibodies used were IgG (Santa Cruz #sc-2027), H3K4me2 (Millipore #07-030), H3K27me3 (Millipore, #07-449) and H3K9ac (Millipore #07-352).

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: Genomic DNA was eluted with elution buffer (1% SDS and 100 mM NaHCO3 ) at 65 °C for 30 min. 5 M NaCl was added to a final concentration of 200 mM for further incubation at 65 °C for 4 h or overnight. .. Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    DNA Purification:

    Article Title: A positive role for polycomb in transcriptional regulation via H4K20me1
    Article Snippet: Genomic DNA was purified with a DNA purification kit (QIAGEN) and sent for real-time PCR or sequencing. .. Antibodies used were rabbit anti-Pc antibody (Santa Cruz), rabbit anti-H3K27me3 antibody (Millipore), rabbit anti-H4K20me1 antibody (Abcam) and control rabbit IgG (Santa Cruz).

    Lysis:

    Article Title: Vertebrate GAGA factor associated insulator elements demarcate homeotic genes in the HOX clusters
    Article Snippet: Fixed cells were washed well with 1 × PBS containing protease inhibitors at 4°C and resuspended in 2 ml lysis buffer supplemented with PMSF, DTT, and protease inhibitor cocktail (Roche). .. ChIP was performed on precleared chromatin from 106 cells using rabbit polyclonal antibodies against core histone H3 (Abcam, #ab1791), H3K4me3 (Millipore, #07–473), H3K27me3 (Millipore, #07–449), Th-POK (Abcam, #ab20985), or CTCF (Abcam, #ab70303).

    Article Title: Polycomb Target Genes Are Silenced in Multiple Myeloma
    Article Snippet: Cells were washed in ice-cold phosphate-buffered saline (PBS), treated with cell lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40 and protease inhibitors) for 10 min on ice and cell nuclei were collected at 2500 rpm for 5 min. .. Antibodies used were: anti-H3K9ac (Millipore, 06-942), anti-H3K27me3 (Millipore, 07-449) and IgG (Negative control, OneDay ChIP Kit™Diagenode).

    Article Title: Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma
    Article Snippet: Cell pellet was resuspended in Cell Lysis Buffer and after 6000rpm centrifugation RIPA buffer were added to perform nuclei lysis. .. Antibodies used in this study were as follows: MYCN (B8.4.B, Santa Cruz Biotechnology), LSD1 (ab17721, ABCAM), H3K4me2 (07-030, Millipore), H3K27me3 (07-449, Millipore), H3Ac (06-599, Millipore).

    Article Title: Recruitment of Pontin/Reptin by E2f1 amplifies E2f transcriptional response during cancer progression
    Article Snippet: Cell lines were collected into 0.5% SDS lysis buffer at a density of 50 × 106 cells per ml and crosslinked in 2% formaldehyde for 10 min. Lysates were sonicated on a BioLogics Model 3000 Ultrasonic Homogenizer for 30 s at 40% power on ice to produce chromatin fragments of ∼400–700 bp. .. Antibodies used for IP are as follows: E2F1 (Santa Cruz, SC-193X), E2F3 (Santa Cruz, SC-878X), H2a.z (Abcam, ab4174), H3k27ac (Abcam, AB4729), H3k27me3 (Millipore, 07-449), H3k9ac (Abcam, AB10812), H3k9me3 (Abcam, AB8898), H3k4me3 (Abcam, AB8580), H3k4me1 (Abcam, AB8895), H4k20me3 (Abcam, AB9053), H3r17me2 (Abcam, AB8284), H3s10ph (Abcam, AB5176).

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  • 79
    Millipore antibodies against histone h3k27me3
    Distribution of bovine <t>H3K27me3</t> reads. (A) The mapping result of H3K27me3 reads. Raw reads were generated by Solexa sequencing, and then clean reads were obtained after filtering dirty reads. All clean reads were mapped to the bovine reference genome and only uniquely matching reads were retained to use for subsequent analysis. Mapping rate is the ratio of mapped reads to clean reads and unique mapping rate is the ratio of unique mapped reads to clean reads. (B) Distribution of H3K27me3 reads among different genomic regions. The bovine genome was divided into five kinds of regions: 20 kb upstream of transcription start site (TSS), exon, intron, 20 kb downstream of transcription end site (TES) and intergenic regions. The histogram described the percentage of unique mapped reads among five genomic regions and the proportion of each region of the total genome. (C) Abundance of H3K27me3 reads among different genomic regions. The percentages of reads distribution were normalized to the abundance values. (D) Coverage depth of H3K27me3 reads among genic regions. For each gene, the tag numbers detected in every 5% of the gene-body region and every 1 kb outside of the gene-body region were summed to obtain methylation levels. These numbers were then normalized by the total number of base pairs in each region [20] .
    Antibodies Against Histone H3k27me3, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against histone h3k27me3/product/Millipore
    Average 79 stars, based on 173 article reviews
    Price from $9.99 to $1999.99
    antibodies against histone h3k27me3 - by Bioz Stars, 2019-10
    79/100 stars
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    99
    Millipore rabbit anti h3k27me3
    Gene classification based on promoter association with PRC and RNAPII at different stages of the neuronal differentiation Related to Fig 5 . Number of genes in each promoter state described in Fig 5 A. Classification based on the presence or absence of RNAPII‐S5p, RNAPII‐S7p, and <t>H3K27me3</t> at the 2‐kb window centered around promoters, shown from ESC to day 30 neurons. Promoter state dynamics during differentiation for all classified genes. Bottom panel shows the promoter state dynamics for genes that are marked by H3K27me3 in at least one time point. Gene expression changes for two selected groups of genes that become Active from Polycomb‐repressed states (highlighted by black boxes in panel B). Colors indicate promoter states in each time point. In boxplots, as default of R graphics package, box center represents the median value; box top and bottom represent third and first quartiles, respectively. Notches extend to ± 1.58 × Interquartile range (IQR)/n. Whiskers extend to the highest point within 1.5 IQR of the third quartile and to the lowest point within 1.5 IQR of the first quartile, respectively. Values outside whiskers are represented as individual points. Promoter state of genes that are marked by PRC in ESCs and become Active in day 30 neurons. Examples of enriched GO terms are shown using as background all genes. Permute P ‐value (GO‐Elite) is shown.
    Rabbit Anti H3k27me3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti h3k27me3/product/Millipore
    Average 99 stars, based on 214 article reviews
    Price from $9.99 to $1999.99
    rabbit anti h3k27me3 - by Bioz Stars, 2019-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    Distribution of bovine H3K27me3 reads. (A) The mapping result of H3K27me3 reads. Raw reads were generated by Solexa sequencing, and then clean reads were obtained after filtering dirty reads. All clean reads were mapped to the bovine reference genome and only uniquely matching reads were retained to use for subsequent analysis. Mapping rate is the ratio of mapped reads to clean reads and unique mapping rate is the ratio of unique mapped reads to clean reads. (B) Distribution of H3K27me3 reads among different genomic regions. The bovine genome was divided into five kinds of regions: 20 kb upstream of transcription start site (TSS), exon, intron, 20 kb downstream of transcription end site (TES) and intergenic regions. The histogram described the percentage of unique mapped reads among five genomic regions and the proportion of each region of the total genome. (C) Abundance of H3K27me3 reads among different genomic regions. The percentages of reads distribution were normalized to the abundance values. (D) Coverage depth of H3K27me3 reads among genic regions. For each gene, the tag numbers detected in every 5% of the gene-body region and every 1 kb outside of the gene-body region were summed to obtain methylation levels. These numbers were then normalized by the total number of base pairs in each region [20] .

    Journal: PLoS ONE

    Article Title: Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

    doi: 10.1371/journal.pone.0039094

    Figure Lengend Snippet: Distribution of bovine H3K27me3 reads. (A) The mapping result of H3K27me3 reads. Raw reads were generated by Solexa sequencing, and then clean reads were obtained after filtering dirty reads. All clean reads were mapped to the bovine reference genome and only uniquely matching reads were retained to use for subsequent analysis. Mapping rate is the ratio of mapped reads to clean reads and unique mapping rate is the ratio of unique mapped reads to clean reads. (B) Distribution of H3K27me3 reads among different genomic regions. The bovine genome was divided into five kinds of regions: 20 kb upstream of transcription start site (TSS), exon, intron, 20 kb downstream of transcription end site (TES) and intergenic regions. The histogram described the percentage of unique mapped reads among five genomic regions and the proportion of each region of the total genome. (C) Abundance of H3K27me3 reads among different genomic regions. The percentages of reads distribution were normalized to the abundance values. (D) Coverage depth of H3K27me3 reads among genic regions. For each gene, the tag numbers detected in every 5% of the gene-body region and every 1 kb outside of the gene-body region were summed to obtain methylation levels. These numbers were then normalized by the total number of base pairs in each region [20] .

    Article Snippet: Chromatin from 5×107 cells were used for ChIP experiment and antibodies against histone H3K27me3 (millipore, Cat. #17–622) were used, that yielded approximately 30 ng of DNA.

    Techniques: Generated, Sequencing, Methylation

    Modifications of H3K27me3 near transcription start sites. Profiles of the H3K27me3 indicated across the TSS for highly active (high), two kinds of intermediately active (medium and low) and silent gene sets were shown. Each gene set included 700 genes according to their expression levels in primary lymphocytes of cow peripheral blood. Here, up and down 20 kb regions of 700 genes in each group were aligned relative to their TSSs (x axis). The y axis shows the detected tag density.

