Structured Review

Millipore h3k27me3
H3K4me3 and <t>H3K27me3</t> enrichment profiles in ZF4 cells. (A) Distinct H3K4me3 and H3K27me3 enrichment profiles on indicated genomic regions. Genomic positioning is indicated by nucleotide number of the first (5′) and last (3′) probe in the tiled region. Gene names or accession numbers as well as their genomic position are shown in blue. (B) 2-D scatter plot of averaged MaxSixty values for H3K4me3 vs. H3K27me3 log 2 signal intensities. Data points (all points being shown in gray) were colored to visualize classification according to peak calling highlighting H3K4me3-enriched promoters (purple; N = 6315), H3K27me3-enriched promoters (green; N = 1079) and H3K4me3/K27me3-co-enriched promoters (blue; N = 2120). Red line is the regression line through all data points. (C) Venn diagram analysis of H3K4me3 and H3K27me3 genes.
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1) Product Images from "Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays"

Article Title: Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays

Journal: PLoS ONE

doi: 10.1371/journal.pone.0015651

H3K4me3 and H3K27me3 enrichment profiles in ZF4 cells. (A) Distinct H3K4me3 and H3K27me3 enrichment profiles on indicated genomic regions. Genomic positioning is indicated by nucleotide number of the first (5′) and last (3′) probe in the tiled region. Gene names or accession numbers as well as their genomic position are shown in blue. (B) 2-D scatter plot of averaged MaxSixty values for H3K4me3 vs. H3K27me3 log 2 signal intensities. Data points (all points being shown in gray) were colored to visualize classification according to peak calling highlighting H3K4me3-enriched promoters (purple; N = 6315), H3K27me3-enriched promoters (green; N = 1079) and H3K4me3/K27me3-co-enriched promoters (blue; N = 2120). Red line is the regression line through all data points. (C) Venn diagram analysis of H3K4me3 and H3K27me3 genes.
Figure Legend Snippet: H3K4me3 and H3K27me3 enrichment profiles in ZF4 cells. (A) Distinct H3K4me3 and H3K27me3 enrichment profiles on indicated genomic regions. Genomic positioning is indicated by nucleotide number of the first (5′) and last (3′) probe in the tiled region. Gene names or accession numbers as well as their genomic position are shown in blue. (B) 2-D scatter plot of averaged MaxSixty values for H3K4me3 vs. H3K27me3 log 2 signal intensities. Data points (all points being shown in gray) were colored to visualize classification according to peak calling highlighting H3K4me3-enriched promoters (purple; N = 6315), H3K27me3-enriched promoters (green; N = 1079) and H3K4me3/K27me3-co-enriched promoters (blue; N = 2120). Red line is the regression line through all data points. (C) Venn diagram analysis of H3K4me3 and H3K27me3 genes.

Techniques Used:

Distribution of H3K4me3 and H3K27me3 on promoters. Metagene analysis of the distribution of H3K4me3 and H3K27me3 occupancy on (A) H3K4me3-only, (B) H3K27me3-only and (C) H3K4me3/K27me3 tiled regions, relative to the TSS (red vertical bar). (D) Sequential ChIP analysis of H3K4me3 and H3K27me3 co-enrichment on the sox3 , sox2 promoters and on bactin1 , downstream of the coding region. Panels on the left show results from the first ChIP using antibodies indicated on the x-axis. The graph on the right shows results of the re-ChIP experiment as indicated on the x-axis.
Figure Legend Snippet: Distribution of H3K4me3 and H3K27me3 on promoters. Metagene analysis of the distribution of H3K4me3 and H3K27me3 occupancy on (A) H3K4me3-only, (B) H3K27me3-only and (C) H3K4me3/K27me3 tiled regions, relative to the TSS (red vertical bar). (D) Sequential ChIP analysis of H3K4me3 and H3K27me3 co-enrichment on the sox3 , sox2 promoters and on bactin1 , downstream of the coding region. Panels on the left show results from the first ChIP using antibodies indicated on the x-axis. The graph on the right shows results of the re-ChIP experiment as indicated on the x-axis.

Techniques Used: Chromatin Immunoprecipitation

Reproducibility of zebrafish ChIP-chip experiments. (A) Two-dimensional scatter plots of MaxSixty values for H3K4me3 and H3K27me3 log 2 signal intensities detected in each of two ChIP-chip replicates from ZF4 cells. Correlation coefficient (R) and regression line are shown. (B) H3K4me3 and H3K27me3 profiles detected by ChIP-chip in two independent replicates through 310 kb of zebrafish chromosome 10. Data are expressed as log 2 ChIP/input ratios. Position of methylation peaks are shown as blue horizontal bars. Tracks representing primary transcripts and tiled regions are also shown. Primary transcripts included in the region are as follows: 1) sin2 ; 2) NM_001003421; 3) NM_200663; 4) NM_001008616; 5) ENSDART00000081978; 6) ripply3 , 7) ENSDART00000081992; 8) dyrk1aa ; 9) ENSDART00000058411; 10) ENSDART00000088605; 11) NM_001037708; 12) hyou1 ; 13) hist2h2l ; 14) znf259 . Red bars in the H3K4me3 tracks indicate probes with out-of-scale signal intensity.
Figure Legend Snippet: Reproducibility of zebrafish ChIP-chip experiments. (A) Two-dimensional scatter plots of MaxSixty values for H3K4me3 and H3K27me3 log 2 signal intensities detected in each of two ChIP-chip replicates from ZF4 cells. Correlation coefficient (R) and regression line are shown. (B) H3K4me3 and H3K27me3 profiles detected by ChIP-chip in two independent replicates through 310 kb of zebrafish chromosome 10. Data are expressed as log 2 ChIP/input ratios. Position of methylation peaks are shown as blue horizontal bars. Tracks representing primary transcripts and tiled regions are also shown. Primary transcripts included in the region are as follows: 1) sin2 ; 2) NM_001003421; 3) NM_200663; 4) NM_001008616; 5) ENSDART00000081978; 6) ripply3 , 7) ENSDART00000081992; 8) dyrk1aa ; 9) ENSDART00000058411; 10) ENSDART00000088605; 11) NM_001037708; 12) hyou1 ; 13) hist2h2l ; 14) znf259 . Red bars in the H3K4me3 tracks indicate probes with out-of-scale signal intensity.

Techniques Used: Chromatin Immunoprecipitation, Methylation

Quantitative PCR validation of ChIP-chip data. (A) ChIP-on-chip profiles of H3K4me3 and H3K27me3 enrichment on indicated genes. Position of primary transcripts and TSS (arrow) are shown. (B) ChIP-qPCR analysis of H3K4me3 and H3K27me3 enrichment on the same genes as in (A) from separate duplicate ChIPs. ChIP DNA was not WGA amplified prior to PCR. Position of amplicons and primer sequences for each gene are shown in Table S3 . Note the correlation between ChIP- chip and ChIP-qPCR data.
Figure Legend Snippet: Quantitative PCR validation of ChIP-chip data. (A) ChIP-on-chip profiles of H3K4me3 and H3K27me3 enrichment on indicated genes. Position of primary transcripts and TSS (arrow) are shown. (B) ChIP-qPCR analysis of H3K4me3 and H3K27me3 enrichment on the same genes as in (A) from separate duplicate ChIPs. ChIP DNA was not WGA amplified prior to PCR. Position of amplicons and primer sequences for each gene are shown in Table S3 . Note the correlation between ChIP- chip and ChIP-qPCR data.

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

Genes marked by H3K4me3 and/or H3K27me3 are linked to distinct functional GO terms. (A) GO term enrichment of genes containing H3K4me3, H3K27me3 or H3K4/K27me3 promoters in ZF4 cells. The twelve most significant GO terms are shown as a function of significance from bottom (highest significance) to top. (B) Representation of all enriched GO terms among H3K4/K27me3 genes. All enriched GO terms are listed in Table S1 . (C) H3K4me3 and H3K27me3 enrichment profiles on the developmentally regulated hoxc locus, expressed as log 2 ChIP/Input (y axis).
Figure Legend Snippet: Genes marked by H3K4me3 and/or H3K27me3 are linked to distinct functional GO terms. (A) GO term enrichment of genes containing H3K4me3, H3K27me3 or H3K4/K27me3 promoters in ZF4 cells. The twelve most significant GO terms are shown as a function of significance from bottom (highest significance) to top. (B) Representation of all enriched GO terms among H3K4/K27me3 genes. All enriched GO terms are listed in Table S1 . (C) H3K4me3 and H3K27me3 enrichment profiles on the developmentally regulated hoxc locus, expressed as log 2 ChIP/Input (y axis).

Techniques Used: Functional Assay, Chromatin Immunoprecipitation

2) Product Images from "miR-125b-1 is repressed by histone modifications in breast cancer cell lines"

Article Title: miR-125b-1 is repressed by histone modifications in breast cancer cell lines

Journal: SpringerPlus

doi: 10.1186/s40064-016-2475-z

H3K9me3 and H3K27me3 are enriched on miR-125b-1 promoters in MDA-MB-231 and MCF7 breast cancer cells, respectively. To determine which histone modification was involved in miR-125b-1 repression, we evaluated H3K9me3 ( a ) and H3K27me3 ( b ) expression in the promoter regions by chromatin immunoprecipitation
Figure Legend Snippet: H3K9me3 and H3K27me3 are enriched on miR-125b-1 promoters in MDA-MB-231 and MCF7 breast cancer cells, respectively. To determine which histone modification was involved in miR-125b-1 repression, we evaluated H3K9me3 ( a ) and H3K27me3 ( b ) expression in the promoter regions by chromatin immunoprecipitation

Techniques Used: Multiple Displacement Amplification, Modification, Expressing, Chromatin Immunoprecipitation

An inhibitor of EZH2 reactivates miR-125b-1 in MCF7 breast cancer cells. To determine the dose of GSK126 that reduces H3K27me3 levels, we treated the breast cell lines with different GSK126 concentrations and performed Western blot analysis ( a ). b Densitometric analysis of triplicate Western blots. We chose the 1000-nM dose to evaluate pri-miR-125b-1 ( c ) and mature miR-125b expression levels ( d )
Figure Legend Snippet: An inhibitor of EZH2 reactivates miR-125b-1 in MCF7 breast cancer cells. To determine the dose of GSK126 that reduces H3K27me3 levels, we treated the breast cell lines with different GSK126 concentrations and performed Western blot analysis ( a ). b Densitometric analysis of triplicate Western blots. We chose the 1000-nM dose to evaluate pri-miR-125b-1 ( c ) and mature miR-125b expression levels ( d )

Techniques Used: Western Blot, Expressing

Elevated levels of miR-125b-1 affect the expression levels of BAK1. The target BAK1 was selected based on an evaluation of H3K27me3 enrichment in MCF7 cells by ENCODE ( a ). Next, we evaluated BAK1 expression levels by qRT-PCR ( b ). Finally, we determined protein levels by Western blotting ( c , d ). n = 3 *p > 0.05
Figure Legend Snippet: Elevated levels of miR-125b-1 affect the expression levels of BAK1. The target BAK1 was selected based on an evaluation of H3K27me3 enrichment in MCF7 cells by ENCODE ( a ). Next, we evaluated BAK1 expression levels by qRT-PCR ( b ). Finally, we determined protein levels by Western blotting ( c , d ). n = 3 *p > 0.05

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

3) Product Images from "Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae"

Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2014.00277

Genome-wide distribution of histone modifications . Distribution of (A) H3K27me3 and (B) H3K27ac with respect to gene features in A. gambiae midguts. The enrichment or depletion is shown relative to chromatin input. The regions in the map comprise gene bodies flanked by a segment of 200 bp at the 5′ end of TSSs and TTSs. Average profiles across gene regions ±200 bp for each histone modification are shown on top. (C) Heatmap of RNA-seq data showing the level gene expression, as read count, profiled along the region. In all heatmaps (A–C) genes were organized into 3 clusters based on their level of H3K27me3. For H3K27ac, cluster genes are ordered by descent but independent to H3K27me3. The color bars indicate the range of intensities based on ChIP enrichment, from red to blues for higher to low enrichment values. Boxplots showing the mean enrichment of H3K27ac and H3K27me3 by cluster (D) , and mean level of gene expression by gene cluster (E) . Significant pairwise comparisons are indicated by asterisks (see text).
Figure Legend Snippet: Genome-wide distribution of histone modifications . Distribution of (A) H3K27me3 and (B) H3K27ac with respect to gene features in A. gambiae midguts. The enrichment or depletion is shown relative to chromatin input. The regions in the map comprise gene bodies flanked by a segment of 200 bp at the 5′ end of TSSs and TTSs. Average profiles across gene regions ±200 bp for each histone modification are shown on top. (C) Heatmap of RNA-seq data showing the level gene expression, as read count, profiled along the region. In all heatmaps (A–C) genes were organized into 3 clusters based on their level of H3K27me3. For H3K27ac, cluster genes are ordered by descent but independent to H3K27me3. The color bars indicate the range of intensities based on ChIP enrichment, from red to blues for higher to low enrichment values. Boxplots showing the mean enrichment of H3K27ac and H3K27me3 by cluster (D) , and mean level of gene expression by gene cluster (E) . Significant pairwise comparisons are indicated by asterisks (see text).

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

Functional analysis of A. gambiae genes . The graphs show GO terms significantly associated with genes that show significant enrichment or depletion of H3K27ac and H3K27me3 at high levels (see Figure 2 ). Bars corresponds to number of sequences associated with each GO term. In the case of genes marked with H3K27ac from cluster 1 due to the number of records only most significant GO terms are shown (see Table S2 for the complete list).
Figure Legend Snippet: Functional analysis of A. gambiae genes . The graphs show GO terms significantly associated with genes that show significant enrichment or depletion of H3K27ac and H3K27me3 at high levels (see Figure 2 ). Bars corresponds to number of sequences associated with each GO term. In the case of genes marked with H3K27ac from cluster 1 due to the number of records only most significant GO terms are shown (see Table S2 for the complete list).