    Journal: PLoS ONE

    Article Title: Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

    doi: 10.1371/journal.pone.0039094

    Figure Lengend Snippet: Modifications of H3K27me3 near transcription start sites. Profiles of the H3K27me3 indicated across the TSS for highly active (high), two kinds of intermediately active (medium and low) and silent gene sets were shown. Each gene set included 700 genes according to their expression levels in primary lymphocytes of cow peripheral blood. Here, up and down 20 kb regions of 700 genes in each group were aligned relative to their TSSs (x axis). The y axis shows the detected tag density.

    Article Snippet: Chromatin from 5×107 cells were used for ChIP experiment and antibodies against histone H3K27me3 (millipore, Cat. #17–622) were used, that yielded approximately 30 ng of DNA.

    Techniques: Expressing

    Distribution of bovine H3K27me3 peaks. (A) H3K27me3 peaks and genes related to peaks. (B) Percentages of genes related to H3K27me3 peaks. The bovine genic region was divided into four kinds of regions: 20 kb upstream of TSS, exon, intron, and 20 kb downstream of TES. The bar chart described the percentage of genes associated with H3K27me3 peaks among kinds of genic regions.

    Journal: PLoS ONE

    Article Title: Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

    doi: 10.1371/journal.pone.0039094

    Figure Lengend Snippet: Distribution of bovine H3K27me3 peaks. (A) H3K27me3 peaks and genes related to peaks. (B) Percentages of genes related to H3K27me3 peaks. The bovine genic region was divided into four kinds of regions: 20 kb upstream of TSS, exon, intron, and 20 kb downstream of TES. The bar chart described the percentage of genes associated with H3K27me3 peaks among kinds of genic regions.

    Article Snippet: Chromatin from 5×107 cells were used for ChIP experiment and antibodies against histone H3K27me3 (millipore, Cat. #17–622) were used, that yielded approximately 30 ng of DNA.

    Techniques:

    Overview of the experimental design and samples characterization. (A) Peripheral blood was collected from cow caudal vein, and primary lymphocytes were isolated. The genome-wide distribution of H3K27me3 was determined with ChIP-seq, and all genes expressions in lymphocytes were performed with Digital Gene Expression technique. (B) Performance testing information about the four cows. † Days_in_Milk is the days from calving to sampling. ‡ Daily milk yield, fat percentage and protein percentage were the average values of annual records. § Somatic cell counts.

    Journal: PLoS ONE

    Article Title: Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

    doi: 10.1371/journal.pone.0039094

    Figure Lengend Snippet: Overview of the experimental design and samples characterization. (A) Peripheral blood was collected from cow caudal vein, and primary lymphocytes were isolated. The genome-wide distribution of H3K27me3 was determined with ChIP-seq, and all genes expressions in lymphocytes were performed with Digital Gene Expression technique. (B) Performance testing information about the four cows. † Days_in_Milk is the days from calving to sampling. ‡ Daily milk yield, fat percentage and protein percentage were the average values of annual records. § Somatic cell counts.

    Article Snippet: Chromatin from 5×107 cells were used for ChIP experiment and antibodies against histone H3K27me3 (millipore, Cat. #17–622) were used, that yielded approximately 30 ng of DNA.

    Techniques: Isolation, Genome Wide, Chromatin Immunoprecipitation, Expressing, Sampling

    Hierarchical clustering of all cytokines and transcription factors genes. (A) Based on annotation for bovine DGE profiles, we identified 138 unique cytokine genes and their related genes. Normalized intensity values of genes (rows) and their H3K27me3 modifications were ordered using Centroid Spearman Rank Correlation and hierarchical clustering in Cluster3.0 software. The dendrogram showed the similarity (distance) of mRNA expression levels and H3K27me3 modifications of genes and was divided into sub-trees as distinguished from different colors. Arrays (columns) were grouped by four different individuals. Yellow and blue colors reflect the high and low intensities, respectively. (B) Based on annotation for bovine DGE profiles, 178 unique transcription factors and related genes were identified. The heatmap and the hierarchical cluster were generated as above described.

    Journal: PLoS ONE

    Article Title: Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

    doi: 10.1371/journal.pone.0039094

    Figure Lengend Snippet: Hierarchical clustering of all cytokines and transcription factors genes. (A) Based on annotation for bovine DGE profiles, we identified 138 unique cytokine genes and their related genes. Normalized intensity values of genes (rows) and their H3K27me3 modifications were ordered using Centroid Spearman Rank Correlation and hierarchical clustering in Cluster3.0 software. The dendrogram showed the similarity (distance) of mRNA expression levels and H3K27me3 modifications of genes and was divided into sub-trees as distinguished from different colors. Arrays (columns) were grouped by four different individuals. Yellow and blue colors reflect the high and low intensities, respectively. (B) Based on annotation for bovine DGE profiles, 178 unique transcription factors and related genes were identified. The heatmap and the hierarchical cluster were generated as above described.

    Article Snippet: Chromatin from 5×107 cells were used for ChIP experiment and antibodies against histone H3K27me3 (millipore, Cat. #17–622) were used, that yielded approximately 30 ng of DNA.

    Techniques: Software, Expressing, Generated

    The genes expressions and H3K27me3 modification of DGE differential genes between the two parity cows. A total of 53 differentially expressed genes between the two parity cows were tested (FDR ≤0.01 and | log2Ratio |≥1), in which 28 genes were up-regulated and other 25 genes were down-regulated in the third parity cows. (A) The heatmap of 25 down-expressed genes for the four individuals. Normalized intensity values of genes (rows) were ordered using Centroid Spearman Rank Correlation and hierarchical clustering in Cluster3.0 software. The dendrogram showed the similarity (distance) of mRNA expression levels and was divided into sub-trees as distinguished from different colors. Arrays (columns) were grouped by four different individuals. Yellow and blue colors reflect the high and low expression intensities, respectively. (B) Modifications of H3K27me3 in 25 down-regulated genes for the four individuals. Profiles of the H3K27me3 covered the region of up5K to TSS of the genes were shown. The tag density (number of tags per base pair) was calculated in 500 bp windows in upstream 5 K regions to TSS. (C) The heatmap of 28 up-expressed genes for the four individuals. (D) Modifications of H3K27me3 in 28 up-expressed genes for the four samples.

    Journal: PLoS ONE

    Article Title: Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

    doi: 10.1371/journal.pone.0039094

    Figure Lengend Snippet: The genes expressions and H3K27me3 modification of DGE differential genes between the two parity cows. A total of 53 differentially expressed genes between the two parity cows were tested (FDR ≤0.01 and | log2Ratio |≥1), in which 28 genes were up-regulated and other 25 genes were down-regulated in the third parity cows. (A) The heatmap of 25 down-expressed genes for the four individuals. Normalized intensity values of genes (rows) were ordered using Centroid Spearman Rank Correlation and hierarchical clustering in Cluster3.0 software. The dendrogram showed the similarity (distance) of mRNA expression levels and was divided into sub-trees as distinguished from different colors. Arrays (columns) were grouped by four different individuals. Yellow and blue colors reflect the high and low expression intensities, respectively. (B) Modifications of H3K27me3 in 25 down-regulated genes for the four individuals. Profiles of the H3K27me3 covered the region of up5K to TSS of the genes were shown. The tag density (number of tags per base pair) was calculated in 500 bp windows in upstream 5 K regions to TSS. (C) The heatmap of 28 up-expressed genes for the four individuals. (D) Modifications of H3K27me3 in 28 up-expressed genes for the four samples.

    Article Snippet: Chromatin from 5×107 cells were used for ChIP experiment and antibodies against histone H3K27me3 (millipore, Cat. #17–622) were used, that yielded approximately 30 ng of DNA.

    Techniques: Modification, Software, Expressing

    Modifications of H3K27me3 across the gene bodies. Profiles of H3K27me3 patterns of the four sets genes were shown across the gene bodies. The classification of four groups is the same as Figure 4 and density plots extend 20 kb of 5’ and 3’ of the gene bodies. For each gene, the tag numbers detected in every 5% of the gene-body region and every 1 kb outside of the gene-body region were summed to obtain methylation levels. These numbers were then normalized by the total number of base pairs in each region.

    Journal: PLoS ONE

    Article Title: Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

    doi: 10.1371/journal.pone.0039094

    Figure Lengend Snippet: Modifications of H3K27me3 across the gene bodies. Profiles of H3K27me3 patterns of the four sets genes were shown across the gene bodies. The classification of four groups is the same as Figure 4 and density plots extend 20 kb of 5’ and 3’ of the gene bodies. For each gene, the tag numbers detected in every 5% of the gene-body region and every 1 kb outside of the gene-body region were summed to obtain methylation levels. These numbers were then normalized by the total number of base pairs in each region.

    Article Snippet: Chromatin from 5×107 cells were used for ChIP experiment and antibodies against histone H3K27me3 (millipore, Cat. #17–622) were used, that yielded approximately 30 ng of DNA.

    Techniques: Methylation

    H3K27 trimethylations of CD4 and IL10 overlaying different regions and genes expressions in bovine lymphocytes. (A–D) The H3K27me3 enrichment and expression for bovine CD4 gene were showed in four individuals (C1, C2, C3 and C4). (A) The histone modification profile was shown in custom track in IGV2.0 (Integrative Genomics Viewer). Regions enriched for H3K27me3 in bovine lymphocytes were shown (blue bar). Four sites of CD4 validated by real-time PCR were indicated (line boxes). The position of the gene was presented on the bottom of the panel, where blue boxes represent exons and the arrow means the transcriptional direction of the gene. (B–C) Real-time PCR results showing enrichment of indicated four sites of CD4 in H3K27me3 ChIP-seq results carried out in the four cows. The negative controls were P1 site in the promoter of GAPDH ( Figure 8B ) and P1 site in the promoter of 18s rRNA ( Figure 8C ), respectively. (D) Real-time RT-PCR were performed on the samples of the four cows for validation of CD4 expression ( GAPDH , 18s rRNA and beta actin were housekeeping genes). The individual whose Cp value corrected was the minimum was selected as a control sample to calculate relative expression of CD4 for all four cows. Error bars indicate standard deviation of three technical replicates. DGE indicates the results of digital gene expression of CD4 (right Y axis for TPM). (E–G) The H3K27me3 enrichment and expression for bovine IL10 gene were showed in the four individuals. The details are similar to CD4 gene. A zero-height bar indicates this gene was not expressed in individual C3.

    Journal: PLoS ONE

    Article Title: Genome-Wide Bovine H3K27me3 Modifications and the Regulatory Effects on Genes Expressions in Peripheral Blood Lymphocytes

    doi: 10.1371/journal.pone.0039094

    Figure Lengend Snippet: H3K27 trimethylations of CD4 and IL10 overlaying different regions and genes expressions in bovine lymphocytes. (A–D) The H3K27me3 enrichment and expression for bovine CD4 gene were showed in four individuals (C1, C2, C3 and C4). (A) The histone modification profile was shown in custom track in IGV2.0 (Integrative Genomics Viewer). Regions enriched for H3K27me3 in bovine lymphocytes were shown (blue bar). Four sites of CD4 validated by real-time PCR were indicated (line boxes). The position of the gene was presented on the bottom of the panel, where blue boxes represent exons and the arrow means the transcriptional direction of the gene. (B–C) Real-time PCR results showing enrichment of indicated four sites of CD4 in H3K27me3 ChIP-seq results carried out in the four cows. The negative controls were P1 site in the promoter of GAPDH ( Figure 8B ) and P1 site in the promoter of 18s rRNA ( Figure 8C ), respectively. (D) Real-time RT-PCR were performed on the samples of the four cows for validation of CD4 expression ( GAPDH , 18s rRNA and beta actin were housekeeping genes). The individual whose Cp value corrected was the minimum was selected as a control sample to calculate relative expression of CD4 for all four cows. Error bars indicate standard deviation of three technical replicates. DGE indicates the results of digital gene expression of CD4 (right Y axis for TPM). (E–G) The H3K27me3 enrichment and expression for bovine IL10 gene were showed in the four individuals. The details are similar to CD4 gene. A zero-height bar indicates this gene was not expressed in individual C3.

    Article Snippet: Chromatin from 5×107 cells were used for ChIP experiment and antibodies against histone H3K27me3 (millipore, Cat. #17–622) were used, that yielded approximately 30 ng of DNA.

    Techniques: Expressing, Modification, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Quantitative RT-PCR, Standard Deviation

    CREB repression inhibits growth of NEPC xenografts, and blocks castration-activated EZH2 axis and angiogenesis in vivo. a NOD/SCID mice were injected with luciferase-labelled LNCaP or NE1.3 cells, which were then treated daily with saline or 2 mg kg −1 propranolol for 25 days, as indicated (6 mice in each group, 2 tumors in each mouse, n = 12). The tumor growth was monitored by bioluminescent imaging (BLI) with IVIS instrument. Presented on Y -axis are average fold changes of BLI signals for each tumor at day 25, relative to BLI signals at day 1 of treatments. b Left 4 panels: representative images of IHC staining for pCREB-S133 and H3K27me3 in LNCaP-derived xenografts treated daily for 25 days with saline or 10 mg kg −1 of ISO (tumors from our previous study 36 ). Right 4 panels: representative images of IHC staining for pCREB-S133 and H3K27me3 in NE1.3-derived xenografts treated daily with saline or 2 mg kg −1 of PRO. c Western blots of xenograft tumors from LNCaP with or without ISO treatments (left) or from NE1.3 with or without PRO treatment (right) for the CREB/EZH2/TSP1 pathway proteins as indicated. d Top two panels: representative IHC staining images of angiogenesis marker CD31 in LNCaP CDX treated with saline or ISO. Bottom 2 panels: representative IHC staining images of CD31 in NE1.3 CDX treated with saline or PRO. The arrows indicated typical CD31 + microvessels. e Left panel: dot plots of tumor weights in the three indicated experimental groups that are collected at the end of the experiment. Right panel: photographic picture of LNCaP xenograft tumors in the three groups at sacrifice. f Western blotting of the proteins on the CREB/EZH2/TSP1/NE pathway in the indicated three groups of LNCaP xenograft tumors. g A summary model of the key findings in this study. ADT activates CREB, in part through PKA, which in turn enhances the PRC2 activity of EZH2. EZH2 is critical for ADT/CREB-induced neuroendocrine differentiation and angiogenesis of prostate cancer cells. EZH2 enhances angiogenesis through epigenetically repressing anti-angiogenic factor TSP1. Additional data related to this Figure are in Supplementary Fig. 5 . Statistical significance was determined by using unpaired two-sided Student’s t test and shown as mean with s.d. * P

    Journal: Nature Communications

    Article Title: Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers

    doi: 10.1038/s41467-018-06177-2

    Figure Lengend Snippet: CREB repression inhibits growth of NEPC xenografts, and blocks castration-activated EZH2 axis and angiogenesis in vivo. a NOD/SCID mice were injected with luciferase-labelled LNCaP or NE1.3 cells, which were then treated daily with saline or 2 mg kg −1 propranolol for 25 days, as indicated (6 mice in each group, 2 tumors in each mouse, n = 12). The tumor growth was monitored by bioluminescent imaging (BLI) with IVIS instrument. Presented on Y -axis are average fold changes of BLI signals for each tumor at day 25, relative to BLI signals at day 1 of treatments. b Left 4 panels: representative images of IHC staining for pCREB-S133 and H3K27me3 in LNCaP-derived xenografts treated daily for 25 days with saline or 10 mg kg −1 of ISO (tumors from our previous study 36 ). Right 4 panels: representative images of IHC staining for pCREB-S133 and H3K27me3 in NE1.3-derived xenografts treated daily with saline or 2 mg kg −1 of PRO. c Western blots of xenograft tumors from LNCaP with or without ISO treatments (left) or from NE1.3 with or without PRO treatment (right) for the CREB/EZH2/TSP1 pathway proteins as indicated. d Top two panels: representative IHC staining images of angiogenesis marker CD31 in LNCaP CDX treated with saline or ISO. Bottom 2 panels: representative IHC staining images of CD31 in NE1.3 CDX treated with saline or PRO. The arrows indicated typical CD31 + microvessels. e Left panel: dot plots of tumor weights in the three indicated experimental groups that are collected at the end of the experiment. Right panel: photographic picture of LNCaP xenograft tumors in the three groups at sacrifice. f Western blotting of the proteins on the CREB/EZH2/TSP1/NE pathway in the indicated three groups of LNCaP xenograft tumors. g A summary model of the key findings in this study. ADT activates CREB, in part through PKA, which in turn enhances the PRC2 activity of EZH2. EZH2 is critical for ADT/CREB-induced neuroendocrine differentiation and angiogenesis of prostate cancer cells. EZH2 enhances angiogenesis through epigenetically repressing anti-angiogenic factor TSP1. Additional data related to this Figure are in Supplementary Fig. 5 . Statistical significance was determined by using unpaired two-sided Student’s t test and shown as mean with s.d. * P

    Article Snippet: The remaining supernatants were incubated overnight with 2 µg anti-H3K27me3 (Millipore) or anti-IgG antibody, followed by 1 h incubation with protein A/G agrose beads at 4 °C.

    Techniques: In Vivo, Mouse Assay, Injection, Luciferase, Imaging, Immunohistochemistry, Staining, Derivative Assay, Western Blot, Marker, Activity Assay

    TSP1 is a novel EZH2 epigenetic target in prostate cancer cells. a , b Changes in TSP1 expression and H3K27me3 levels upon EZH2 cDNA overexpression ( a ) and shRNA silencing ( b ) in prostate cancer cells. c Marked reduction of H3K27me3 and up-regulation of TSP1 in PC3, RWPE, and NE1.3 cells treated with 5 μM of EZH2 inhibitor DZNEP or GSK126 for 24 h. d PC3 cells were treated with 2.5 μM DZNEP or vehicle control (DMSO) for 24 h. A ChIP assay was conducted using IgG control or anti-H3K27me3 antibody. The level of H3K27me3 mark on TSP1 promoter was presented as % of the inputs ( Y -axis), which was confirmed through DNA gel electrophoresis. e PC3 cells were transfected with pGL3 firefly luciferase vector with TSP1 promoter along with a pRL-TK renilla luciferase control vector, then treated with DZNEP 2.5 μM and 5 μM for 24 h, followed by Dual-luciferase reporter assay. Plotted on Y -axis are the ratios of firefly luciferase signals vs renilla luciferase control signals in each treatment. f PC3 cells carrying an empty vector or siRNA-resistant EZH2 cDNA were transfected with control siRNA (siCNL) or siEZH2. Total RNA was extracted 72 h after siRNA treatment, and RT-qPCR for EZH2 and TSP1 was carried out, using GAPDH as the internal control. Plotted on Y -axis are average fold changes with s.d.