Techniques Used: Functional Assay

Histone modification levels in selected genes. (A) Enrichment profile of histone PTMs in two Hox genes of A. gambiae (Antennapedia homeotic proteins, AGAP004659-AGAP004660). These are classic examples of Drosophila developmental genes where H3K27me3 is highly enriched and distributed in large domains that encompass both genic and intergenic regions in cells where the Antennapedia gene is silenced. Examples of various histone modification profiles, based on the level of enrichment or depletion in H3K27ac or H3K27me3, in two candidate genes: (B) Rel2 (AGAP006747, GO:0005515), and (C) Dscam (AGAP007092, GO: GO:0005515) (Tables S2, S3). The scale of the tracks is proportional to the number of sequencing reads for each histone modification. Gene expression in terms number of RNA reads mapped to each gene is also displayed.
Figure Legend Snippet: Histone modification levels in selected genes. (A) Enrichment profile of histone PTMs in two Hox genes of A. gambiae (Antennapedia homeotic proteins, AGAP004659-AGAP004660). These are classic examples of Drosophila developmental genes where H3K27me3 is highly enriched and distributed in large domains that encompass both genic and intergenic regions in cells where the Antennapedia gene is silenced. Examples of various histone modification profiles, based on the level of enrichment or depletion in H3K27ac or H3K27me3, in two candidate genes: (B) Rel2 (AGAP006747, GO:0005515), and (C) Dscam (AGAP007092, GO: GO:0005515) (Tables S2, S3). The scale of the tracks is proportional to the number of sequencing reads for each histone modification. Gene expression in terms number of RNA reads mapped to each gene is also displayed.

Techniques Used: Modification, Sequencing, Expressing

4) Product Images from "Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae"

Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2014.00277

Genome-wide distribution of histone modifications . Distribution of (A) H3K27me3 and (B) H3K27ac with respect to gene features in A. gambiae midguts. The enrichment or depletion is shown relative to chromatin input. The regions in the map comprise gene bodies flanked by a segment of 200 bp at the 5′ end of TSSs and TTSs. Average profiles across gene regions ±200 bp for each histone modification are shown on top. (C) Heatmap of RNA-seq data showing the level gene expression, as read count, profiled along the region. In all heatmaps (A–C) genes were organized into 3 clusters based on their level of H3K27me3. For H3K27ac, cluster genes are ordered by descent but independent to H3K27me3. The color bars indicate the range of intensities based on ChIP enrichment, from red to blues for higher to low enrichment values. Boxplots showing the mean enrichment of H3K27ac and H3K27me3 by cluster (D) , and mean level of gene expression by gene cluster (E) . Significant pairwise comparisons are indicated by asterisks (see text).
Figure Legend Snippet: Genome-wide distribution of histone modifications . Distribution of (A) H3K27me3 and (B) H3K27ac with respect to gene features in A. gambiae midguts. The enrichment or depletion is shown relative to chromatin input. The regions in the map comprise gene bodies flanked by a segment of 200 bp at the 5′ end of TSSs and TTSs. Average profiles across gene regions ±200 bp for each histone modification are shown on top. (C) Heatmap of RNA-seq data showing the level gene expression, as read count, profiled along the region. In all heatmaps (A–C) genes were organized into 3 clusters based on their level of H3K27me3. For H3K27ac, cluster genes are ordered by descent but independent to H3K27me3. The color bars indicate the range of intensities based on ChIP enrichment, from red to blues for higher to low enrichment values. Boxplots showing the mean enrichment of H3K27ac and H3K27me3 by cluster (D) , and mean level of gene expression by gene cluster (E) . Significant pairwise comparisons are indicated by asterisks (see text).

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

Functional analysis of A. gambiae genes . The graphs show GO terms significantly associated with genes that show significant enrichment or depletion of H3K27ac and H3K27me3 at high levels (see Figure 2 ). Bars corresponds to number of sequences associated with each GO term. In the case of genes marked with H3K27ac from cluster 1 due to the number of records only most significant GO terms are shown (see Table S2 for the complete list).
Figure Legend Snippet: Functional analysis of A. gambiae genes . The graphs show GO terms significantly associated with genes that show significant enrichment or depletion of H3K27ac and H3K27me3 at high levels (see Figure 2 ). Bars corresponds to number of sequences associated with each GO term. In the case of genes marked with H3K27ac from cluster 1 due to the number of records only most significant GO terms are shown (see Table S2 for the complete list).

Techniques Used: Functional Assay

Histone modification levels in selected genes. (A) Enrichment profile of histone PTMs in two Hox genes of A. gambiae (Antennapedia homeotic proteins, AGAP004659-AGAP004660). These are classic examples of Drosophila developmental genes where H3K27me3 is highly enriched and distributed in large domains that encompass both genic and intergenic regions in cells where the Antennapedia gene is silenced. Examples of various histone modification profiles, based on the level of enrichment or depletion in H3K27ac or H3K27me3, in two candidate genes: (B) Rel2 (AGAP006747, GO:0005515), and (C) Dscam (AGAP007092, GO: GO:0005515) (Tables S2, S3). The scale of the tracks is proportional to the number of sequencing reads for each histone modification. Gene expression in terms number of RNA reads mapped to each gene is also displayed.
Figure Legend Snippet: Histone modification levels in selected genes. (A) Enrichment profile of histone PTMs in two Hox genes of A. gambiae (Antennapedia homeotic proteins, AGAP004659-AGAP004660). These are classic examples of Drosophila developmental genes where H3K27me3 is highly enriched and distributed in large domains that encompass both genic and intergenic regions in cells where the Antennapedia gene is silenced. Examples of various histone modification profiles, based on the level of enrichment or depletion in H3K27ac or H3K27me3, in two candidate genes: (B) Rel2 (AGAP006747, GO:0005515), and (C) Dscam (AGAP007092, GO: GO:0005515) (Tables S2, S3). The scale of the tracks is proportional to the number of sequencing reads for each histone modification. Gene expression in terms number of RNA reads mapped to each gene is also displayed.

Techniques Used: Modification, Sequencing, Expressing

5) Product Images from "Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae"

Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2014.00277

Genome-wide distribution of histone modifications . Distribution of (A) H3K27me3 and (B) H3K27ac with respect to gene features in A. gambiae midguts. The enrichment or depletion is shown relative to chromatin input. The regions in the map comprise gene bodies flanked by a segment of 200 bp at the 5′ end of TSSs and TTSs. Average profiles across gene regions ±200 bp for each histone modification are shown on top. (C) Heatmap of RNA-seq data showing the level gene expression, as read count, profiled along the region. In all heatmaps (A–C) genes were organized into 3 clusters based on their level of H3K27me3. For H3K27ac, cluster genes are ordered by descent but independent to H3K27me3. The color bars indicate the range of intensities based on ChIP enrichment, from red to blues for higher to low enrichment values. Boxplots showing the mean enrichment of H3K27ac and H3K27me3 by cluster (D) , and mean level of gene expression by gene cluster (E) . Significant pairwise comparisons are indicated by asterisks (see text).
Figure Legend Snippet: Genome-wide distribution of histone modifications . Distribution of (A) H3K27me3 and (B) H3K27ac with respect to gene features in A. gambiae midguts. The enrichment or depletion is shown relative to chromatin input. The regions in the map comprise gene bodies flanked by a segment of 200 bp at the 5′ end of TSSs and TTSs. Average profiles across gene regions ±200 bp for each histone modification are shown on top. (C) Heatmap of RNA-seq data showing the level gene expression, as read count, profiled along the region. In all heatmaps (A–C) genes were organized into 3 clusters based on their level of H3K27me3. For H3K27ac, cluster genes are ordered by descent but independent to H3K27me3. The color bars indicate the range of intensities based on ChIP enrichment, from red to blues for higher to low enrichment values. Boxplots showing the mean enrichment of H3K27ac and H3K27me3 by cluster (D) , and mean level of gene expression by gene cluster (E) . Significant pairwise comparisons are indicated by asterisks (see text).

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

Functional analysis of A. gambiae genes . The graphs show GO terms significantly associated with genes that show significant enrichment or depletion of H3K27ac and H3K27me3 at high levels (see Figure 2 ). Bars corresponds to number of sequences associated with each GO term. In the case of genes marked with H3K27ac from cluster 1 due to the number of records only most significant GO terms are shown (see Table S2 for the complete list).
Figure Legend Snippet: Functional analysis of A. gambiae genes . The graphs show GO terms significantly associated with genes that show significant enrichment or depletion of H3K27ac and H3K27me3 at high levels (see Figure 2 ). Bars corresponds to number of sequences associated with each GO term. In the case of genes marked with H3K27ac from cluster 1 due to the number of records only most significant GO terms are shown (see Table S2 for the complete list).

Techniques Used: Functional Assay

Histone modification levels in selected genes. (A) Enrichment profile of histone PTMs in two Hox genes of A. gambiae (Antennapedia homeotic proteins, AGAP004659-AGAP004660). These are classic examples of Drosophila developmental genes where H3K27me3 is highly enriched and distributed in large domains that encompass both genic and intergenic regions in cells where the Antennapedia gene is silenced. Examples of various histone modification profiles, based on the level of enrichment or depletion in H3K27ac or H3K27me3, in two candidate genes: (B) Rel2 (AGAP006747, GO:0005515), and (C) Dscam (AGAP007092, GO: GO:0005515) (Tables S2, S3). The scale of the tracks is proportional to the number of sequencing reads for each histone modification. Gene expression in terms number of RNA reads mapped to each gene is also displayed.
Figure Legend Snippet: Histone modification levels in selected genes. (A) Enrichment profile of histone PTMs in two Hox genes of A. gambiae (Antennapedia homeotic proteins, AGAP004659-AGAP004660). These are classic examples of Drosophila developmental genes where H3K27me3 is highly enriched and distributed in large domains that encompass both genic and intergenic regions in cells where the Antennapedia gene is silenced. Examples of various histone modification profiles, based on the level of enrichment or depletion in H3K27ac or H3K27me3, in two candidate genes: (B) Rel2 (AGAP006747, GO:0005515), and (C) Dscam (AGAP007092, GO: GO:0005515) (Tables S2, S3). The scale of the tracks is proportional to the number of sequencing reads for each histone modification. Gene expression in terms number of RNA reads mapped to each gene is also displayed.

Techniques Used: Modification, Sequencing, Expressing

6) Product Images from "ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity"

Article Title: ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr416

A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).
Figure Legend Snippet: A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).

Techniques Used: Chromatin Immunoprecipitation, Magnetic Cell Separation

Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.
Figure Legend Snippet: Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.

Techniques Used: Expressing

K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).
Figure Legend Snippet: K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).

Techniques Used: Expressing

Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).
Figure Legend Snippet: Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).

Techniques Used:

TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.
Figure Legend Snippet: TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.

Techniques Used: Expressing, Chromatin Immunoprecipitation

The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.
Figure Legend Snippet: The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.

Techniques Used:

7) Product Images from "ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity"

Article Title: ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr416

A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).
Figure Legend Snippet: A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).

Techniques Used: Chromatin Immunoprecipitation, Magnetic Cell Separation

Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.
Figure Legend Snippet: Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.

Techniques Used: Expressing

K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).
Figure Legend Snippet: K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).

Techniques Used: Expressing

Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).
Figure Legend Snippet: Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).

Techniques Used:

TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.
Figure Legend Snippet: TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.

Techniques Used: Expressing, Chromatin Immunoprecipitation

The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.
Figure Legend Snippet: The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.

Techniques Used:

8) Product Images from "ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity"

Article Title: ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr416

A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).
Figure Legend Snippet: A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).

Techniques Used: Chromatin Immunoprecipitation, Magnetic Cell Separation

Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.
Figure Legend Snippet: Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.

Techniques Used: Expressing

K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).
Figure Legend Snippet: K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).

Techniques Used: Expressing

Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).
Figure Legend Snippet: Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).

Techniques Used:

TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.
Figure Legend Snippet: TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.

Techniques Used: Expressing, Chromatin Immunoprecipitation

The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.
Figure Legend Snippet: The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.