    Journal: Nature Communications

    Article Title: Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers

    doi: 10.1038/s41467-018-06177-2

    Figure Lengend Snippet: TSP1 is a novel EZH2 epigenetic target in prostate cancer cells. a , b Changes in TSP1 expression and H3K27me3 levels upon EZH2 cDNA overexpression ( a ) and shRNA silencing ( b ) in prostate cancer cells. c Marked reduction of H3K27me3 and up-regulation of TSP1 in PC3, RWPE, and NE1.3 cells treated with 5 μM of EZH2 inhibitor DZNEP or GSK126 for 24 h. d PC3 cells were treated with 2.5 μM DZNEP or vehicle control (DMSO) for 24 h. A ChIP assay was conducted using IgG control or anti-H3K27me3 antibody. The level of H3K27me3 mark on TSP1 promoter was presented as % of the inputs ( Y -axis), which was confirmed through DNA gel electrophoresis. e PC3 cells were transfected with pGL3 firefly luciferase vector with TSP1 promoter along with a pRL-TK renilla luciferase control vector, then treated with DZNEP 2.5 μM and 5 μM for 24 h, followed by Dual-luciferase reporter assay. Plotted on Y -axis are the ratios of firefly luciferase signals vs renilla luciferase control signals in each treatment. f PC3 cells carrying an empty vector or siRNA-resistant EZH2 cDNA were transfected with control siRNA (siCNL) or siEZH2. Total RNA was extracted 72 h after siRNA treatment, and RT-qPCR for EZH2 and TSP1 was carried out, using GAPDH as the internal control. Plotted on Y -axis are average fold changes with s.d.

    Article Snippet: The remaining supernatants were incubated overnight with 2 µg anti-H3K27me3 (Millipore) or anti-IgG antibody, followed by 1 h incubation with protein A/G agrose beads at 4 °C.

    Techniques: Expressing, Over Expression, shRNA, Chromatin Immunoprecipitation, DNA Gel Electrophoresis, Transfection, Luciferase, Plasmid Preparation, Reporter Assay, Quantitative RT-PCR

    ADT and CREB activation downregulate TSP1 through EZH2-mediated epigenetic repression. a Western blotting for TSP1 and p-CREB levels in 22Rv1 cells treated with 10 μM MDV3100 for 72 h without or with 5 nM of DHT for 24 h. b LNCaP cells were grown in CSS medium for 48 h, without or with subsequent treatment of 10 μM PKA inhibitor PKI for 5 h. TSP1 and p-CREB protein levels were assessed by western blotting. c Protein level changes of p-CREB, TSP1, and NE markers in LNCaP-Dox-ACREB cells treated by MDV3100 with and without Dox for 72 h. ACREB, a CREB inhibitory polypeptide, is induced by Dox. d TSP1 expression in PC3 cells overexpressing empty vector, wild-type (WT) or constitutively active mutant (Y134F) of CREB cDNA. e PC3 cells expressing empty vector or CREB-Y134F were infected with pLKO.1 lentivirus for shEZH2, followed by the examination of TSP1 expression. f PC3 cells were treated with 10 μM PKA/CREB activator forskolin (FSK) with or without EZH2 inhibitor GSK126 (5 μM) for 24 h. Levels of indicated proteins were measured by western blotting. g PC3 cells expressing shEZH2 were treated with 10 μM PKA/CREB activator ISO for 24 h. PC3 cells expressing Scramble control shRNA were used as control. h LNCaP and PC3 cells were treated with 10 μM PKA/CREB signaling activator ISO, with or without 10 μM PKA/CREB signaling inhibitor PRO, for 24 h, followed by ChIP assay using anti-H3K27me3 or IgG control antibody. The amount of H3K27me3 histone marks on TSP1 promoter was measured by quantitative PCR and presented as % of the input ( Y -axis). DNA gel electrophoresis was performed to visualize the PCR results. i RT-PCR for TSP1mRNA changes in PC3 and LNCaP cells that were treated similarly as in the ChIP-qPCR above. j LNCaP cells were treated either with DMSO, 10 μM MDV3100 without or with 5 μM GSK126 for 72 h. RT-PCR was done for TSP1 and beta actin

    Journal: Nature Communications

    Article Title: Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers

    doi: 10.1038/s41467-018-06177-2

    Figure Lengend Snippet: ADT and CREB activation downregulate TSP1 through EZH2-mediated epigenetic repression. a Western blotting for TSP1 and p-CREB levels in 22Rv1 cells treated with 10 μM MDV3100 for 72 h without or with 5 nM of DHT for 24 h. b LNCaP cells were grown in CSS medium for 48 h, without or with subsequent treatment of 10 μM PKA inhibitor PKI for 5 h. TSP1 and p-CREB protein levels were assessed by western blotting. c Protein level changes of p-CREB, TSP1, and NE markers in LNCaP-Dox-ACREB cells treated by MDV3100 with and without Dox for 72 h. ACREB, a CREB inhibitory polypeptide, is induced by Dox. d TSP1 expression in PC3 cells overexpressing empty vector, wild-type (WT) or constitutively active mutant (Y134F) of CREB cDNA. e PC3 cells expressing empty vector or CREB-Y134F were infected with pLKO.1 lentivirus for shEZH2, followed by the examination of TSP1 expression. f PC3 cells were treated with 10 μM PKA/CREB activator forskolin (FSK) with or without EZH2 inhibitor GSK126 (5 μM) for 24 h. Levels of indicated proteins were measured by western blotting. g PC3 cells expressing shEZH2 were treated with 10 μM PKA/CREB activator ISO for 24 h. PC3 cells expressing Scramble control shRNA were used as control. h LNCaP and PC3 cells were treated with 10 μM PKA/CREB signaling activator ISO, with or without 10 μM PKA/CREB signaling inhibitor PRO, for 24 h, followed by ChIP assay using anti-H3K27me3 or IgG control antibody. The amount of H3K27me3 histone marks on TSP1 promoter was measured by quantitative PCR and presented as % of the input ( Y -axis). DNA gel electrophoresis was performed to visualize the PCR results. i RT-PCR for TSP1mRNA changes in PC3 and LNCaP cells that were treated similarly as in the ChIP-qPCR above. j LNCaP cells were treated either with DMSO, 10 μM MDV3100 without or with 5 μM GSK126 for 72 h. RT-PCR was done for TSP1 and beta actin

    Article Snippet: The remaining supernatants were incubated overnight with 2 µg anti-H3K27me3 (Millipore) or anti-IgG antibody, followed by 1 h incubation with protein A/G agrose beads at 4 °C.

    Techniques: Activation Assay, Western Blot, Expressing, Plasmid Preparation, Mutagenesis, Infection, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, DNA Gel Electrophoresis, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

    EZH2 is activated in neuroendocrine prostate cancer (NEPC). a Western blotting for EZH2 catalytic product H3K27me3 level and NE marker CHGA in LNCaP and 22Rv1 cells treated with either CSS or 10 μM MDV3100 for 72 h, without or with 5 nM DHT in the last 24 h. b Higher levels of H3K27me3 marks in NEPC NE1.3, NCI-H660, and 144-13 cells, as compared to LNCaP cells. H3K27me3 levels were also examined in patient-derived xenograft (PDX) NEPC MDA-PCA-144-13 tumor and adenocarcinoma MDA-PCA-133 tumor. Beta actin and total histone 3 (H3) were also examined as loading controls. c , d Downregulation of known EZH2 targets, SLIT2, DAB2IP, and ADRB2 in c NEPC NE1.3 and NCI-H660 cells as compared to LNCaP cells; and in d NEPC PDX tumor MDA-PCA-144-13 as compared to adenocarcinoma PDX tumor MDA-PCA-133. Y -axis shows relative fold changes in mRNA expression, normalized to GAPDH. Error bars in PCR results represent standard deviation (s.d.) of triplicate experiments

    Journal: Nature Communications

    Article Title: Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers

    doi: 10.1038/s41467-018-06177-2

    Figure Lengend Snippet: EZH2 is activated in neuroendocrine prostate cancer (NEPC). a Western blotting for EZH2 catalytic product H3K27me3 level and NE marker CHGA in LNCaP and 22Rv1 cells treated with either CSS or 10 μM MDV3100 for 72 h, without or with 5 nM DHT in the last 24 h. b Higher levels of H3K27me3 marks in NEPC NE1.3, NCI-H660, and 144-13 cells, as compared to LNCaP cells. H3K27me3 levels were also examined in patient-derived xenograft (PDX) NEPC MDA-PCA-144-13 tumor and adenocarcinoma MDA-PCA-133 tumor. Beta actin and total histone 3 (H3) were also examined as loading controls. c , d Downregulation of known EZH2 targets, SLIT2, DAB2IP, and ADRB2 in c NEPC NE1.3 and NCI-H660 cells as compared to LNCaP cells; and in d NEPC PDX tumor MDA-PCA-144-13 as compared to adenocarcinoma PDX tumor MDA-PCA-133. Y -axis shows relative fold changes in mRNA expression, normalized to GAPDH. Error bars in PCR results represent standard deviation (s.d.) of triplicate experiments

    Article Snippet: The remaining supernatants were incubated overnight with 2 µg anti-H3K27me3 (Millipore) or anti-IgG antibody, followed by 1 h incubation with protein A/G agrose beads at 4 °C.

    Techniques: Western Blot, Marker, Derivative Assay, Multiple Displacement Amplification, Expressing, Polymerase Chain Reaction, Standard Deviation

    EZH2 is a critical for CREB to induce NE phenotypes. a H3K27me3 level (left), as well as expression of EZH2 and NE markers (right), are up-regulated in PC3 and LNCaP cells overexpressing EZH2 cDNA. b RT-qPCR measures the expression of NE markers CHGA, CHGB in NE1.3 cells treated with EZH2 inhibitors GSK126 (5 μM) or DZNEP (5 μM) for 48 h (left). Western blotting confirmed the reduction of H3K27me3 in NE1.3 cells treated with these EZH2 inhibitors (right). Error bars in PCR results represent standard deviation (s.d.) of triplicate experiments. c RT-qPCR result shows that EZH2 knockdown by a validated shRNA downregulates the expression of NE markers CHGA and CHGB in NE1.3 cells. d PC3 cells carrying Dox-inducible EZH2 cDNA were infected with scramble shRNA or shEZH2 lentiviruses, then treated without or with 1 μg ml −1 of Dox for 72 h. e RT-qPCR analysis of the changes in NE marker expression in LNCaP cells after treating with 10 μM forskolin (FSK) with or without 5 μM EZH2 inhibitor GSK126 for 24 h. f LNCaP cells expressing either control shRNA or shEZH2 were treated with 10 μM FSK for 5 h. CHGA, CHGB, and ENO2 levels were measured by RT-qPCR, using GAPDH as the loading control

    Journal: Nature Communications

    Article Title: Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers

    doi: 10.1038/s41467-018-06177-2

    Figure Lengend Snippet: EZH2 is a critical for CREB to induce NE phenotypes. a H3K27me3 level (left), as well as expression of EZH2 and NE markers (right), are up-regulated in PC3 and LNCaP cells overexpressing EZH2 cDNA. b RT-qPCR measures the expression of NE markers CHGA, CHGB in NE1.3 cells treated with EZH2 inhibitors GSK126 (5 μM) or DZNEP (5 μM) for 48 h (left). Western blotting confirmed the reduction of H3K27me3 in NE1.3 cells treated with these EZH2 inhibitors (right). Error bars in PCR results represent standard deviation (s.d.) of triplicate experiments. c RT-qPCR result shows that EZH2 knockdown by a validated shRNA downregulates the expression of NE markers CHGA and CHGB in NE1.3 cells. d PC3 cells carrying Dox-inducible EZH2 cDNA were infected with scramble shRNA or shEZH2 lentiviruses, then treated without or with 1 μg ml −1 of Dox for 72 h. e RT-qPCR analysis of the changes in NE marker expression in LNCaP cells after treating with 10 μM forskolin (FSK) with or without 5 μM EZH2 inhibitor GSK126 for 24 h. f LNCaP cells expressing either control shRNA or shEZH2 were treated with 10 μM FSK for 5 h. CHGA, CHGB, and ENO2 levels were measured by RT-qPCR, using GAPDH as the loading control

    Article Snippet: The remaining supernatants were incubated overnight with 2 µg anti-H3K27me3 (Millipore) or anti-IgG antibody, followed by 1 h incubation with protein A/G agrose beads at 4 °C.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Polymerase Chain Reaction, Standard Deviation, shRNA, Infection, Marker

    ADT activates EZH2 through PKA/CREB signaling. a Western blotting for LNCaP-Dox-shCREB cells treated with 10 μM MDV3100 without or with 1 μg ml −1 Dox for 72 h. b Elevated H3K27me3 levels in PC3 cells overexpressing wild-type (WT) or constitutively active mutant (Y134F) of CREB cDNA. c LNCaP cells were treated with PKA/CREB signaling activators, i.e., 10 μM forskolin (FSK, adenylyl cyclase activator) with 0.5 mM IBMX (phosphodiesterase inhibitor) for 24 h (left). Similar experiments for three other cell lines (PC3, NE1.3, and RWPE) treated with these PKA/CREB activators are shown in Supplementary Fig. 1a . Conversely, NEPC cells 144-13 were treated with PKA inhibitor PKI, beta-adrenergic antagonist ICI or propranolol (PRO) (all 10 μM, 48 h) (right). Similar experiment on another NEPC line NE1.3 was shown in Supplementary Fig. 1b . d , e RT-qPCR analyses of EZH2 target genes DAB2IP and ADRB2 in LNCaP cells treated with 10 μM PKA signaling activator forskolin or ISO ( d ), and in NE1.3 cells treated with PKA signaling inhibitor ICI, or PRO ( e ) (all 10 μM, 48 h). f Western blotting of LNCaP cells treated as indicated for p-CREB, H3K27me3, and NE marker ENO2. 10 μM MDV3100 with or without PRO + ICI (CREB signaling inhibitor, 10 μM each) or 5 μM EPZ6438 (EZH2 inhibitor) for 72 h. 5 nM of DHT was added in one of the MDV3100 treated cells 24 h before sample collection. Similar results were observed in another AR-positive cell line 22Rv1 (Supplementary Fig. 2a ). g A tissue microarray with 78 cases of human prostate cancer and normal samples was IHC stained with antibodies against pS133-CREB and H3K27me3. IHC pictures for two representative cases are shown (scale bar = 50 μm). The summary of IHC data is in Supplementary Table 1

    Journal: Nature Communications

    Article Title: Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers

    doi: 10.1038/s41467-018-06177-2

    Figure Lengend Snippet: ADT activates EZH2 through PKA/CREB signaling. a Western blotting for LNCaP-Dox-shCREB cells treated with 10 μM MDV3100 without or with 1 μg ml −1 Dox for 72 h. b Elevated H3K27me3 levels in PC3 cells overexpressing wild-type (WT) or constitutively active mutant (Y134F) of CREB cDNA. c LNCaP cells were treated with PKA/CREB signaling activators, i.e., 10 μM forskolin (FSK, adenylyl cyclase activator) with 0.5 mM IBMX (phosphodiesterase inhibitor) for 24 h (left). Similar experiments for three other cell lines (PC3, NE1.3, and RWPE) treated with these PKA/CREB activators are shown in Supplementary Fig. 1a . Conversely, NEPC cells 144-13 were treated with PKA inhibitor PKI, beta-adrenergic antagonist ICI or propranolol (PRO) (all 10 μM, 48 h) (right). Similar experiment on another NEPC line NE1.3 was shown in Supplementary Fig. 1b . d , e RT-qPCR analyses of EZH2 target genes DAB2IP and ADRB2 in LNCaP cells treated with 10 μM PKA signaling activator forskolin or ISO ( d ), and in NE1.3 cells treated with PKA signaling inhibitor ICI, or PRO ( e ) (all 10 μM, 48 h). f Western blotting of LNCaP cells treated as indicated for p-CREB, H3K27me3, and NE marker ENO2. 10 μM MDV3100 with or without PRO + ICI (CREB signaling inhibitor, 10 μM each) or 5 μM EPZ6438 (EZH2 inhibitor) for 72 h. 5 nM of DHT was added in one of the MDV3100 treated cells 24 h before sample collection. Similar results were observed in another AR-positive cell line 22Rv1 (Supplementary Fig. 2a ). g A tissue microarray with 78 cases of human prostate cancer and normal samples was IHC stained with antibodies against pS133-CREB and H3K27me3. IHC pictures for two representative cases are shown (scale bar = 50 μm). The summary of IHC data is in Supplementary Table 1

    Article Snippet: The remaining supernatants were incubated overnight with 2 µg anti-H3K27me3 (Millipore) or anti-IgG antibody, followed by 1 h incubation with protein A/G agrose beads at 4 °C.

    Techniques: Western Blot, Mutagenesis, Quantitative RT-PCR, Marker, Microarray, Immunohistochemistry, Staining

    Gene classification based on promoter association with PRC and RNAPII at different stages of the neuronal differentiation Related to Fig 5 . Number of genes in each promoter state described in Fig 5 A. Classification based on the presence or absence of RNAPII‐S5p, RNAPII‐S7p, and H3K27me3 at the 2‐kb window centered around promoters, shown from ESC to day 30 neurons. Promoter state dynamics during differentiation for all classified genes. Bottom panel shows the promoter state dynamics for genes that are marked by H3K27me3 in at least one time point. Gene expression changes for two selected groups of genes that become Active from Polycomb‐repressed states (highlighted by black boxes in panel B). Colors indicate promoter states in each time point. In boxplots, as default of R graphics package, box center represents the median value; box top and bottom represent third and first quartiles, respectively. Notches extend to ± 1.58 × Interquartile range (IQR)/n. Whiskers extend to the highest point within 1.5 IQR of the third quartile and to the lowest point within 1.5 IQR of the first quartile, respectively. Values outside whiskers are represented as individual points. Promoter state of genes that are marked by PRC in ESCs and become Active in day 30 neurons. Examples of enriched GO terms are shown using as background all genes. Permute P ‐value (GO‐Elite) is shown.

    Journal: Molecular Systems Biology

    Article Title: RNA polymerase II primes Polycomb‐repressed developmental genes throughout terminal neuronal differentiation

    doi: 10.15252/msb.20177754

    Figure Lengend Snippet: Gene classification based on promoter association with PRC and RNAPII at different stages of the neuronal differentiation Related to Fig 5 . Number of genes in each promoter state described in Fig 5 A. Classification based on the presence or absence of RNAPII‐S5p, RNAPII‐S7p, and H3K27me3 at the 2‐kb window centered around promoters, shown from ESC to day 30 neurons. Promoter state dynamics during differentiation for all classified genes. Bottom panel shows the promoter state dynamics for genes that are marked by H3K27me3 in at least one time point. Gene expression changes for two selected groups of genes that become Active from Polycomb‐repressed states (highlighted by black boxes in panel B). Colors indicate promoter states in each time point. In boxplots, as default of R graphics package, box center represents the median value; box top and bottom represent third and first quartiles, respectively. Notches extend to ± 1.58 × Interquartile range (IQR)/n. Whiskers extend to the highest point within 1.5 IQR of the third quartile and to the lowest point within 1.5 IQR of the first quartile, respectively. Values outside whiskers are represented as individual points. Promoter state of genes that are marked by PRC in ESCs and become Active in day 30 neurons. Examples of enriched GO terms are shown using as background all genes. Permute P ‐value (GO‐Elite) is shown.