Techniques Used:

9) Product Images from "A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas"

Article Title: A sensitive and specific histopathologic prognostic marker for H3F3A K27M mutant pediatric glioblastomas

Journal: Acta Neuropathologica

doi: 10.1007/s00401-014-1338-3

Comparison of H3K27me3 and H3.3 K27M in adult brain tumors. a , b Representative images of H3K27me3 (40×, a ) and H3.3 K27M (40×, b ) in diffuse astrocytoma. c , d Representative images of H3K27me3 (40×, c ) and H3.3 K27M (40×, d ) in anaplastic oligodendroglioma e and f. Representative images of H3K27me3 (40×, e ) and H3.3 K27M (40×, f ) in anaplastic astrocytoma. g , h Representative images of H3K27me3 (40×, g ) and H3.3 K27M (40×, h ) in adult GBM
Figure Legend Snippet: Comparison of H3K27me3 and H3.3 K27M in adult brain tumors. a , b Representative images of H3K27me3 (40×, a ) and H3.3 K27M (40×, b ) in diffuse astrocytoma. c , d Representative images of H3K27me3 (40×, c ) and H3.3 K27M (40×, d ) in anaplastic oligodendroglioma e and f. Representative images of H3K27me3 (40×, e ) and H3.3 K27M (40×, f ) in anaplastic astrocytoma. g , h Representative images of H3K27me3 (40×, g ) and H3.3 K27M (40×, h ) in adult GBM

Techniques Used:

Comparison of H3K27me3 and H3.3 K27M in non-neoplastic brain tissues. a , b Representative images of H3K27me3 (5×, a , inset 40×) and H3.3 K27M (5×, b , inset 40×) in focal cortical dysplasia (FCD). c , d Representative images of H3K27me3 (10×, c , inset 40×) and H3.3 K27M (10×, d , inset 40×) in hippocampal (Hip) sclerosis. e , f Representative images of H3K27me3 (5×, e , inset 40×) and H3.3 K27M (5×, f , inset 40×) in hemorrhage. g , h Representative images of H3K27me3 (5×, g , inset 40×) and H3.3 K27M (5×, h , inset 40×) in abscess, inset shows abscess wall. i , j Representative images of H3K27me3 (5×, i , inset 40×) and H3.3 K27M (5×, j , inset 40×) in infarction, inset shows macrophages
Figure Legend Snippet: Comparison of H3K27me3 and H3.3 K27M in non-neoplastic brain tissues. a , b Representative images of H3K27me3 (5×, a , inset 40×) and H3.3 K27M (5×, b , inset 40×) in focal cortical dysplasia (FCD). c , d Representative images of H3K27me3 (10×, c , inset 40×) and H3.3 K27M (10×, d , inset 40×) in hippocampal (Hip) sclerosis. e , f Representative images of H3K27me3 (5×, e , inset 40×) and H3.3 K27M (5×, f , inset 40×) in hemorrhage. g , h Representative images of H3K27me3 (5×, g , inset 40×) and H3.3 K27M (5×, h , inset 40×) in abscess, inset shows abscess wall. i , j Representative images of H3K27me3 (5×, i , inset 40×) and H3.3 K27M (5×, j , inset 40×) in infarction, inset shows macrophages

Techniques Used:

Comparison of immunostaining for H3K27me3 and H3.3 K27M in wild-type and H3F3A K27M mutant pediatric GBM. a , b Representative images from a H3F3A K27M wild-type tumor, H3K27me3 (40×, a ) and H3.3 K27M (40×, b ). c – f . Representative images from two different H3F3A K27M mutant tumors, H3K27me3 (40×, c and 60×, e ) and H3.3 K27M (40×, d and 60×, f ). Arrowheads indicate endothelial cells in blood vessels
Figure Legend Snippet: Comparison of immunostaining for H3K27me3 and H3.3 K27M in wild-type and H3F3A K27M mutant pediatric GBM. a , b Representative images from a H3F3A K27M wild-type tumor, H3K27me3 (40×, a ) and H3.3 K27M (40×, b ). c – f . Representative images from two different H3F3A K27M mutant tumors, H3K27me3 (40×, c and 60×, e ) and H3.3 K27M (40×, d and 60×, f ). Arrowheads indicate endothelial cells in blood vessels

Techniques Used: Immunostaining, Mutagenesis

Quantification of H3K27me3 and H3.3 K27M immunostaining in adult and pediatric brain tumors and survival analyses in pediatric high-grade gliomas. a , c Quantification of H3K27me3 ( a ) and H3.3 K27M ( c ) IHC in pediatric brain tumors. *** p
Figure Legend Snippet: Quantification of H3K27me3 and H3.3 K27M immunostaining in adult and pediatric brain tumors and survival analyses in pediatric high-grade gliomas. a , c Quantification of H3K27me3 ( a ) and H3.3 K27M ( c ) IHC in pediatric brain tumors. *** p

Techniques Used: Immunostaining, Immunohistochemistry

Comparison of H3K27me3 and H3.3 K27M in pediatric brain tumors. a , b Representative images of H3K27me3 (40×, a ) and H3.3 K27M (40×, b ) in choroid plexus papilloma (CPC). c , d Representative images of H3K27me3 (40×, c ) and H3.3 K27M (40×, d ) in meningioma. e , f Representative images of H3K27me3 (40×, e ) and H3.3 K27M (40×, f ) in craniopharyngioma. g , h Representative images of H3K27me3 (40×, g ) and H3.3 K27M (40×, h ) in neurocytoma
Figure Legend Snippet: Comparison of H3K27me3 and H3.3 K27M in pediatric brain tumors. a , b Representative images of H3K27me3 (40×, a ) and H3.3 K27M (40×, b ) in choroid plexus papilloma (CPC). c , d Representative images of H3K27me3 (40×, c ) and H3.3 K27M (40×, d ) in meningioma. e , f Representative images of H3K27me3 (40×, e ) and H3.3 K27M (40×, f ) in craniopharyngioma. g , h Representative images of H3K27me3 (40×, g ) and H3.3 K27M (40×, h ) in neurocytoma

Techniques Used:

Comparison of H3K27me3 and H3.3 K27M in CNS embryonal tumors. a , b Representative images of H3K27me3 (40×, a ) and H3.3 K27M (40×, b ) in medulloblastoma. c , d Representative images of H3K27me3 (40×, c ) and H3.3 K27M (40×, d ) in an AT/RT case with high H3K27me3. e , f Representative images of H3K27me3 (40×, e ) and H3.3 K27M (40×, f ) in subset of AT/RT with low H3K27me3
Figure Legend Snippet: Comparison of H3K27me3 and H3.3 K27M in CNS embryonal tumors. a , b Representative images of H3K27me3 (40×, a ) and H3.3 K27M (40×, b ) in medulloblastoma. c , d Representative images of H3K27me3 (40×, c ) and H3.3 K27M (40×, d ) in an AT/RT case with high H3K27me3. e , f Representative images of H3K27me3 (40×, e ) and H3.3 K27M (40×, f ) in subset of AT/RT with low H3K27me3

Techniques Used:

10) Product Images from "A comparison of control samples for ChIP-seq of histone modifications"

Article Title: A comparison of control samples for ChIP-seq of histone modifications

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2014.00329

Peak scores from a MACS analysis of enriched regions . H3 and H3K27me3 replicates are merged before analysis. (A) Distributions of scores of peaks in WCE and H3. The peaks in each control are split into peaks that overlap between the two background samples (marked with “o”) or do not overlap (marked with “n”). (B) Scatter plot of scores of overlapping peaks from the WCE and H3 sample. (C) Distributions of scores of peaks in H3K27me3 with either WCE or H3 as a control. Labels on the x-axis refer to the control sample. (D) Scatter plot of scores of overlapping peaks from H3K27me3 with either WCE or H3 as control.
Figure Legend Snippet: Peak scores from a MACS analysis of enriched regions . H3 and H3K27me3 replicates are merged before analysis. (A) Distributions of scores of peaks in WCE and H3. The peaks in each control are split into peaks that overlap between the two background samples (marked with “o”) or do not overlap (marked with “n”). (B) Scatter plot of scores of overlapping peaks from the WCE and H3 sample. (C) Distributions of scores of peaks in H3K27me3 with either WCE or H3 as a control. Labels on the x-axis refer to the control sample. (D) Scatter plot of scores of overlapping peaks from H3K27me3 with either WCE or H3 as control.

Techniques Used: Magnetic Cell Separation

Average read density in RPKM over genes (A,C,E) and around the transcription start site (B,D,F) for H3K27me3 (A,B), WCE (C,D), and H3 (E,F) . Ratio of average read densities of H3K27me3 to the background sample over genes (G) and around the TSS (H) . The genes are divided up into four equal groups based on expression (RPKM), shown by thin lines for lowest expression and thicker lines for higher expression. The two H3 samples and three H3K27me3 samples are merged.
Figure Legend Snippet: Average read density in RPKM over genes (A,C,E) and around the transcription start site (B,D,F) for H3K27me3 (A,B), WCE (C,D), and H3 (E,F) . Ratio of average read densities of H3K27me3 to the background sample over genes (G) and around the TSS (H) . The genes are divided up into four equal groups based on expression (RPKM), shown by thin lines for lowest expression and thicker lines for higher expression. The two H3 samples and three H3K27me3 samples are merged.

Techniques Used: Expressing

11) Product Images from "Integrative epigenomic mapping defines four main chromatin states in Arabidopsis"

Article Title: Integrative epigenomic mapping defines four main chromatin states in Arabidopsis

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.103

Analysis of genes co-marked with H3K27me3 and H3K4me3 in whole seedlings. ( A ) The 3433 genes co-marked in whole seedlings were split into different classes according to their marking in roots (R; this study) and aerial parts (AP; Oh et al, 2008 ). ‘Others' indicate genes with other marking patterns in the two plant parts. This class, which is not expected based on the co-marking observed in whole seedlings, can be explained in part by the fact that the different data sets were not all generated using the same conditions and methodologies. ( B ) Expression analysis in roots and shoots ( Schmid et al, 2005 ) for the 284 genes showing opposite marking in roots and aerial parts. Brown dots indicate genes with H3K4me3 in roots and H3K27me3 in aerial parts, green dots indicate genes with the opposite marking pattern. ( C ) Expression analysis in roots and shoots ( Schmid et al, 2005 ) for the 224 genes showing persistent co-marking in roots and aerial parts.
Figure Legend Snippet: Analysis of genes co-marked with H3K27me3 and H3K4me3 in whole seedlings. ( A ) The 3433 genes co-marked in whole seedlings were split into different classes according to their marking in roots (R; this study) and aerial parts (AP; Oh et al, 2008 ). ‘Others' indicate genes with other marking patterns in the two plant parts. This class, which is not expected based on the co-marking observed in whole seedlings, can be explained in part by the fact that the different data sets were not all generated using the same conditions and methodologies. ( B ) Expression analysis in roots and shoots ( Schmid et al, 2005 ) for the 284 genes showing opposite marking in roots and aerial parts. Brown dots indicate genes with H3K4me3 in roots and H3K27me3 in aerial parts, green dots indicate genes with the opposite marking pattern. ( C ) Expression analysis in roots and shoots ( Schmid et al, 2005 ) for the 224 genes showing persistent co-marking in roots and aerial parts.

Techniques Used: Generated, Expressing

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: 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: 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 "DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter"

Article Title: DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddr356

Active histone modifications mark active non-CpG promoters; inactive non-CpG island promoters lack inactive histone marks. ChIP was performed using antibodies for H3K4me3, acetylated H3, H2A.Z, phosphorylated Pol II, H3K27me3 and H3K9me3. Y -axis represents IP-IgG/Input-IgG of two biological experiments + the range between the two experiments. FAM84a and CDKN2A Exon 2 are used as positive controls for H3K27me3 and H3K9me3, respectively.
Figure Legend Snippet: Active histone modifications mark active non-CpG promoters; inactive non-CpG island promoters lack inactive histone marks. ChIP was performed using antibodies for H3K4me3, acetylated H3, H2A.Z, phosphorylated Pol II, H3K27me3 and H3K9me3. Y -axis represents IP-IgG/Input-IgG of two biological experiments + the range between the two experiments. FAM84a and CDKN2A Exon 2 are used as positive controls for H3K27me3 and H3K9me3, respectively.

Techniques Used: Chromatin Immunoprecipitation

15) Product Images from "Damage-responsive elements in Drosophila regeneration"

Article Title: Damage-responsive elements in Drosophila regeneration

Journal: Genome Research

doi: 10.1101/gr.233098.117

Accessible chromatin landscape after induction of cell death. ( A ) Schematic overview of peak distribution in the genome. ( B ) Bar plot showing the number of more accessible peaks at each time point falling in each genomic region: first intron, proximal, and distal. ( C ) Genome Browser screenshot and schematic drawing of iDRRE and eDRRE. ( D ) Heatmaps showing nucleosome-free (NF) and mononucleosome (MN) enrichment around ±500 bp from the peak summit of DRREs at the early stage of control and regeneration. Sites are ordered by genomic distribution (shown on the left ), and by peak height based on ATAC-seq regeneration sample. ( E ) Venn diagrams showing the intersection of H3K4me1, H3K27ac, and Pol II at DRREs in regeneration. ( F ) Average profile of H3K4me1, H3K27ac, and Pol II at DRREs. A solid line denotes DRREs with the presence of at least one ChIP-seq signal; and a dashed line denotes the absence of any ChIP-seq signal. ( G ) Genome Browser screenshot showing ATAC-seq and ChIP-seq profiles (control and regeneration) of the DRREs tested by ChIP-qPCR. ( H ) ChIP-qPCR analysis of H3K4me1, H3K27ac, H3K27me3, and Pol II-ser5P on individual DRREs at the early stage. ChIP results are presented as fold change enrichment between control and regeneration. Error bars represent the standard error of the mean from two biological replicates.
Figure Legend Snippet: Accessible chromatin landscape after induction of cell death. ( A ) Schematic overview of peak distribution in the genome. ( B ) Bar plot showing the number of more accessible peaks at each time point falling in each genomic region: first intron, proximal, and distal. ( C ) Genome Browser screenshot and schematic drawing of iDRRE and eDRRE. ( D ) Heatmaps showing nucleosome-free (NF) and mononucleosome (MN) enrichment around ±500 bp from the peak summit of DRREs at the early stage of control and regeneration. Sites are ordered by genomic distribution (shown on the left ), and by peak height based on ATAC-seq regeneration sample. ( E ) Venn diagrams showing the intersection of H3K4me1, H3K27ac, and Pol II at DRREs in regeneration. ( F ) Average profile of H3K4me1, H3K27ac, and Pol II at DRREs. A solid line denotes DRREs with the presence of at least one ChIP-seq signal; and a dashed line denotes the absence of any ChIP-seq signal. ( G ) Genome Browser screenshot showing ATAC-seq and ChIP-seq profiles (control and regeneration) of the DRREs tested by ChIP-qPCR. ( H ) ChIP-qPCR analysis of H3K4me1, H3K27ac, H3K27me3, and Pol II-ser5P on individual DRREs at the early stage. ChIP results are presented as fold change enrichment between control and regeneration. Error bars represent the standard error of the mean from two biological replicates.

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

16) Product Images from "The Scaffold attachment factor b1 (Safb1) regulates myogenic differentiation by facilitating the transition of myogenic gene chromatin from a repressed to an activated state"

Article Title: The Scaffold attachment factor b1 (Safb1) regulates myogenic differentiation by facilitating the transition of myogenic gene chromatin from a repressed to an activated state

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt285

shRNA-mediated knockdown of Safb1 does not alter MyoD recruitment to myogenic sequences but increases Polycomb repressive marks. Proliferating (GM) or 72 h differentiated (DM) C2C12 cells expressing control shRNA or shRNA against Safb1 were fixed and processed for ChIP assays using ( A ) antibodies against MyoD and Safb1 (top panel) or ( B ) antibodies against Ezh2 and H3K27me3 at the indicated gene regulatory sequences. H3K27me3 enrichment is presented on the right-hand y-axis in the bottom panel. Data represent the mean of three independent experiments ± SEM. t test analyses indicate that when comparing binding in sh-Safb1-treated differentiated cells with sh-Control differentiated cells, the binding of Safb1 was significantly reduced ( P
Figure Legend Snippet: shRNA-mediated knockdown of Safb1 does not alter MyoD recruitment to myogenic sequences but increases Polycomb repressive marks. Proliferating (GM) or 72 h differentiated (DM) C2C12 cells expressing control shRNA or shRNA against Safb1 were fixed and processed for ChIP assays using ( A ) antibodies against MyoD and Safb1 (top panel) or ( B ) antibodies against Ezh2 and H3K27me3 at the indicated gene regulatory sequences. H3K27me3 enrichment is presented on the right-hand y-axis in the bottom panel. Data represent the mean of three independent experiments ± SEM. t test analyses indicate that when comparing binding in sh-Safb1-treated differentiated cells with sh-Control differentiated cells, the binding of Safb1 was significantly reduced ( P

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

17) Product Images from "Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2"

Article Title: Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2

Journal: Nature Communications

doi: 10.1038/ncomms14922

Loss of histone H3 lysine 27 trimethylation accompanies SCC transition. ( a ) Western blotting analysis performed on whole-cell extracts from tumours of the indicated genotypes and histologies. Histone H3 lysine 27 tri-methylation (H3K27me3) is markedly lower in SCC lesions; total histone H3 is the loading control. ( b ) Western blotting analysis performed on whole-cell extracts from tumours of the indicated genotypes and histologies. p63 and LKB1 confirm the histologies and genotypes of the lysates, and while EZH2 in more highly expressed in SCC tumours, the essential PRC2 subunit EED is absent in the SCC lesions. β-Actin is the loading control. ( c ) Immunohistochemisty for H3K27me3 on a panel of mouse lung tumours of the indicated histologies from KRAS /Lkb1 , Pten/Lkb1/p53 , and Pten/Lkb1 mice, scale bar, 50 μm. ( d ) Quantification of nuclear staining by dot analysis with Nikon software; data are mean±s.e.m. measured on serially stained sections, n =6–10. ( e ) Immunohistochemisty for H3K27me3 on a panel of human mixed adenocarcionomas, pure ADC and pure SCCs. Scale bar, 50 μm. ( f ) Quantification of nuclear staining, for H3K27me3 n =6 ADSCC, 14 ADC, 9 SCC, for EZH2 n =6 ADSCC, 7 ADC, 5 SCC, data are mean±s.e.m. measured on serially stained sections. P values represent 2 tailed t-test. See also Supplementary Fig. 3a–c .
Figure Legend Snippet: Loss of histone H3 lysine 27 trimethylation accompanies SCC transition. ( a ) Western blotting analysis performed on whole-cell extracts from tumours of the indicated genotypes and histologies. Histone H3 lysine 27 tri-methylation (H3K27me3) is markedly lower in SCC lesions; total histone H3 is the loading control. ( b ) Western blotting analysis performed on whole-cell extracts from tumours of the indicated genotypes and histologies. p63 and LKB1 confirm the histologies and genotypes of the lysates, and while EZH2 in more highly expressed in SCC tumours, the essential PRC2 subunit EED is absent in the SCC lesions. β-Actin is the loading control. ( c ) Immunohistochemisty for H3K27me3 on a panel of mouse lung tumours of the indicated histologies from KRAS /Lkb1 , Pten/Lkb1/p53 , and Pten/Lkb1 mice, scale bar, 50 μm. ( d ) Quantification of nuclear staining by dot analysis with Nikon software; data are mean±s.e.m. measured on serially stained sections, n =6–10. ( e ) Immunohistochemisty for H3K27me3 on a panel of human mixed adenocarcionomas, pure ADC and pure SCCs. Scale bar, 50 μm. ( f ) Quantification of nuclear staining, for H3K27me3 n =6 ADSCC, 14 ADC, 9 SCC, for EZH2 n =6 ADSCC, 7 ADC, 5 SCC, data are mean±s.e.m. measured on serially stained sections. P values represent 2 tailed t-test. See also Supplementary Fig. 3a–c .

Techniques Used: Western Blot, Methylation, Mouse Assay, Staining, Software

Chromatin landscapes of ADC and SCCs reveal de-repression of squamous genes. ( a ) Heatmap depicting global analysis of H3K4me3 marked chromatin in ADC and SCC from KRAS /Lkb1 mice. ( b ) Genome browser snapshots of the squamous loci Sox2 , Ngfr and Krt6b/6a/5 with the indicated ChIP-sequencing peaks for H3K27ac, H3K27me3 and H3K4me3 in the ADC (blue) and SCC (red) KRAS /Lkb1 tumour samples. ( c ) GREAT analysis of genes adjacent to H3K4me3-enriched sites specific to SCC showed that this gene set is enriched for genes (from MSigDB) that are normally repressed by Polycomb in other cell types. Graphed as -log10 ( P value represents hypergeometric probability test.) Level of H3K27me3 marks at all loci enriched for H3K27ac (black), loci with H3K27ac unique to ADC (blue) or loci with H3K27ac unique to SCC (red) in either ADC (left panel) or SCC (right panel) from KRAS /Lkb1 mice. See also Supplementary Fig. 5a–c .
Figure Legend Snippet: Chromatin landscapes of ADC and SCCs reveal de-repression of squamous genes. ( a ) Heatmap depicting global analysis of H3K4me3 marked chromatin in ADC and SCC from KRAS /Lkb1 mice. ( b ) Genome browser snapshots of the squamous loci Sox2 , Ngfr and Krt6b/6a/5 with the indicated ChIP-sequencing peaks for H3K27ac, H3K27me3 and H3K4me3 in the ADC (blue) and SCC (red) KRAS /Lkb1 tumour samples. ( c ) GREAT analysis of genes adjacent to H3K4me3-enriched sites specific to SCC showed that this gene set is enriched for genes (from MSigDB) that are normally repressed by Polycomb in other cell types. Graphed as -log10 ( P value represents hypergeometric probability test.) Level of H3K27me3 marks at all loci enriched for H3K27ac (black), loci with H3K27ac unique to ADC (blue) or loci with H3K27ac unique to SCC (red) in either ADC (left panel) or SCC (right panel) from KRAS /Lkb1 mice. See also Supplementary Fig. 5a–c .

Techniques Used: Mouse Assay, Chromatin Immunoprecipitation, Sequencing

Lkb1 deletion drives squamous transition of club cell- and BASC-derived KRAS+ populations. ( a ) Schematic of Lkb1 deletion in three-dimensional KRAS+ cultures derived from non-basal tracheal cells, bronchioalveolar stem cells (BASCs) or alveolar type II (AT2) cells. ( b ) Representative brightfield images of KRAS+ colonies from alveolar type II cells treated with placebo or 4-hydroxy tamoxifen for 7 days, scale bar, 200 μm. ( c ) RT-qPCR for Lkb1 and Sox2 in tumour organoid 3D cultures treated with 100 nM tamoxifen for 9–12 days, mean±s.e.m. on log 2 scale is graphed, n varies by sample. ( d ) Representative haematoxylin and eosin staining from non-basal cell derived subcutaneous tumour (left) and BASC-derived orthotopic tumours (right), tumour histologies are indicated, scale bar, 100 μm. ( e ) Model: Here we combined Cre and FlpO recombinase technologies to temporally delete Lkb1 in established KRAS-driven lung adenocarcinomas. Serially transplanted KRAS+ adenocarcinoma could transdifferentiate into squamous disease when Lkb1 was deleted in the transplanted tumours. The Polycomb Repressive Complex 2 (PRC2), which represses genes through the histone H3K27me3 mark, was abrogated in the KRAS /Lkb1 squamous tumours through loss of expression of the EED subunit. This led to derepression of key squamous genes including Sox2 , ΔNp63 and Ngfr . See also Supplementary Fig. 7a–c .
Figure Legend Snippet: Lkb1 deletion drives squamous transition of club cell- and BASC-derived KRAS+ populations. ( a ) Schematic of Lkb1 deletion in three-dimensional KRAS+ cultures derived from non-basal tracheal cells, bronchioalveolar stem cells (BASCs) or alveolar type II (AT2) cells. ( b ) Representative brightfield images of KRAS+ colonies from alveolar type II cells treated with placebo or 4-hydroxy tamoxifen for 7 days, scale bar, 200 μm. ( c ) RT-qPCR for Lkb1 and Sox2 in tumour organoid 3D cultures treated with 100 nM tamoxifen for 9–12 days, mean±s.e.m. on log 2 scale is graphed, n varies by sample. ( d ) Representative haematoxylin and eosin staining from non-basal cell derived subcutaneous tumour (left) and BASC-derived orthotopic tumours (right), tumour histologies are indicated, scale bar, 100 μm. ( e ) Model: Here we combined Cre and FlpO recombinase technologies to temporally delete Lkb1 in established KRAS-driven lung adenocarcinomas. Serially transplanted KRAS+ adenocarcinoma could transdifferentiate into squamous disease when Lkb1 was deleted in the transplanted tumours. The Polycomb Repressive Complex 2 (PRC2), which represses genes through the histone H3K27me3 mark, was abrogated in the KRAS /Lkb1 squamous tumours through loss of expression of the EED subunit. This led to derepression of key squamous genes including Sox2 , ΔNp63 and Ngfr . See also Supplementary Fig. 7a–c .

Techniques Used: Derivative Assay, Quantitative RT-PCR, Staining, Expressing

18) Product Images from "EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers"

Article Title: EZH2-Mediated H3K27me3 Is Involved in Epigenetic Repression of Deleted in Liver Cancer 1 in Human Cancers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068226

Promoter DNA methylation and histone modification of DLC1 in HCC cell lines. ( A ) Schematic diagram showing CpG island located at DLC1 locus. DNA methylation status of 45 CpG dinucleotides (BS-1 region included 35 CpG dinulceotides and BS-2 region included 10 CpG dinucleotides) were subject to bisulfite sequencing analysis. MIHA showed complete unmethylation, MHCC97L showed partial methylation and SMMC-7721 showed complete methylation. Open circle represents unmethylated CpG dinucleotides and closed circle represents methylated CpG dinucleotides. Each column represents a single clone being sequenced. ( B ) RT-PCR (upper panel) and qRT-PCR (bottom panel) showing detectable DLC1 mRNA expression in MIHA, but not in MHCC97L and SMMC-7721 cells. ( C ) Chromatin immunoprecipitation (ChIP) assay coupled with qPCR (qChIP) analysis revealed the relative enrichment of H3K9me3 and H3K27me3 on DLC1 promoter region in MIHA, MHCC97L and SMMC-7721 cells. Fold of enrichment of ChIP assay was calculated with reference to IgG control after normalized with the input DNA. Data are represented as mean ± SEM from three independent experiments.
Figure Legend Snippet: Promoter DNA methylation and histone modification of DLC1 in HCC cell lines. ( A ) Schematic diagram showing CpG island located at DLC1 locus. DNA methylation status of 45 CpG dinucleotides (BS-1 region included 35 CpG dinulceotides and BS-2 region included 10 CpG dinucleotides) were subject to bisulfite sequencing analysis. MIHA showed complete unmethylation, MHCC97L showed partial methylation and SMMC-7721 showed complete methylation. Open circle represents unmethylated CpG dinucleotides and closed circle represents methylated CpG dinucleotides. Each column represents a single clone being sequenced. ( B ) RT-PCR (upper panel) and qRT-PCR (bottom panel) showing detectable DLC1 mRNA expression in MIHA, but not in MHCC97L and SMMC-7721 cells. ( C ) Chromatin immunoprecipitation (ChIP) assay coupled with qPCR (qChIP) analysis revealed the relative enrichment of H3K9me3 and H3K27me3 on DLC1 promoter region in MIHA, MHCC97L and SMMC-7721 cells. Fold of enrichment of ChIP assay was calculated with reference to IgG control after normalized with the input DNA. Data are represented as mean ± SEM from three independent experiments.

Techniques Used: DNA Methylation Assay, Modification, Methylation Sequencing, Methylation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

EZH2-mediated H3K27me3 was involved in epigenetic repression of DLC1 in HCC and multiple other human cancers. ( A ) DLC1 was transcriptionally induced upon stable knockdown of EZH2 in MHCC97L cells. ( B ) qChIP analysis confirmed the depletion of H3K27me3 enrichment on DLC1 promoter upon EZH2 knockdown in MHCC97L cells. Data are represented as mean ± SEM from three independent experiments. ( C ) DLC1 was transcriptionally repressed in Huh7 cells after transient overexpression of GFP-EZH2. ( D ) qChIP analysis revealed a concomitant enrichment of EZH2 at DLC1’s promoter locus upon GFP-EZH2 overexpression in Huh7 cells. Data are represented as mean ± SEM from three independent experiments. ( E ) EZH2 and H3K27me3 expression was reduced upon 1µM DZNep treatment for 48 hours in MHCC97L cells (left panel). DMSO was used as mock treatment. Pan H3 and α-tubulin were loading control of the immunoblot. DZNep treatment transcriptionally induced DLC1 expression in MHCC97L as indicated by qPCR analysis (right panel). ( F ) Different human cancer cells, including the nasopharyngeal carcinoma cell line CNE2, the colorectal carcinoma cell line HCT116 and the cervical adenocarcinoma cell line HeLa were examined for DLC1 re-expression upon DZNep treatment. Treatment of cells with 10µM DZNep reactivated DLC1 expression. P -values obtained from t -test.
Figure Legend Snippet: EZH2-mediated H3K27me3 was involved in epigenetic repression of DLC1 in HCC and multiple other human cancers. ( A ) DLC1 was transcriptionally induced upon stable knockdown of EZH2 in MHCC97L cells. ( B ) qChIP analysis confirmed the depletion of H3K27me3 enrichment on DLC1 promoter upon EZH2 knockdown in MHCC97L cells. Data are represented as mean ± SEM from three independent experiments. ( C ) DLC1 was transcriptionally repressed in Huh7 cells after transient overexpression of GFP-EZH2. ( D ) qChIP analysis revealed a concomitant enrichment of EZH2 at DLC1’s promoter locus upon GFP-EZH2 overexpression in Huh7 cells. Data are represented as mean ± SEM from three independent experiments. ( E ) EZH2 and H3K27me3 expression was reduced upon 1µM DZNep treatment for 48 hours in MHCC97L cells (left panel). DMSO was used as mock treatment. Pan H3 and α-tubulin were loading control of the immunoblot. DZNep treatment transcriptionally induced DLC1 expression in MHCC97L as indicated by qPCR analysis (right panel). ( F ) Different human cancer cells, including the nasopharyngeal carcinoma cell line CNE2, the colorectal carcinoma cell line HCT116 and the cervical adenocarcinoma cell line HeLa were examined for DLC1 re-expression upon DZNep treatment. Treatment of cells with 10µM DZNep reactivated DLC1 expression. P -values obtained from t -test.