    Article Snippet: For rabbit anti‐H3K27me3 (Millipore, Cat# 07‐449) antibodies, magnetic beads were just washed with sonication buffer.

    Techniques: Expressing

    PRC occupancy at gene promoters is highly dynamic throughout all stages of the neuronal differentiation Numbers of PRC + promoters from ESC to day 30. In terminally differentiated neurons, 2,178 genes are marked by H3K27me3. Dynamic changes in H3K27me3 presence at gene promoters. Vertical lines represent each of the promoters marked by H3K27me3 + in at least one time point. Throughout differentiation, 1,408 genes are marked by H3K27me3 (PRC Maintained), 2,699 lose H3K27me3 (PRC Lost), and 1,565 gain H3K27me3 (PRC Acquired). Color represents number of reads in transcription start site (TSS) window, scaled for visualization. Examples of enriched GO terms and gene examples among the groups of genes classified as PRC Maintained, Lost, and Acquired, using as background all genes.

    Journal: Molecular Systems Biology

    Article Title: RNA polymerase II primes Polycomb‐repressed developmental genes throughout terminal neuronal differentiation

    doi: 10.15252/msb.20177754

    Figure Lengend Snippet: PRC occupancy at gene promoters is highly dynamic throughout all stages of the neuronal differentiation Numbers of PRC + promoters from ESC to day 30. In terminally differentiated neurons, 2,178 genes are marked by H3K27me3. Dynamic changes in H3K27me3 presence at gene promoters. Vertical lines represent each of the promoters marked by H3K27me3 + in at least one time point. Throughout differentiation, 1,408 genes are marked by H3K27me3 (PRC Maintained), 2,699 lose H3K27me3 (PRC Lost), and 1,565 gain H3K27me3 (PRC Acquired). Color represents number of reads in transcription start site (TSS) window, scaled for visualization. Examples of enriched GO terms and gene examples among the groups of genes classified as PRC Maintained, Lost, and Acquired, using as background all genes.

    Article Snippet: For rabbit anti‐H3K27me3 (Millipore, Cat# 07‐449) antibodies, magnetic beads were just washed with sonication buffer.

    Techniques:

    RNAPII ‐S5p overlaps with Polycomb at all stages of differentiation Schematic summarizing the strategy used to define promoter states through differentiation based on the interplay between PRC and RNAPII. Examples of single‐gene ChIP‐seq profiles across differentiation. Eif2b2 and Brs3 are always Active and Inactive, respectively. Adcy5 and Pkp1 are PRC/S5p in ESC and days 1/3, but become Active or PRC Only, respectively. Pitx1 maintains the poised PRC/S5p conformation from ESCs until day 30 neurons. Ajuba is Active in ESCs but acquires H3K27me3 at day 16 while becoming downregulated. Proportion of H3K27me3 + promoters (dark red) marked by RNAPII‐S5p (purple) throughout differentiation. The vast majority of H3K27me3 + promoters are bound by RNAPII‐S5p in all time points. Proportion of H3K27me3 + and S5p + promoters (purple) marked by S7p (green) throughout differentiation. The number of promoters marked by H3K27me3 + S5p + S7p + increases during differentiation. Proportion of H3K27me3 and RNAPII‐S5p promoters (purple) that are also transcribed (gold; TPM > 1). The number of actively transcribed promoters increases during differentiation, in agreement with the increased presence of S7p.

    Journal: Molecular Systems Biology

    Article Title: RNA polymerase II primes Polycomb‐repressed developmental genes throughout terminal neuronal differentiation

    doi: 10.15252/msb.20177754

    Figure Lengend Snippet: RNAPII ‐S5p overlaps with Polycomb at all stages of differentiation Schematic summarizing the strategy used to define promoter states through differentiation based on the interplay between PRC and RNAPII. Examples of single‐gene ChIP‐seq profiles across differentiation. Eif2b2 and Brs3 are always Active and Inactive, respectively. Adcy5 and Pkp1 are PRC/S5p in ESC and days 1/3, but become Active or PRC Only, respectively. Pitx1 maintains the poised PRC/S5p conformation from ESCs until day 30 neurons. Ajuba is Active in ESCs but acquires H3K27me3 at day 16 while becoming downregulated. Proportion of H3K27me3 + promoters (dark red) marked by RNAPII‐S5p (purple) throughout differentiation. The vast majority of H3K27me3 + promoters are bound by RNAPII‐S5p in all time points. Proportion of H3K27me3 + and S5p + promoters (purple) marked by S7p (green) throughout differentiation. The number of promoters marked by H3K27me3 + S5p + S7p + increases during differentiation. Proportion of H3K27me3 and RNAPII‐S5p promoters (purple) that are also transcribed (gold; TPM > 1). The number of actively transcribed promoters increases during differentiation, in agreement with the increased presence of S7p.

    Article Snippet: For rabbit anti‐H3K27me3 (Millipore, Cat# 07‐449) antibodies, magnetic beads were just washed with sonication buffer.

    Techniques: Chromatin Immunoprecipitation

    Promoter state changes involving PRC loss or acquisition in intermediate or late stages of neuronal differentiation Related to Fig 5 . Most genes that acquire H3K27me3 de novo during the early phases of differentiation (days 1 or 3) have Active promoters in ESCs and in day 30 neurons. Acquisition of PRC by genes with Active promoters in ESCs is accompanied by a decrease of gene expression, as exemplified for genes that acquire H3K27me3 at day 1 and become Active again in day 30 ( n = 358). P ‐values were calculated using Wilcoxon signed rank (paired) test. Genes that acquire H3K27me3 de novo in day 16 or 30 neurons tend to have Active promoters in ESCs. Active genes in ESCs that gain PRC in day 30 show decreased mRNA expression ( n = 311). P ‐values were calculated using Wilcoxon signed rank (paired) test.

    Journal: Molecular Systems Biology

    Article Title: RNA polymerase II primes Polycomb‐repressed developmental genes throughout terminal neuronal differentiation

    doi: 10.15252/msb.20177754

    Figure Lengend Snippet: Promoter state changes involving PRC loss or acquisition in intermediate or late stages of neuronal differentiation Related to Fig 5 . Most genes that acquire H3K27me3 de novo during the early phases of differentiation (days 1 or 3) have Active promoters in ESCs and in day 30 neurons. Acquisition of PRC by genes with Active promoters in ESCs is accompanied by a decrease of gene expression, as exemplified for genes that acquire H3K27me3 at day 1 and become Active again in day 30 ( n = 358). P ‐values were calculated using Wilcoxon signed rank (paired) test. Genes that acquire H3K27me3 de novo in day 16 or 30 neurons tend to have Active promoters in ESCs. Active genes in ESCs that gain PRC in day 30 show decreased mRNA expression ( n = 311). P ‐values were calculated using Wilcoxon signed rank (paired) test.

    Article Snippet: For rabbit anti‐H3K27me3 (Millipore, Cat# 07‐449) antibodies, magnetic beads were just washed with sonication buffer.

    Techniques: Expressing

    Genes that maintain the PRC /S5p promoter state throughout differentiation are often morphogenic TF s linked to different tissue identities and show high coverage by CpG islands Related to Fig 7 . Similar low levels of mRNA expression are detected in ESC, day 16, and day 30 neurons for genes that are PRC/S5p in ESCs and remain silent through differentiation either by retaining the PRC/S5p promoter state, or by becoming PRC Only or Inactive. Colors indicate gene groups according to panel (B). Examples of enriched Gene Ontologies and associated genes of the three groups described in panel (A), using as background all genes. PRC/S5p genes that retain the PRC/S5p state throughout differentiation include important morphogenetic TFs for different cell lineages. Permute P ‐value (GO‐Elite) is shown. G/C composition of promoter windows for different groups of genes. Always PRC/S5p promoters are more enriched in G/C nucleotides compared to PRC/S5p genes that resolve to Inactive, or to the group of additional genes marked by H3K27me3 in day 30. Inactive genes in day 30 are shown for comparison. P ‐values were calculated using Wilcoxon rank‐sum test. Overlap of CpG islands with gene bodies for different groups of genes. The gene bodies of Always PRC/S5p are significantly more covered with CpG islands than PRC/S5p genes that become PRC Only or Inactive, or than the group of other H3K27me3 + genes in day 30. Inactive genes in day 30 are shown for comparison. P ‐values were calculated using Wilcoxon rank‐sum test.

    Journal: Molecular Systems Biology

    Article Title: RNA polymerase II primes Polycomb‐repressed developmental genes throughout terminal neuronal differentiation

    doi: 10.15252/msb.20177754

    Figure Lengend Snippet: Genes that maintain the PRC /S5p promoter state throughout differentiation are often morphogenic TF s linked to different tissue identities and show high coverage by CpG islands Related to Fig 7 . Similar low levels of mRNA expression are detected in ESC, day 16, and day 30 neurons for genes that are PRC/S5p in ESCs and remain silent through differentiation either by retaining the PRC/S5p promoter state, or by becoming PRC Only or Inactive. Colors indicate gene groups according to panel (B). Examples of enriched Gene Ontologies and associated genes of the three groups described in panel (A), using as background all genes. PRC/S5p genes that retain the PRC/S5p state throughout differentiation include important morphogenetic TFs for different cell lineages. Permute P ‐value (GO‐Elite) is shown. G/C composition of promoter windows for different groups of genes. Always PRC/S5p promoters are more enriched in G/C nucleotides compared to PRC/S5p genes that resolve to Inactive, or to the group of additional genes marked by H3K27me3 in day 30. Inactive genes in day 30 are shown for comparison. P ‐values were calculated using Wilcoxon rank‐sum test. Overlap of CpG islands with gene bodies for different groups of genes. The gene bodies of Always PRC/S5p are significantly more covered with CpG islands than PRC/S5p genes that become PRC Only or Inactive, or than the group of other H3K27me3 + genes in day 30. Inactive genes in day 30 are shown for comparison. P ‐values were calculated using Wilcoxon rank‐sum test.