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

19) Product Images from "Patient-derived DIPG cells preserve stem-like characteristics and generate orthotopic tumors"

Article Title: Patient-derived DIPG cells preserve stem-like characteristics and generate orthotopic tumors

Journal: Oncotarget

doi: 10.18632/oncotarget.19656

Histopathological characteristic of TT10603 patient specimen and mouse xenograft (20X) H E staining of all xenografts revealed high cell density similar to human DIPG tissues, representing the characteristic of high grade glioma. Immunohistochemistry showed that all xenografts as well as their human DIPG counterparts were positive for Nestin, Sox2, Olig2, PDGFRα and GFAP. Notably, serial xenograft demonstrated higher percent of immunoreactivity for these markers than direct orthotopic xenograft. Most tumor cells of xenografts were negative in H3K27me3 staining, indicating that they retained the loss of trimethylation at position 27 of histone H3 from human tumor tissues. (Scale bars: 200μm).
Figure Legend Snippet: Histopathological characteristic of TT10603 patient specimen and mouse xenograft (20X) H E staining of all xenografts revealed high cell density similar to human DIPG tissues, representing the characteristic of high grade glioma. Immunohistochemistry showed that all xenografts as well as their human DIPG counterparts were positive for Nestin, Sox2, Olig2, PDGFRα and GFAP. Notably, serial xenograft demonstrated higher percent of immunoreactivity for these markers than direct orthotopic xenograft. Most tumor cells of xenografts were negative in H3K27me3 staining, indicating that they retained the loss of trimethylation at position 27 of histone H3 from human tumor tissues. (Scale bars: 200μm).

Techniques Used: Staining, Immunohistochemistry

20) Product Images from "Lnc RNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination"

Article Title: Lnc RNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination

Journal: EMBO Reports

doi: 10.15252/embr.201643668

GAS 5 suppresses IRF 4 transcription by binding PRC 2 and inhibiting microglial M2 polarization RNA IP analysis of the binding between EZH2 and GAS5, n = 3 experiments. RNA pull‐down analysis of the binding between EZH2 and GAS5. Quantitative PCR analysis of M1 and M2 markers in microglia transduced with the EZH2i lentivirus versus the control, n ≥ 4 experiments. ChIP analysis of microglia transduced with the CtrlOE or GAS5OE lentivirus. A relatively high enrichment was detected on the IRF4 promoter regions in MG GAS5OE versus the control using the anti‐EZH2 antibody, n = 3 experiments. ChIRP analysis of the binding between the IRF4 promoter and GAS5, n = 3 experiments. IGF1 served as a negative control. ChIP analysis of microglia transduced with the CtrlOE/GAS5OE (F) or Ctrli/GAS5i (G) lentivirus on the IRF4 promoter regions using the anti‐H3K27me3 antibody, n = 3 experiments. Quantitative PCR analysis of IRF4 in microglia transduced with the GAS5OE (H), GAS5i (I) or EZH2i (J) lentivirus, n ≥ 3 experiments per group. Western blotting analysis of IRF4 in microglia transduced with the GAS5OE (K), GAS5i (L), or EZH2i (M) lentivirus, n ≥ 3 experiments per group. Data information: * P
Figure Legend Snippet: GAS 5 suppresses IRF 4 transcription by binding PRC 2 and inhibiting microglial M2 polarization RNA IP analysis of the binding between EZH2 and GAS5, n = 3 experiments. RNA pull‐down analysis of the binding between EZH2 and GAS5. Quantitative PCR analysis of M1 and M2 markers in microglia transduced with the EZH2i lentivirus versus the control, n ≥ 4 experiments. ChIP analysis of microglia transduced with the CtrlOE or GAS5OE lentivirus. A relatively high enrichment was detected on the IRF4 promoter regions in MG GAS5OE versus the control using the anti‐EZH2 antibody, n = 3 experiments. ChIRP analysis of the binding between the IRF4 promoter and GAS5, n = 3 experiments. IGF1 served as a negative control. ChIP analysis of microglia transduced with the CtrlOE/GAS5OE (F) or Ctrli/GAS5i (G) lentivirus on the IRF4 promoter regions using the anti‐H3K27me3 antibody, n = 3 experiments. Quantitative PCR analysis of IRF4 in microglia transduced with the GAS5OE (H), GAS5i (I) or EZH2i (J) lentivirus, n ≥ 3 experiments per group. Western blotting analysis of IRF4 in microglia transduced with the GAS5OE (K), GAS5i (L), or EZH2i (M) lentivirus, n ≥ 3 experiments per group. Data information: * P

Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Transduction, Chromatin Immunoprecipitation, Negative Control, Western Blot

21) Product Images from "Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations"

Article Title: Investigation into the role of the germline epigenome in the transmission of glucocorticoid-programmed effects across generations

Journal: Genome Biology

doi: 10.1186/s13059-018-1422-4

Dex treatment does not induce detectable changes in histone methylation. a Enrichment of methylated H3K IP over unmodified H3 IP for annotated features. Only features with at least 1.2-fold enrichment or depletion in one or more sample are shown. Error bars represent range for the three replicates. No significant differences were observed (Student’s t-test, Benjamini-Hochberg adjusted p -value, 10% FDR). b Enrichment of methylated H3K IP over unmodified H3 IP centred over transcription start sites ( TSS ) and transcription termination site ( TTS ) ± 3000 bp. Each replicate is shown as a separate line. c Hierarchical clustering of samples and peaks by average enrichment of methylated H3K IP over unmodified H3 IP. Samples clearly cluster by histone mark but do not cluster by Veh vs Dex for any histone mark. Vertical colour bar indicates six clusters following k-means clustering. Gold and purple clusters show higher H3K9me3 enrichment. Bivalent enrichment observed for H3K4me3 and H3K9me3 ( turquoise ) and H3K4me3 and H3K27me3 ( green ). Blue cluster represents inactive enhancers marked by H3K4me1 and H3K9me3
Figure Legend Snippet: Dex treatment does not induce detectable changes in histone methylation. a Enrichment of methylated H3K IP over unmodified H3 IP for annotated features. Only features with at least 1.2-fold enrichment or depletion in one or more sample are shown. Error bars represent range for the three replicates. No significant differences were observed (Student’s t-test, Benjamini-Hochberg adjusted p -value, 10% FDR). b Enrichment of methylated H3K IP over unmodified H3 IP centred over transcription start sites ( TSS ) and transcription termination site ( TTS ) ± 3000 bp. Each replicate is shown as a separate line. c Hierarchical clustering of samples and peaks by average enrichment of methylated H3K IP over unmodified H3 IP. Samples clearly cluster by histone mark but do not cluster by Veh vs Dex for any histone mark. Vertical colour bar indicates six clusters following k-means clustering. Gold and purple clusters show higher H3K9me3 enrichment. Bivalent enrichment observed for H3K4me3 and H3K9me3 ( turquoise ) and H3K4me3 and H3K27me3 ( green ). Blue cluster represents inactive enhancers marked by H3K4me1 and H3K9me3

Techniques Used: Methylation

22) Product Images from "Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis"

Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis

Journal: Genome Biology

doi: 10.1186/gb-2010-11-4-r42

Monovalent chromatin signature is prevalent in undifferentiated cells of bam testis . (a) UCSC genome browser screenshot showing the H3K4me3 monovalency at the E(z) gene locus in undifferentiated cells. The read counts are labeled on the y-axis and the genomic region used to calculate the enrichment of modified histones (0 to +500 bp with respect to the TSS) is shaded in grey. (b) Transcription level of two representative monovalent genes, E(z) and sa , in bam and wild-type ( wt ) testis, respectively. According to the RNA-seq data, the RPKM for E(z) mRNA is four-fold higher in bam testis, and that for sa mRNA is 113-fold higher in wild-typetestis. (c) UCSC genome browser snapshot showing the H3K27me3 monovalency at the sa gene locus in undifferentiated cells. The read counts are labeled on the y-axis and the genomic region used to calculate the enrichment of modified histones (0 to +500 bp with respect to the TSS) is shaded in grey. (d) Scatter plot for H3K4me3 and H3K27me3 enrichment of all annotated genes. A common window (0 to +500 bp with respect to the TSS) was used to calculate H3K4me3 and H3K27me3 reads. The blue dashed lines indicate the statistical cutoff line - P
Figure Legend Snippet: Monovalent chromatin signature is prevalent in undifferentiated cells of bam testis . (a) UCSC genome browser screenshot showing the H3K4me3 monovalency at the E(z) gene locus in undifferentiated cells. The read counts are labeled on the y-axis and the genomic region used to calculate the enrichment of modified histones (0 to +500 bp with respect to the TSS) is shaded in grey. (b) Transcription level of two representative monovalent genes, E(z) and sa , in bam and wild-type ( wt ) testis, respectively. According to the RNA-seq data, the RPKM for E(z) mRNA is four-fold higher in bam testis, and that for sa mRNA is 113-fold higher in wild-typetestis. (c) UCSC genome browser snapshot showing the H3K27me3 monovalency at the sa gene locus in undifferentiated cells. The read counts are labeled on the y-axis and the genomic region used to calculate the enrichment of modified histones (0 to +500 bp with respect to the TSS) is shaded in grey. (d) Scatter plot for H3K4me3 and H3K27me3 enrichment of all annotated genes. A common window (0 to +500 bp with respect to the TSS) was used to calculate H3K4me3 and H3K27me3 reads. The blue dashed lines indicate the statistical cutoff line - P

Techniques Used: Labeling, Modification, RNA Sequencing Assay

Cartoons comparing ESCs and undifferentiated cells of bam testis . (a) During ESC differentiation, bivalent genes resolve into monovalent genes according to cell type specificity in somatic lineages. In the Drosophila male germline lineage, monovalent genes in undifferentiated cells may either retain their chromatin signature or switch to another pattern. Differentiation genes that are required for spermatogenesis require additional activation mechanisms to turn on their expression robustly, in addition to the removal of the repressive H3K27me3 mark. (b) A potential molecular mechanism that renders the bivalency and poised status dispensable in the Drosophila germline stem cell lineage, due to the lack of endogenous DNA methylase activity. DNMT, DNA methyltransferase.
Figure Legend Snippet: Cartoons comparing ESCs and undifferentiated cells of bam testis . (a) During ESC differentiation, bivalent genes resolve into monovalent genes according to cell type specificity in somatic lineages. In the Drosophila male germline lineage, monovalent genes in undifferentiated cells may either retain their chromatin signature or switch to another pattern. Differentiation genes that are required for spermatogenesis require additional activation mechanisms to turn on their expression robustly, in addition to the removal of the repressive H3K27me3 mark. (b) A potential molecular mechanism that renders the bivalency and poised status dispensable in the Drosophila germline stem cell lineage, due to the lack of endogenous DNA methylase activity. DNMT, DNA methyltransferase.

Techniques Used: Activation Assay, Expressing, Activity Assay

Summary of the ChIP-seq results using bam testis . (a) The four groups of genes were classified according to their RPKM value based on the RNA-seq results [ 21 ]. The numbers in brackets denote genes used for H3K36me3 (K36) analysis. *See Materials and methods for gene selection criteria. Antibodies used for ChIP-seq were: (b) anti-RNA Pol II (Pol II); (c) anti-H3K4me3 (K4); (d) anti-H3K36me3 (K36); and (e) anti-H3K27me3 (K27). Enrichment of each histone modification and RNA Pol II is plotted over a -3-kb to +3-kb region with respect (w.r.t.) to genes' TSSs, except for K36, for which a -5-kb to +5-kb region is used.
Figure Legend Snippet: Summary of the ChIP-seq results using bam testis . (a) The four groups of genes were classified according to their RPKM value based on the RNA-seq results [ 21 ]. The numbers in brackets denote genes used for H3K36me3 (K36) analysis. *See Materials and methods for gene selection criteria. Antibodies used for ChIP-seq were: (b) anti-RNA Pol II (Pol II); (c) anti-H3K4me3 (K4); (d) anti-H3K36me3 (K36); and (e) anti-H3K27me3 (K27). Enrichment of each histone modification and RNA Pol II is plotted over a -3-kb to +3-kb region with respect (w.r.t.) to genes' TSSs, except for K36, for which a -5-kb to +5-kb region is used.

Techniques Used: Chromatin Immunoprecipitation, RNA Sequencing Assay, Selection, Modification

Comparison of RNA-seq and ChIP-seq data using S2cells . (a) Scatter plot for H3K4me3 and H3K27me3 enrichment of all annotated genes. A common 0 to +500-bp window with respect to the TSS was used to calculate both H3K4me3 and H3K27me3 reads. The blue dashed lines indicate the statistical cutoff line - P
Figure Legend Snippet: Comparison of RNA-seq and ChIP-seq data using S2cells . (a) Scatter plot for H3K4me3 and H3K27me3 enrichment of all annotated genes. A common 0 to +500-bp window with respect to the TSS was used to calculate both H3K4me3 and H3K27me3 reads. The blue dashed lines indicate the statistical cutoff line - P

Techniques Used: RNA Sequencing Assay, Chromatin Immunoprecipitation

23) Product Images from "The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia"

Article Title: The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia

Journal: Nature Communications

doi: 10.1038/s41467-018-03854-0

SHARP1 binds to actively transcribed genes and positively regulates target genes. a Integrated view of SHARP1 binding sites in conjunction with H3K4me3, H3K27ac, and H3K27me3 profiles across promoters, introns and intergenic regions. Top enriched motifs within SHARP1 ChIP-seq peaks in ML-2 cells are shown according to their genomic location discovered by peak-motifs module from the RSAT suite, using oligomer length ranging from 6 to 8 nucleotides and the 'merge lengths for assembly' option. b Box plot showing the expression levels in microarray analysis of ML-2 cells for the all genes (25293 genes) and genes enriched with SHARP1 + H3K4me3 (6459 genes), SHARP1 + H3K27ac (5840 genes), and SHARP1 + H3K27me3 (1055 genes) identified in ChIP-seq analysis. The box extends from the 25 th to 75 th percentiles and the whisker extends from the minimum level to the maximum. Median value is plotted in the box. c Representative SHARP1 binding peaks in the known target gene loci (circadian clock genes and MLH1 ). d Pathway analysis for the genes in the SHARP1 and H3K4me3 co-bounded regions within the promoter and gene body
Figure Legend Snippet: SHARP1 binds to actively transcribed genes and positively regulates target genes. a Integrated view of SHARP1 binding sites in conjunction with H3K4me3, H3K27ac, and H3K27me3 profiles across promoters, introns and intergenic regions. Top enriched motifs within SHARP1 ChIP-seq peaks in ML-2 cells are shown according to their genomic location discovered by peak-motifs module from the RSAT suite, using oligomer length ranging from 6 to 8 nucleotides and the 'merge lengths for assembly' option. b Box plot showing the expression levels in microarray analysis of ML-2 cells for the all genes (25293 genes) and genes enriched with SHARP1 + H3K4me3 (6459 genes), SHARP1 + H3K27ac (5840 genes), and SHARP1 + H3K27me3 (1055 genes) identified in ChIP-seq analysis. The box extends from the 25 th to 75 th percentiles and the whisker extends from the minimum level to the maximum. Median value is plotted in the box. c Representative SHARP1 binding peaks in the known target gene loci (circadian clock genes and MLH1 ). d Pathway analysis for the genes in the SHARP1 and H3K4me3 co-bounded regions within the promoter and gene body

Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Expressing, Microarray, Whisker Assay

24) Product Images from "Polycomb Repressive Complex 2 and H3K27me3 Cooperate with H3K9 Methylation To Maintain Heterochromatin Protein 1α at Chromatin"

Article Title: Polycomb Repressive Complex 2 and H3K27me3 Cooperate with H3K9 Methylation To Maintain Heterochromatin Protein 1α at Chromatin

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00205-14

Overexpressed H3K27me2/3 demethylases remove HP1α from chromatin. Immunofluorescence analyses of H3K27me3 (A), H3K9me3 (B), or HP1α (C) (green) in HT1080 cells transiently transfected (24 h) with either the HA-JMJD3 construct or the HA
Figure Legend Snippet: Overexpressed H3K27me2/3 demethylases remove HP1α from chromatin. Immunofluorescence analyses of H3K27me3 (A), H3K9me3 (B), or HP1α (C) (green) in HT1080 cells transiently transfected (24 h) with either the HA-JMJD3 construct or the HA

Techniques Used: Immunofluorescence, Transfection, Construct

Binding of human cell extract-derived HP1α to H3 histone tails is increased in the presence of both K9me3 and K27me3 marks. (A) Pulldown experiments with biotinylated H3 tail peptides (H3, unmodified; K9, H3K9me3; K27, H3K27me3; K9;K27, H3K9me3K27me3)
Figure Legend Snippet: Binding of human cell extract-derived HP1α to H3 histone tails is increased in the presence of both K9me3 and K27me3 marks. (A) Pulldown experiments with biotinylated H3 tail peptides (H3, unmodified; K9, H3K9me3; K27, H3K27me3; K9;K27, H3K9me3K27me3)

Techniques Used: Binding Assay, Derivative Assay

The H3K27me3-H3K9me3 cooperation model. We propose that H3K27me3-bound PRC2 stabilizes H3K9me3-anchored HP1α, either directly (interactions with SUZ12) or indirectly (interactions with another factor illustrated by a question mark). In this model,
Figure Legend Snippet: The H3K27me3-H3K9me3 cooperation model. We propose that H3K27me3-bound PRC2 stabilizes H3K9me3-anchored HP1α, either directly (interactions with SUZ12) or indirectly (interactions with another factor illustrated by a question mark). In this model,

Techniques Used:

25) Product Images from "Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis"

Article Title: Monovalent and unpoised status of most genes in undifferentiated cell-enriched Drosophila testis

Journal: Genome Biology

doi: 10.1186/gb-2010-11-4-r42

Monovalent chromatin signature is prevalent in undifferentiated cells of bam testis . (a) UCSC genome browser screenshot showing the H3K4me3 monovalency at the E(z) gene locus in undifferentiated cells. The read counts are labeled on the y-axis and the genomic region used to calculate the enrichment of modified histones (0 to +500 bp with respect to the TSS) is shaded in grey. (b) Transcription level of two representative monovalent genes, E(z) and sa , in bam and wild-type ( wt ) testis, respectively. According to the RNA-seq data, the RPKM for E(z) mRNA is four-fold higher in bam testis, and that for sa mRNA is 113-fold higher in wild-typetestis. (c) UCSC genome browser snapshot showing the H3K27me3 monovalency at the sa gene locus in undifferentiated cells. The read counts are labeled on the y-axis and the genomic region used to calculate the enrichment of modified histones (0 to +500 bp with respect to the TSS) is shaded in grey. (d) Scatter plot for H3K4me3 and H3K27me3 enrichment of all annotated genes. A common window (0 to +500 bp with respect to the TSS) was used to calculate H3K4me3 and H3K27me3 reads. The blue dashed lines indicate the statistical cutoff line - P
Figure Legend Snippet: Monovalent chromatin signature is prevalent in undifferentiated cells of bam testis . (a) UCSC genome browser screenshot showing the H3K4me3 monovalency at the E(z) gene locus in undifferentiated cells. The read counts are labeled on the y-axis and the genomic region used to calculate the enrichment of modified histones (0 to +500 bp with respect to the TSS) is shaded in grey. (b) Transcription level of two representative monovalent genes, E(z) and sa , in bam and wild-type ( wt ) testis, respectively. According to the RNA-seq data, the RPKM for E(z) mRNA is four-fold higher in bam testis, and that for sa mRNA is 113-fold higher in wild-typetestis. (c) UCSC genome browser snapshot showing the H3K27me3 monovalency at the sa gene locus in undifferentiated cells. The read counts are labeled on the y-axis and the genomic region used to calculate the enrichment of modified histones (0 to +500 bp with respect to the TSS) is shaded in grey. (d) Scatter plot for H3K4me3 and H3K27me3 enrichment of all annotated genes. A common window (0 to +500 bp with respect to the TSS) was used to calculate H3K4me3 and H3K27me3 reads. The blue dashed lines indicate the statistical cutoff line - P

Techniques Used: Labeling, Modification, RNA Sequencing Assay

Cartoons comparing ESCs and undifferentiated cells of bam testis . (a) During ESC differentiation, bivalent genes resolve into monovalent genes according to cell type specificity in somatic lineages. In the Drosophila male germline lineage, monovalent genes in undifferentiated cells may either retain their chromatin signature or switch to another pattern. Differentiation genes that are required for spermatogenesis require additional activation mechanisms to turn on their expression robustly, in addition to the removal of the repressive H3K27me3 mark. (b) A potential molecular mechanism that renders the bivalency and poised status dispensable in the Drosophila germline stem cell lineage, due to the lack of endogenous DNA methylase activity. DNMT, DNA methyltransferase.
Figure Legend Snippet: Cartoons comparing ESCs and undifferentiated cells of bam testis . (a) During ESC differentiation, bivalent genes resolve into monovalent genes according to cell type specificity in somatic lineages. In the Drosophila male germline lineage, monovalent genes in undifferentiated cells may either retain their chromatin signature or switch to another pattern. Differentiation genes that are required for spermatogenesis require additional activation mechanisms to turn on their expression robustly, in addition to the removal of the repressive H3K27me3 mark. (b) A potential molecular mechanism that renders the bivalency and poised status dispensable in the Drosophila germline stem cell lineage, due to the lack of endogenous DNA methylase activity. DNMT, DNA methyltransferase.

Techniques Used: Activation Assay, Expressing, Activity Assay

Summary of the ChIP-seq results using bam testis . (a) The four groups of genes were classified according to their RPKM value based on the RNA-seq results [ 21 ]. The numbers in brackets denote genes used for H3K36me3 (K36) analysis. *See Materials and methods for gene selection criteria. Antibodies used for ChIP-seq were: (b) anti-RNA Pol II (Pol II); (c) anti-H3K4me3 (K4); (d) anti-H3K36me3 (K36); and (e) anti-H3K27me3 (K27). Enrichment of each histone modification and RNA Pol II is plotted over a -3-kb to +3-kb region with respect (w.r.t.) to genes' TSSs, except for K36, for which a -5-kb to +5-kb region is used.
Figure Legend Snippet: Summary of the ChIP-seq results using bam testis . (a) The four groups of genes were classified according to their RPKM value based on the RNA-seq results [ 21 ]. The numbers in brackets denote genes used for H3K36me3 (K36) analysis. *See Materials and methods for gene selection criteria. Antibodies used for ChIP-seq were: (b) anti-RNA Pol II (Pol II); (c) anti-H3K4me3 (K4); (d) anti-H3K36me3 (K36); and (e) anti-H3K27me3 (K27). Enrichment of each histone modification and RNA Pol II is plotted over a -3-kb to +3-kb region with respect (w.r.t.) to genes' TSSs, except for K36, for which a -5-kb to +5-kb region is used.

Techniques Used: Chromatin Immunoprecipitation, RNA Sequencing Assay, Selection, Modification

Comparison of RNA-seq and ChIP-seq data using S2cells . (a) Scatter plot for H3K4me3 and H3K27me3 enrichment of all annotated genes. A common 0 to +500-bp window with respect to the TSS was used to calculate both H3K4me3 and H3K27me3 reads. The blue dashed lines indicate the statistical cutoff line - P
Figure Legend Snippet: Comparison of RNA-seq and ChIP-seq data using S2cells . (a) Scatter plot for H3K4me3 and H3K27me3 enrichment of all annotated genes. A common 0 to +500-bp window with respect to the TSS was used to calculate both H3K4me3 and H3K27me3 reads. The blue dashed lines indicate the statistical cutoff line - P

Techniques Used: RNA Sequencing Assay, Chromatin Immunoprecipitation

26) Product Images from "Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications"

Article Title: Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications

Journal: Carcinogenesis

doi: 10.1093/carcin/bgp336

Effect of the loss of H3K27me3 on CLDN3 and CLDN4 expression in ovarian cancer cells. ( A ) Effect of EZH2 knockdown by EZH2 siRNA on CLDN3 and CLDN4 expression. Knockdown of EZH2 transcripts ( EZH2 var1 and EZH2 var2) and the decrease in protein levels of H3K27me3 were assessed in TOV-112D cells 72 h after siRNA treatment (100 nM). The effect of EZH2 knockdown on H3K27me3 enrichment in the CLDN3 and CLDN4 promoters was also evaluated by quantitative ChIP in TOV-112D cells. * P
Figure Legend Snippet: Effect of the loss of H3K27me3 on CLDN3 and CLDN4 expression in ovarian cancer cells. ( A ) Effect of EZH2 knockdown by EZH2 siRNA on CLDN3 and CLDN4 expression. Knockdown of EZH2 transcripts ( EZH2 var1 and EZH2 var2) and the decrease in protein levels of H3K27me3 were assessed in TOV-112D cells 72 h after siRNA treatment (100 nM). The effect of EZH2 knockdown on H3K27me3 enrichment in the CLDN3 and CLDN4 promoters was also evaluated by quantitative ChIP in TOV-112D cells. * P

Techniques Used: Expressing, Chromatin Immunoprecipitation

27) Product Images from "Developmental silencing of the AtTERT gene is associated with increased H3K27me3 loading and maintenance of its euchromatic environment"

Article Title: Developmental silencing of the AtTERT gene is associated with increased H3K27me3 loading and maintenance of its euchromatic environment

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/ers107

Analysis of histone modifications in the AtTERT upstream region and in exon 5 by ChIP. DNAs from immunoprecipitated fractions of chromatin were purified and a 336 bp region upstream of the ATG signal and a 476 bp region of the fifth exon were amplified using classical (A) or quantitative (B) PCR (qPCR). (A) A representative example of PCR amplification of the AtTERT upstream region and of exon 5 in immunoprecipitated fractions. Signals of euchromatin-specific marks (H3K4me2, H3K9Ac) were strong in both tissues analysed; signals for the modification typical for constitutive heterochromatin (H3K9me2) were below the detection limit. Note the distinct H3K27me3 band in the leaf samples. (B) Two biological replicates of wild-type seedlings and mature leaves were immunoprecipitated and subjected to quantitative PCR. Signal from the immunoprecipitated fractions was expressed relative to that from the total input chromatin. The amount of the H3K27me3 mark increased in the telomerase-negative tissue (leaf) in both regions analysed ( P
Figure Legend Snippet: Analysis of histone modifications in the AtTERT upstream region and in exon 5 by ChIP. DNAs from immunoprecipitated fractions of chromatin were purified and a 336 bp region upstream of the ATG signal and a 476 bp region of the fifth exon were amplified using classical (A) or quantitative (B) PCR (qPCR). (A) A representative example of PCR amplification of the AtTERT upstream region and of exon 5 in immunoprecipitated fractions. Signals of euchromatin-specific marks (H3K4me2, H3K9Ac) were strong in both tissues analysed; signals for the modification typical for constitutive heterochromatin (H3K9me2) were below the detection limit. Note the distinct H3K27me3 band in the leaf samples. (B) Two biological replicates of wild-type seedlings and mature leaves were immunoprecipitated and subjected to quantitative PCR. Signal from the immunoprecipitated fractions was expressed relative to that from the total input chromatin. The amount of the H3K27me3 mark increased in the telomerase-negative tissue (leaf) in both regions analysed ( P

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

28) Product Images from "The rice enhancer of zeste [E(z)] genes SDG711 and SDG718 are respectively involved in long day and short day signaling to mediate the accurate photoperiod control of flowering time"

Article Title: The rice enhancer of zeste [E(z)] genes SDG711 and SDG718 are respectively involved in long day and short day signaling to mediate the accurate photoperiod control of flowering time

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2014.00591

SDG711 and SDG718 function in H3K27me3 and H3K4me3 on key flowering genes. (A) chromatin immunoprecipitation (ChIP) analysis of H3K27me3 on the indicated flowering genes in WT, SDG711 RNAi and OX plants (mature leaves pooled from 3 T3 lines). (B) ChIP analysis of H3K4me3 on the indicated flowering genes in WT, SDG711 RNAi and OX plants (pooled from 3 T3 lines). H3K27me3 and H3K4me3 enrichments on the 5′ end (P1) and the coding region (P2) of the flowering genes were detected by quantitative PCR. Three biological repeats were performed, one repetition is shown. One other biological replicate is shown in Figure S7 . Bars = mean ± SD from three technical repeats. (C) ChIP analysis of H3K27me3 on the indicated flowering genes in WT and SDG718 RNAi plants pooled from 3 T3 lines. Leaves of SDG711 transgenic plants were harvested from 8 week-old LD plants grown, and leaves of SDG718 transgenic plants were harvested from 6 week-old SD plants grown.
Figure Legend Snippet: SDG711 and SDG718 function in H3K27me3 and H3K4me3 on key flowering genes. (A) chromatin immunoprecipitation (ChIP) analysis of H3K27me3 on the indicated flowering genes in WT, SDG711 RNAi and OX plants (mature leaves pooled from 3 T3 lines). (B) ChIP analysis of H3K4me3 on the indicated flowering genes in WT, SDG711 RNAi and OX plants (pooled from 3 T3 lines). H3K27me3 and H3K4me3 enrichments on the 5′ end (P1) and the coding region (P2) of the flowering genes were detected by quantitative PCR. Three biological repeats were performed, one repetition is shown. One other biological replicate is shown in Figure S7 . Bars = mean ± SD from three technical repeats. (C) ChIP analysis of H3K27me3 on the indicated flowering genes in WT and SDG718 RNAi plants pooled from 3 T3 lines. Leaves of SDG711 transgenic plants were harvested from 8 week-old LD plants grown, and leaves of SDG718 transgenic plants were harvested from 6 week-old SD plants grown.

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

29) Product Images from "The rice enhancer of zeste [E(z)] genes SDG711 and SDG718 are respectively involved in long day and short day signaling to mediate the accurate photoperiod control of flowering time"

Article Title: The rice enhancer of zeste [E(z)] genes SDG711 and SDG718 are respectively involved in long day and short day signaling to mediate the accurate photoperiod control of flowering time

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2014.00591

SDG711 and SDG718 function in H3K27me3 and H3K4me3 on key flowering genes. (A) chromatin immunoprecipitation (ChIP) analysis of H3K27me3 on the indicated flowering genes in WT, SDG711 RNAi and OX plants (mature leaves pooled from 3 T3 lines). (B) ChIP analysis of H3K4me3 on the indicated flowering genes in WT, SDG711 RNAi and OX plants (pooled from 3 T3 lines). H3K27me3 and H3K4me3 enrichments on the 5′ end (P1) and the coding region (P2) of the flowering genes were detected by quantitative PCR. Three biological repeats were performed, one repetition is shown. One other biological replicate is shown in Figure S7 . Bars = mean ± SD from three technical repeats. (C) ChIP analysis of H3K27me3 on the indicated flowering genes in WT and SDG718 RNAi plants pooled from 3 T3 lines. Leaves of SDG711 transgenic plants were harvested from 8 week-old LD plants grown, and leaves of SDG718 transgenic plants were harvested from 6 week-old SD plants grown.
Figure Legend Snippet: SDG711 and SDG718 function in H3K27me3 and H3K4me3 on key flowering genes. (A) chromatin immunoprecipitation (ChIP) analysis of H3K27me3 on the indicated flowering genes in WT, SDG711 RNAi and OX plants (mature leaves pooled from 3 T3 lines). (B) ChIP analysis of H3K4me3 on the indicated flowering genes in WT, SDG711 RNAi and OX plants (pooled from 3 T3 lines). H3K27me3 and H3K4me3 enrichments on the 5′ end (P1) and the coding region (P2) of the flowering genes were detected by quantitative PCR. Three biological repeats were performed, one repetition is shown. One other biological replicate is shown in Figure S7 . Bars = mean ± SD from three technical repeats. (C) ChIP analysis of H3K27me3 on the indicated flowering genes in WT and SDG718 RNAi plants pooled from 3 T3 lines. Leaves of SDG711 transgenic plants were harvested from 8 week-old LD plants grown, and leaves of SDG718 transgenic plants were harvested from 6 week-old SD plants grown.

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

30) Product Images from ""

Article Title:

Journal: Molecular Pharmacology

doi: 10.1124/mol.117.108225

Effects of silencing PXR expression on alterations of histone methylation and acetylation levels as well as enrichment of NCOA6 and p300 in the CYP3A4 promoter. Knockdown of PXR was performed by transiently transfecting shRNA plasmids into LS174T cells followed by treatment with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μ M) for 48 hours, and cells were harvested for ChIP analysis. The relative levels of H3K4me3 (A–C), H3K27me3 (D–F), H3 acetylation (G–I) in the CYP3A4 promoter in control and treated groups. The relative enrichment of NCOA6 (J–L) and p300 (M–O) in the CYP3A4 promoter in control and treated groups. Data are shown as mean ± SD of three independent experiments. * p
Figure Legend Snippet: Effects of silencing PXR expression on alterations of histone methylation and acetylation levels as well as enrichment of NCOA6 and p300 in the CYP3A4 promoter. Knockdown of PXR was performed by transiently transfecting shRNA plasmids into LS174T cells followed by treatment with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μ M) for 48 hours, and cells were harvested for ChIP analysis. The relative levels of H3K4me3 (A–C), H3K27me3 (D–F), H3 acetylation (G–I) in the CYP3A4 promoter in control and treated groups. The relative enrichment of NCOA6 (J–L) and p300 (M–O) in the CYP3A4 promoter in control and treated groups. Data are shown as mean ± SD of three independent experiments. * p

Techniques Used: Expressing, Methylation, shRNA, Chromatin Immunoprecipitation

Effects of silencing NCOA6 or p300 expression on alteration of histone methylation or acetylation levels of the CYP3A4 promoter. Knockdown of NCOA6 or p300 was performed by transfecting shRNA plasmids into LS174T cells followed by treatment with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μ M) for 48 hours, and cells were harvested for ChIP analysis. The relative levels of H3K4me3 (A–C) or H3K27me3 (D–F) in the CYP3A4 promoter in the control and treated groups. The relative levels of H3 acetylation (G–I) in the CYP3A4 promoter in control and treated groups. Data are shown as mean ± S.D. of three independent experiments. * p
Figure Legend Snippet: Effects of silencing NCOA6 or p300 expression on alteration of histone methylation or acetylation levels of the CYP3A4 promoter. Knockdown of NCOA6 or p300 was performed by transfecting shRNA plasmids into LS174T cells followed by treatment with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μ M) for 48 hours, and cells were harvested for ChIP analysis. The relative levels of H3K4me3 (A–C) or H3K27me3 (D–F) in the CYP3A4 promoter in the control and treated groups. The relative levels of H3 acetylation (G–I) in the CYP3A4 promoter in control and treated groups. Data are shown as mean ± S.D. of three independent experiments. * p

Techniques Used: Expressing, Methylation, shRNA, Chromatin Immunoprecipitation

Effect of rifampicin on the levels of histone modifications around the PXR response elements ( PXRE ) in the CYP3A4 promoter. LS174T cells were treated with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μ M) for 48 or 96 hours, followed by ChIP analysis. Enrichment was calculated as percentage of chromatin input. ( A ) qPCR primers that cover different sites near the PXRE in the CYP3A4 promoter, a pair of primers located in intron 2 was performed as a negative control. The relative levels of H3K4me3 (B–D), H3K27me3 (E–G), and H3 acetylation (H–J) in the CYP3A4 promoter region and were determined. Data are shown as mean ± S.D. of three independent experiments. * p
Figure Legend Snippet: Effect of rifampicin on the levels of histone modifications around the PXR response elements ( PXRE ) in the CYP3A4 promoter. LS174T cells were treated with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μ M) for 48 or 96 hours, followed by ChIP analysis. Enrichment was calculated as percentage of chromatin input. ( A ) qPCR primers that cover different sites near the PXRE in the CYP3A4 promoter, a pair of primers located in intron 2 was performed as a negative control. The relative levels of H3K4me3 (B–D), H3K27me3 (E–G), and H3 acetylation (H–J) in the CYP3A4 promoter region and were determined. Data are shown as mean ± S.D. of three independent experiments. * p

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

31) Product Images from "ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity"

Article Title: ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr416

A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).
Figure Legend Snippet: A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).

Techniques Used: Chromatin Immunoprecipitation, Magnetic Cell Separation

Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.
Figure Legend Snippet: Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.

Techniques Used: Expressing

K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).
Figure Legend Snippet: K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).

Techniques Used: Expressing

Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).
Figure Legend Snippet: Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).

Techniques Used:

TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.
Figure Legend Snippet: TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.

Techniques Used: Expressing, Chromatin Immunoprecipitation

The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.
Figure Legend Snippet: The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.

Techniques Used:

32) Product Images from "ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity"

Article Title: ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr416

A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).
Figure Legend Snippet: A comparison of H3K27me3 ChIP-seq data sets from ES cells. ( A ) The number of mapped reads in H3K27me3 data was assessed in 5 kb intervals across the entire genome in ES cells for both our data (WEHI) and the public data (Mikkelsen). The number of reads in each interval was expressed as a proportion of total mapped reads. The data are plotted on a log 2 scale and show a strong positive correlation (Pearson correlation co-efficient 0.92). ( B ) A Venn diagram showing the number of genes identified as marked with H3K27me3 in our data using different calling methods (MACS or Poisson). We recovered 93.3% of genes previously characterized as marked, as well as 3549 new genes. The majority of genes identified by Mikkelsen et al. (2007) were identified in our data using MACS (90%), whereas a much smaller proportion was identified using the Poisson test (58%). This difference may indicate a bias towards shorter domains of H3K27me3 in the genes defined by Mikkelsen et al. (2007).

Techniques Used: Chromatin Immunoprecipitation, Magnetic Cell Separation

Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.
Figure Legend Snippet: Classification of profiles. ASE plots for each H3K27me3 enrichment profile are shown for ES cells ( B ) and G1ME cells ( F ). The number of genes classified into each enrichment profile is shown in the adjoining bar plots ( A and E ). ASE plots of RNApol-II for each class of genes in ES ( C ) and G1ME cells ( G ). Promoter genes show strong enrichment for RNApol-II, while TSS and broad genes do not. Box plots of expression levels are shown for each class of gene in ES cells ( D ) and G1ME cells ( H ). Genes classified with the promoter profile show high levels of expression, whereas genes with the broad profile have the lowest expression levels. Genes classified as TSS have intermediate expression levels, but are still repressed relative to the average of all genes on the array.

Techniques Used: Expressing

K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).
Figure Legend Snippet: K -means clustering of genic H3K27me3 profiles in G1ME cells. The signal intensity is shown as a spectrogram, with red reflecting a high enrichment signal and blue reflecting no signal. All genes were scaled to have the same length, and position relative to the TSS is shown in percentage terms. Genes were sorted first by cluster, then by classification (black: broad; green: promoter; blue: TSS; grey: marked but unclassified). The expression level of all genes is shown on the far right. Additional cluster profiles are provided for the other cell types ( Supplementary Figure S8 ).

Techniques Used: Expressing

Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).
Figure Legend Snippet: Profiles of genes exclusive and commonly marked by H3K27me3 in ES and G1ME cells. ( A ) The Venn diagram shows the overlap between the marked genes in ES cells and G1MEs. ( B and C ) ASE plots of H3K27me3 signal for the 2689 genes that are enriched for H3K27me3 in ES and G1ME. In each plot, the red lines denote the boundaries of the gene and the level of signal in the input control is plotted (grey line). ( D and E ) ASE plots of H3K27me3 signal for cell type-specific genes (the number of genes differs for each cell line: ES cells = 3180, G1MEs = 3887).

Techniques Used:

TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.
Figure Legend Snippet: TSS centered, ASE and expression stratified ASE plots of H3K27me3 and RNApol-II. Comparison of the TSS centered averaged plots ( A , D and G ) with the ASE plots ( B , E and H ) for genes marked by RNApol-II in ES cells and H3K27me3 in ES cells and G1MEs. There is information contained in the scaled version that is not observed from the TSS centered view. ( C , F and I ) ASE plots of all genes stratified by expression. The average level is lower as these include ChIP-seq data for all genes on the expression array, not just marked genes.

Techniques Used: Expressing, Chromatin Immunoprecipitation

The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.
Figure Legend Snippet: The relationship between H3K4me3, H3K36me3 and H3K27me3 enrichment in ES cells. H3K36me3 enrichment across the gene ( A ) and H3K4me3 enrichment around the TSS ( B ) for the promoter, TSS and broad classes of genes in ES cells. ASE plots of the H3K27me3 signal in ES cells, where genes have been separated based on being called marked by H3K27me3 and H3K4me3 ( C ). The solid line is the profile for bivalent genes (marked by both H3K27me3 and H3K4me3). The dashed line corresponds to genes marked with only H3K27me3 and the dotted line is the H3K27me3 profile for genes marked with only H3K4me3.