    Article Snippet: For rabbit anti‐H3K27me3 (Millipore, Cat# 07‐449) antibodies, magnetic beads were just washed with sonication buffer.

    Techniques: Expressing

    RNAPII ‐S5p co‐localizes with H3K27me3 in terminally differentiated neurons Schematic of gene classification according to the occupancy of H3K27me3, S5p, and S7p at promoter regions (2‐kb windows centered on Transcription Start Sites; TSSs). Proportion of genes with each promoter state during terminal neuronal differentiation. Dark red: H3K27me3 + genes. PRC/S5p promoters in ESCs and their promoter state in day 30 neu rons. Examples of enriched GO terms (determined using as background all PRC/S5p genes in ESCs) and genes in each category. Expression level of PRC/S5p promoters in ESCs and day 30 neurons. Acquisition of S7p correlates with increased expression level. Genes with PRC/S5p, PRC Only, or Inactive state remain repressed in day 30 neurons. Sequential ChIP (re‐ChIP) was performed using day 30 neurons to test RNAPII and H3K27me3 co‐occupancy, as described in the schematic. The first ChIP with anti‐RNAPII‐S5p antibody was followed by a second ChIP using antibodies against RNAPII‐S5p (positive control), H3K27me3, RNAPII‐S7p, or Digoxigenin (carryover negative control “Ctrl”). Sequential ChIP shows RNAPII‐S5p co‐association with H3K27me3 at PRC/S5p ( Pitx1 and Gata4 ) and PRC/Active ( Skap2 and Ajuba ) promoters but not at Active ( Actb ) or Inactive ( Myf5 ) promoters. The mean and SD are from two biological replicates.

    Journal: Molecular Systems Biology

    Article Title: RNA polymerase II primes Polycomb‐repressed developmental genes throughout terminal neuronal differentiation

    doi: 10.15252/msb.20177754

    Figure Lengend Snippet: RNAPII ‐S5p co‐localizes with H3K27me3 in terminally differentiated neurons Schematic of gene classification according to the occupancy of H3K27me3, S5p, and S7p at promoter regions (2‐kb windows centered on Transcription Start Sites; TSSs). Proportion of genes with each promoter state during terminal neuronal differentiation. Dark red: H3K27me3 + genes. PRC/S5p promoters in ESCs and their promoter state in day 30 neu rons. Examples of enriched GO terms (determined using as background all PRC/S5p genes in ESCs) and genes in each category. Expression level of PRC/S5p promoters in ESCs and day 30 neurons. Acquisition of S7p correlates with increased expression level. Genes with PRC/S5p, PRC Only, or Inactive state remain repressed in day 30 neurons. Sequential ChIP (re‐ChIP) was performed using day 30 neurons to test RNAPII and H3K27me3 co‐occupancy, as described in the schematic. The first ChIP with anti‐RNAPII‐S5p antibody was followed by a second ChIP using antibodies against RNAPII‐S5p (positive control), H3K27me3, RNAPII‐S7p, or Digoxigenin (carryover negative control “Ctrl”). Sequential ChIP shows RNAPII‐S5p co‐association with H3K27me3 at PRC/S5p ( Pitx1 and Gata4 ) and PRC/Active ( Skap2 and Ajuba ) promoters but not at Active ( Actb ) or Inactive ( Myf5 ) promoters. The mean and SD are from two biological replicates.

    Article Snippet: For rabbit anti‐H3K27me3 (Millipore, Cat# 07‐449) antibodies, magnetic beads were just washed with sonication buffer.

    Techniques: Expressing, Chromatin Immunoprecipitation, Positive Control, Negative Control

    PRC /S5p genes that maintain the poised state throughout differentiation exhibit broader distributions of H3K27me3 and RNAPII ‐S5p which coincide with DMV s PRC/S5p genes are kept silenced through different mechanisms during neuronal differentiation by maintaining PRC/S5p (Always PRC/S5p; purple), resolving to PRC Only (light blue), or resolving to Inactive (orange). Colors indicate promoter states in each time point. Average ChIP‐seq enrichment plots of H3K27me3 and S5p in 10‐kb genomic windows centered on transcription start site (TSS) and transcription end sites (TES), in ESCs. Average ChIP‐seq enrichment plots of H3K27me3 and S5p in 10‐kb genomic windows centered on TSS and TES, in day 30 neurons. Boxplot of the length of the genomic regions enriched for H3K27me3 that overlap with promoters in ESC or in day 30 neurons. The length of H3K27me3‐enriched regions covering the remaining H3K27me3 + genes is shown for comparison. P ‐values are calculated with Wilcoxon rank‐sum test. Percentage of TSS window covered by CpG islands of PRC/S5p that maintain their state is significantly larger compared to the other gene subgroups. P ‐values are calculated with Wilcoxon rank‐sum test. UCSC Browser tracks of genes classified in day 30 neurons as Always PRC/S5p, PRC Only, Inactive, and Active genes. First row shows methylated (gold) or hypomethylated DNA methylation valleys (DMVs, dark blue; and smaller regions of hypomethylated DNA, light blue) in mouse frontal cortex. Proportion of promoters in the three gene groups which overlap with methylated and hypomethylated DNA. Always PRC/S5p genes are preferentially located at DMVs. All H3K27me3 + and Inactive genes in day 30 neurons are shown for comparison.

    Journal: Molecular Systems Biology

    Article Title: RNA polymerase II primes Polycomb‐repressed developmental genes throughout terminal neuronal differentiation

    doi: 10.15252/msb.20177754

    Figure Lengend Snippet: PRC /S5p genes that maintain the poised state throughout differentiation exhibit broader distributions of H3K27me3 and RNAPII ‐S5p which coincide with DMV s PRC/S5p genes are kept silenced through different mechanisms during neuronal differentiation by maintaining PRC/S5p (Always PRC/S5p; purple), resolving to PRC Only (light blue), or resolving to Inactive (orange). Colors indicate promoter states in each time point. Average ChIP‐seq enrichment plots of H3K27me3 and S5p in 10‐kb genomic windows centered on transcription start site (TSS) and transcription end sites (TES), in ESCs. Average ChIP‐seq enrichment plots of H3K27me3 and S5p in 10‐kb genomic windows centered on TSS and TES, in day 30 neurons. Boxplot of the length of the genomic regions enriched for H3K27me3 that overlap with promoters in ESC or in day 30 neurons. The length of H3K27me3‐enriched regions covering the remaining H3K27me3 + genes is shown for comparison. P ‐values are calculated with Wilcoxon rank‐sum test. Percentage of TSS window covered by CpG islands of PRC/S5p that maintain their state is significantly larger compared to the other gene subgroups. P ‐values are calculated with Wilcoxon rank‐sum test. UCSC Browser tracks of genes classified in day 30 neurons as Always PRC/S5p, PRC Only, Inactive, and Active genes. First row shows methylated (gold) or hypomethylated DNA methylation valleys (DMVs, dark blue; and smaller regions of hypomethylated DNA, light blue) in mouse frontal cortex. Proportion of promoters in the three gene groups which overlap with methylated and hypomethylated DNA. Always PRC/S5p genes are preferentially located at DMVs. All H3K27me3 + and Inactive genes in day 30 neurons are shown for comparison.

    Article Snippet: For rabbit anti‐H3K27me3 (Millipore, Cat# 07‐449) antibodies, magnetic beads were just washed with sonication buffer.

    Techniques: Chromatin Immunoprecipitation, Methylation, DNA Methylation Assay

    Genes with the poised PRC /S5p promoter state in terminally differentiated neurons are enriched for TF s with trans‐differentiation potential Cells can trans‐differentiate into other cell types by forced expression of lineage‐specific TFs. The Mogrify algorithm identifies candidate TFs that drive cell conversion as exemplified in the schematic (Rackham et al , 2016 ). Promoter state of TFs in day 30 which are predicted by Mogrify to drive trans‐differentiation toward non‐neuronal or neuronal cell types. H3K27me3 + promoters are enriched in the Mogrify lists of trans‐differentiation candidates. PRC/S5p and the PRC/Active genes are the mostly enriched promoter classes. Inactive genes are depleted. Enrichment and depletion were tested with the hypergeometric test. Mogrify ranks all TFs by the likelihood to induce conversion from cell types A to B. By averaging these ranks, it is possible to identify those TFs most frequently required for a conversion into a certain cell type. The ranks are used to estimate the importance of a given TF to drive the conversion. TFs important for driving trans‐differentiation toward different cell types were grouped according to their promoter state and tested for enrichment in high or low importance factors (upwards/downwards arrows). Purple and white backgrounds correspond to enrichment tested for promoter states in day 30 or maintained along all differentiation, respectively. Enrichment in high or low importance was tested with Gene Set Enrichment Analysis, considered significant when false discovery rate (FDR) was