Techniques Used:

33) Product Images from "Targets and genomic constraints of ectopic Dnmt3b expression"

Article Title: Targets and genomic constraints of ectopic Dnmt3b expression

Journal: eLife

doi: 10.7554/eLife.40757

Cross-validation performance of logistic regression at predicting hypermethylated CGIs. ( A ) Precision recall curves for DMR CGI prediction using logistic regression, Naive Bayes, decision tree, random forest, and gradient boosted trees with five-fold crossvalidation. DMRs were CGIs with gain in methylation of 0.15 or greater and FDR q-value of 0.05, while the negative set was CGIs that gained less than 0.05. Features used for classification were methylation in control liver, DNase-seq enrichment, H3K4me3 enrichment, H3K27me3 enrichment, raw CpG density, normalized CpG density ( Saxonov et al., 2006 ), and GC content. The random forest classifier showed the best performance. ( B ) Fraction of CGIs that are DMRs across the dynamic range of each feature. ( C ) Pearson correlation coefficients between features used for DMR prediction of CGIs.
Figure Legend Snippet: Cross-validation performance of logistic regression at predicting hypermethylated CGIs. ( A ) Precision recall curves for DMR CGI prediction using logistic regression, Naive Bayes, decision tree, random forest, and gradient boosted trees with five-fold crossvalidation. DMRs were CGIs with gain in methylation of 0.15 or greater and FDR q-value of 0.05, while the negative set was CGIs that gained less than 0.05. Features used for classification were methylation in control liver, DNase-seq enrichment, H3K4me3 enrichment, H3K27me3 enrichment, raw CpG density, normalized CpG density ( Saxonov et al., 2006 ), and GC content. The random forest classifier showed the best performance. ( B ) Fraction of CGIs that are DMRs across the dynamic range of each feature. ( C ) Pearson correlation coefficients between features used for DMR prediction of CGIs.

Techniques Used: Methylation

Ectopic Dnmt3b expression in MEFs. ( A ) Correlation of DMRs to gene expression status, H3K4me3 and H3K27me3 enrichment in MEFs (1 d and 7 d dox) for all captured low CpG-density promoters (LCPs). ( B ) For DMR CGIs and 200 bp tiles that were highly methylated in the 1 d control sample (mean methylation > 0.8), H3K27me3 enrichment for control and Dnmt3b overexpression 1 d and 7 d MEFs is displayed. ( C ) Browser shot displaying H3K27me3 enrichment and DNA methylation levels from each immunoprecipitated read (ChIP-BS) for control and induced MEFs. Globally, a small depletion in H3K27me3 was observed after 7 days in culture for both control and induced MEFs, however backgroud (non DMRs) with both H3K27me3 and DNA methylation in control cells showed no further gain in methylation and therefore no distinct depletion as is observed at DMRs ( Figure 4F ). ( D ) Volcano plot of RNA-seq data showing expression changes in MEFs after 7 d induction. Differentially expressed genes (adjusted P -value
Figure Legend Snippet: Ectopic Dnmt3b expression in MEFs. ( A ) Correlation of DMRs to gene expression status, H3K4me3 and H3K27me3 enrichment in MEFs (1 d and 7 d dox) for all captured low CpG-density promoters (LCPs). ( B ) For DMR CGIs and 200 bp tiles that were highly methylated in the 1 d control sample (mean methylation > 0.8), H3K27me3 enrichment for control and Dnmt3b overexpression 1 d and 7 d MEFs is displayed. ( C ) Browser shot displaying H3K27me3 enrichment and DNA methylation levels from each immunoprecipitated read (ChIP-BS) for control and induced MEFs. Globally, a small depletion in H3K27me3 was observed after 7 days in culture for both control and induced MEFs, however backgroud (non DMRs) with both H3K27me3 and DNA methylation in control cells showed no further gain in methylation and therefore no distinct depletion as is observed at DMRs ( Figure 4F ). ( D ) Volcano plot of RNA-seq data showing expression changes in MEFs after 7 d induction. Differentially expressed genes (adjusted P -value

Techniques Used: Expressing, Methylation, Over Expression, DNA Methylation Assay, Immunoprecipitation, Chromatin Immunoprecipitation, RNA Sequencing Assay

Eed knockdown in MEFs. ( A ) Schematic for the target design of two shRNAs against the Eed transcript. Both shRNA4 (exon 11) and shRNA5 (exon 3 and 4) were cloned into the pSicoR-ef1a-GFP vector. ( B ) Western blot analysis for DNMT3B and H3K27me3 in control and Dnmt3b MEFs (7 d dox) expressing the Eed shRNAs. ( C ) RT-qPCR analysis for Eed transcript levels in control and Dnmt3b OE MEFs expressing the shRNAs. Data are represented as mean ±standard deviation. Primer sequences are shown underneath. ( D ) Scatter plots showing DNA methylation levels of CGIs ( n = 13,357) in control MEFs and MEFs expressing the Eed shRNAs as well as MEFs that contain both the shRNA and inducible Dnmt3b.
Figure Legend Snippet: Eed knockdown in MEFs. ( A ) Schematic for the target design of two shRNAs against the Eed transcript. Both shRNA4 (exon 11) and shRNA5 (exon 3 and 4) were cloned into the pSicoR-ef1a-GFP vector. ( B ) Western blot analysis for DNMT3B and H3K27me3 in control and Dnmt3b MEFs (7 d dox) expressing the Eed shRNAs. ( C ) RT-qPCR analysis for Eed transcript levels in control and Dnmt3b OE MEFs expressing the shRNAs. Data are represented as mean ±standard deviation. Primer sequences are shown underneath. ( D ) Scatter plots showing DNA methylation levels of CGIs ( n = 13,357) in control MEFs and MEFs expressing the Eed shRNAs as well as MEFs that contain both the shRNA and inducible Dnmt3b.

Techniques Used: Clone Assay, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Standard Deviation, DNA Methylation Assay, shRNA

34) Product Images from "Targets and genomic constraints of ectopic Dnmt3b expression"

Article Title: Targets and genomic constraints of ectopic Dnmt3b expression

Journal: eLife

doi: 10.7554/eLife.40757

Cross-validation performance of logistic regression at predicting hypermethylated CGIs. ( A ) Precision recall curves for DMR CGI prediction using logistic regression, Naive Bayes, decision tree, random forest, and gradient boosted trees with five-fold crossvalidation. DMRs were CGIs with gain in methylation of 0.15 or greater and FDR q-value of 0.05, while the negative set was CGIs that gained less than 0.05. Features used for classification were methylation in control liver, DNase-seq enrichment, H3K4me3 enrichment, H3K27me3 enrichment, raw CpG density, normalized CpG density ( Saxonov et al., 2006 ), and GC content. The random forest classifier showed the best performance. ( B ) Fraction of CGIs that are DMRs across the dynamic range of each feature. ( C ) Pearson correlation coefficients between features used for DMR prediction of CGIs.
Figure Legend Snippet: Cross-validation performance of logistic regression at predicting hypermethylated CGIs. ( A ) Precision recall curves for DMR CGI prediction using logistic regression, Naive Bayes, decision tree, random forest, and gradient boosted trees with five-fold crossvalidation. DMRs were CGIs with gain in methylation of 0.15 or greater and FDR q-value of 0.05, while the negative set was CGIs that gained less than 0.05. Features used for classification were methylation in control liver, DNase-seq enrichment, H3K4me3 enrichment, H3K27me3 enrichment, raw CpG density, normalized CpG density ( Saxonov et al., 2006 ), and GC content. The random forest classifier showed the best performance. ( B ) Fraction of CGIs that are DMRs across the dynamic range of each feature. ( C ) Pearson correlation coefficients between features used for DMR prediction of CGIs.

Techniques Used: Methylation

Ectopic Dnmt3b expression in MEFs. ( A ) Correlation of DMRs to gene expression status, H3K4me3 and H3K27me3 enrichment in MEFs (1 d and 7 d dox) for all captured low CpG-density promoters (LCPs). ( B ) For DMR CGIs and 200 bp tiles that were highly methylated in the 1 d control sample (mean methylation > 0.8), H3K27me3 enrichment for control and Dnmt3b overexpression 1 d and 7 d MEFs is displayed. ( C ) Browser shot displaying H3K27me3 enrichment and DNA methylation levels from each immunoprecipitated read (ChIP-BS) for control and induced MEFs. Globally, a small depletion in H3K27me3 was observed after 7 days in culture for both control and induced MEFs, however backgroud (non DMRs) with both H3K27me3 and DNA methylation in control cells showed no further gain in methylation and therefore no distinct depletion as is observed at DMRs ( Figure 4F ). ( D ) Volcano plot of RNA-seq data showing expression changes in MEFs after 7 d induction. Differentially expressed genes (adjusted P -value
Figure Legend Snippet: Ectopic Dnmt3b expression in MEFs. ( A ) Correlation of DMRs to gene expression status, H3K4me3 and H3K27me3 enrichment in MEFs (1 d and 7 d dox) for all captured low CpG-density promoters (LCPs). ( B ) For DMR CGIs and 200 bp tiles that were highly methylated in the 1 d control sample (mean methylation > 0.8), H3K27me3 enrichment for control and Dnmt3b overexpression 1 d and 7 d MEFs is displayed. ( C ) Browser shot displaying H3K27me3 enrichment and DNA methylation levels from each immunoprecipitated read (ChIP-BS) for control and induced MEFs. Globally, a small depletion in H3K27me3 was observed after 7 days in culture for both control and induced MEFs, however backgroud (non DMRs) with both H3K27me3 and DNA methylation in control cells showed no further gain in methylation and therefore no distinct depletion as is observed at DMRs ( Figure 4F ). ( D ) Volcano plot of RNA-seq data showing expression changes in MEFs after 7 d induction. Differentially expressed genes (adjusted P -value

Techniques Used: Expressing, Methylation, Over Expression, DNA Methylation Assay, Immunoprecipitation, Chromatin Immunoprecipitation, RNA Sequencing Assay

Eed knockdown in MEFs. ( A ) Schematic for the target design of two shRNAs against the Eed transcript. Both shRNA4 (exon 11) and shRNA5 (exon 3 and 4) were cloned into the pSicoR-ef1a-GFP vector. ( B ) Western blot analysis for DNMT3B and H3K27me3 in control and Dnmt3b MEFs (7 d dox) expressing the Eed shRNAs. ( C ) RT-qPCR analysis for Eed transcript levels in control and Dnmt3b OE MEFs expressing the shRNAs. Data are represented as mean ±standard deviation. Primer sequences are shown underneath. ( D ) Scatter plots showing DNA methylation levels of CGIs ( n = 13,357) in control MEFs and MEFs expressing the Eed shRNAs as well as MEFs that contain both the shRNA and inducible Dnmt3b.
Figure Legend Snippet: Eed knockdown in MEFs. ( A ) Schematic for the target design of two shRNAs against the Eed transcript. Both shRNA4 (exon 11) and shRNA5 (exon 3 and 4) were cloned into the pSicoR-ef1a-GFP vector. ( B ) Western blot analysis for DNMT3B and H3K27me3 in control and Dnmt3b MEFs (7 d dox) expressing the Eed shRNAs. ( C ) RT-qPCR analysis for Eed transcript levels in control and Dnmt3b OE MEFs expressing the shRNAs. Data are represented as mean ±standard deviation. Primer sequences are shown underneath. ( D ) Scatter plots showing DNA methylation levels of CGIs ( n = 13,357) in control MEFs and MEFs expressing the Eed shRNAs as well as MEFs that contain both the shRNA and inducible Dnmt3b.

Techniques Used: Clone Assay, Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Standard Deviation, DNA Methylation Assay, shRNA

35) Product Images from "Polycomb recruitment attenuates retinoic acid-induced transcription of the bivalent NR2F1 gene"

Article Title: Polycomb recruitment attenuates retinoic acid-induced transcription of the bivalent NR2F1 gene

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt367

Chromatin Signatures of Nr2F1 and Hoxa5 in F9 WT and RAR knockout cells. ( A ) Schematic of Nr2F1 IEE and promoter regions. Nr2F1 putative TSSs are indicated by angled arrows (P1, P2 and P3). The proximal nucleotide sequences are shown for the Nr2F1 promoter and IEE region, and the putative enhancer element is underlined. The GC content of the evaluated regions is illustrated in the background graphs, with CpG islands marked in gray. Note the local depletion of CpGs proximal to the Nr2F1 RefSeq promoter (P3). White arrows indicate genomic regions included in the Nr2F1 reporter constructs ( Fig. 2 B, 0.5 and 1.0 kb, respectively). The gray bars specify primary transcripts from the indicated TSSs. ( B ) ChIP analysis shows that Polycomb repressive marks (Suz12, H3K27me3 and Ring1B) decrease at the Hoxa5 promoter and RARE, but increase at the Nr2F1 promoter and IEE in F9 WT cells in response to a 24 h RA treatment (left, boxed). Similar patterns of Nr2F1 epigenetic changes were observed in RAR knockout cells (KO, right). ( C ) ChIP analysis shows that transcriptional permissive marks (H3K9/K14ac, H3K4me3 and polII-CTD) increase at the Hoxa5 promoter and RARE, and at the Nr2F1 promoter and IEE in response to a 24 h RA treatment (left). Similar patterns of Nr2F1 epigenetic changes were observed in RARα knockout cells, whereas H3K9/14ac levels did not increase in RARβ 2 and RARγ knockout cells (right). ( D ) ChIP analysis shows Histone 3 occupancy (H3) and the non-specific background signal (IgG). The ChIP signals are depicted relative to the total chromatin input in each ChIP. Statistical significance ( P
Figure Legend Snippet: Chromatin Signatures of Nr2F1 and Hoxa5 in F9 WT and RAR knockout cells. ( A ) Schematic of Nr2F1 IEE and promoter regions