    Journal: Molecular Systems Biology

    Article Title: RNA polymerase II primes Polycomb‐repressed developmental genes throughout terminal neuronal differentiation

    doi: 10.15252/msb.20177754

    Figure Lengend Snippet: Genes with the poised PRC /S5p promoter state in terminally differentiated neurons are enriched for TF s with trans‐differentiation potential Cells can trans‐differentiate into other cell types by forced expression of lineage‐specific TFs. The Mogrify algorithm identifies candidate TFs that drive cell conversion as exemplified in the schematic (Rackham et al , 2016 ). Promoter state of TFs in day 30 which are predicted by Mogrify to drive trans‐differentiation toward non‐neuronal or neuronal cell types. H3K27me3 + promoters are enriched in the Mogrify lists of trans‐differentiation candidates. PRC/S5p and the PRC/Active genes are the mostly enriched promoter classes. Inactive genes are depleted. Enrichment and depletion were tested with the hypergeometric test. Mogrify ranks all TFs by the likelihood to induce conversion from cell types A to B. By averaging these ranks, it is possible to identify those TFs most frequently required for a conversion into a certain cell type. The ranks are used to estimate the importance of a given TF to drive the conversion. TFs important for driving trans‐differentiation toward different cell types were grouped according to their promoter state and tested for enrichment in high or low importance factors (upwards/downwards arrows). Purple and white backgrounds correspond to enrichment tested for promoter states in day 30 or maintained along all differentiation, respectively. Enrichment in high or low importance was tested with Gene Set Enrichment Analysis, considered significant when false discovery rate (FDR) was

    Article Snippet: For rabbit anti‐H3K27me3 (Millipore, Cat# 07‐449) antibodies, magnetic beads were just washed with sonication buffer.

    Techniques: Expressing

    Correlation between genes in normal NSPCS enriched with H3K27me3 or H4K20me3 without detectable transcripts and genes altered in MRI-classified group I and group II GBM A heatmap for differential expression of genes of human GBM specimens. Genes used

    Journal:

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    doi: 10.1016/j.nepig.2016.04.001

    Figure Lengend Snippet: Correlation between genes in normal NSPCS enriched with H3K27me3 or H4K20me3 without detectable transcripts and genes altered in MRI-classified group I and group II GBM A heatmap for differential expression of genes of human GBM specimens. Genes used

    Article Snippet: Following blocking, cells were incubated for 60 min. at 4°C with primary antibodies anti- GFAP (mouse – Millipore #MAB3402; 1:500; rat – Invitrogen #130300; 1:500), Nestin (rabbit – Abcam #AB27952; 1:500, mouse – DHSB #rat-401, 1:500), DCX (Santa Cruz #sc-8066, 1:500), H3K27me3 (rabbit – Millipore #07-449; 1:500; mouse – Active Motif #61017;1:500), and/or H4K20me3 (Abcam #ab9053; 1:500).

    Techniques: Magnetic Resonance Imaging, Expressing

    Comparison among genes in normal NSPCS enriched with H3K27me3 or H4K20me3, genes without detectable transcripts in normal NSPCs, and genes elevated in MRI-classified group I and group II GBM

    Journal:

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    doi: 10.1016/j.nepig.2016.04.001

    Figure Lengend Snippet: Comparison among genes in normal NSPCS enriched with H3K27me3 or H4K20me3, genes without detectable transcripts in normal NSPCs, and genes elevated in MRI-classified group I and group II GBM

    Article Snippet: Following blocking, cells were incubated for 60 min. at 4°C with primary antibodies anti- GFAP (mouse – Millipore #MAB3402; 1:500; rat – Invitrogen #130300; 1:500), Nestin (rabbit – Abcam #AB27952; 1:500, mouse – DHSB #rat-401, 1:500), DCX (Santa Cruz #sc-8066, 1:500), H3K27me3 (rabbit – Millipore #07-449; 1:500; mouse – Active Motif #61017;1:500), and/or H4K20me3 (Abcam #ab9053; 1:500).

    Techniques: Magnetic Resonance Imaging

    Quantification of H3K27me3 or H4K20me3 co-localization with cell type markers in baboon SVZ by flow cytometry

    Journal:

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    doi: 10.1016/j.nepig.2016.04.001

    Figure Lengend Snippet: Quantification of H3K27me3 or H4K20me3 co-localization with cell type markers in baboon SVZ by flow cytometry

    Article Snippet: Following blocking, cells were incubated for 60 min. at 4°C with primary antibodies anti- GFAP (mouse – Millipore #MAB3402; 1:500; rat – Invitrogen #130300; 1:500), Nestin (rabbit – Abcam #AB27952; 1:500, mouse – DHSB #rat-401, 1:500), DCX (Santa Cruz #sc-8066, 1:500), H3K27me3 (rabbit – Millipore #07-449; 1:500; mouse – Active Motif #61017;1:500), and/or H4K20me3 (Abcam #ab9053; 1:500).

    Techniques: Flow Cytometry, Cytometry

    H3K27me3 and H4K20me3 distributions across SVZ subpopulations. Co-immunostaining of H3K27me3 or H4K20me3 with cell-type specific markers GFAP, Nestin, and DCX. Left panel presents coronal section of baboon brain and right panel presents coronal section

    Journal:

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    doi: 10.1016/j.nepig.2016.04.001

    Figure Lengend Snippet: H3K27me3 and H4K20me3 distributions across SVZ subpopulations. Co-immunostaining of H3K27me3 or H4K20me3 with cell-type specific markers GFAP, Nestin, and DCX. Left panel presents coronal section of baboon brain and right panel presents coronal section

    Article Snippet: Following blocking, cells were incubated for 60 min. at 4°C with primary antibodies anti- GFAP (mouse – Millipore #MAB3402; 1:500; rat – Invitrogen #130300; 1:500), Nestin (rabbit – Abcam #AB27952; 1:500, mouse – DHSB #rat-401, 1:500), DCX (Santa Cruz #sc-8066, 1:500), H3K27me3 (rabbit – Millipore #07-449; 1:500; mouse – Active Motif #61017;1:500), and/or H4K20me3 (Abcam #ab9053; 1:500).

    Techniques: Immunostaining

    H3K27me3 and H4K20me3 genome-wide enrichment patterns in baboon SVZ

    Journal:

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    doi: 10.1016/j.nepig.2016.04.001

    Figure Lengend Snippet: H3K27me3 and H4K20me3 genome-wide enrichment patterns in baboon SVZ

    Article Snippet: Following blocking, cells were incubated for 60 min. at 4°C with primary antibodies anti- GFAP (mouse – Millipore #MAB3402; 1:500; rat – Invitrogen #130300; 1:500), Nestin (rabbit – Abcam #AB27952; 1:500, mouse – DHSB #rat-401, 1:500), DCX (Santa Cruz #sc-8066, 1:500), H3K27me3 (rabbit – Millipore #07-449; 1:500; mouse – Active Motif #61017;1:500), and/or H4K20me3 (Abcam #ab9053; 1:500).

    Techniques: Genome Wide

    Colocalization of H3K27me3 and H4K20me3 in NSPCs of baboon SVZ

    Journal:

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    doi: 10.1016/j.nepig.2016.04.001

    Figure Lengend Snippet: Colocalization of H3K27me3 and H4K20me3 in NSPCs of baboon SVZ

    Article Snippet: Following blocking, cells were incubated for 60 min. at 4°C with primary antibodies anti- GFAP (mouse – Millipore #MAB3402; 1:500; rat – Invitrogen #130300; 1:500), Nestin (rabbit – Abcam #AB27952; 1:500, mouse – DHSB #rat-401, 1:500), DCX (Santa Cruz #sc-8066, 1:500), H3K27me3 (rabbit – Millipore #07-449; 1:500; mouse – Active Motif #61017;1:500), and/or H4K20me3 (Abcam #ab9053; 1:500).

    Techniques:

    Experimental Design for Correlation between genes in normal NSPCS enriched with H3K27me3 or H4K20me3 without detectable transcripts and genes altered in MRI-classified group I and group II GBM

    Journal:

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    doi: 10.1016/j.nepig.2016.04.001

    Figure Lengend Snippet: Experimental Design for Correlation between genes in normal NSPCS enriched with H3K27me3 or H4K20me3 without detectable transcripts and genes altered in MRI-classified group I and group II GBM

    Article Snippet: Following blocking, cells were incubated for 60 min. at 4°C with primary antibodies anti- GFAP (mouse – Millipore #MAB3402; 1:500; rat – Invitrogen #130300; 1:500), Nestin (rabbit – Abcam #AB27952; 1:500, mouse – DHSB #rat-401, 1:500), DCX (Santa Cruz #sc-8066, 1:500), H3K27me3 (rabbit – Millipore #07-449; 1:500; mouse – Active Motif #61017;1:500), and/or H4K20me3 (Abcam #ab9053; 1:500).

    Techniques: Magnetic Resonance Imaging

    EZH2/H3K27me3 influence cell cycle in the SVZ cells

    Journal:

    Article Title: Cross-species Analyses Unravel the Complexity of H3K27me3 and H4K20me3 in the Context of Neural Stem Progenitor Cells

    doi: 10.1016/j.nepig.2016.04.001

    Figure Lengend Snippet: EZH2/H3K27me3 influence cell cycle in the SVZ cells

    Article Snippet: Following blocking, cells were incubated for 60 min. at 4°C with primary antibodies anti- GFAP (mouse – Millipore #MAB3402; 1:500; rat – Invitrogen #130300; 1:500), Nestin (rabbit – Abcam #AB27952; 1:500, mouse – DHSB #rat-401, 1:500), DCX (Santa Cruz #sc-8066, 1:500), H3K27me3 (rabbit – Millipore #07-449; 1:500; mouse – Active Motif #61017;1:500), and/or H4K20me3 (Abcam #ab9053; 1:500).

    Techniques: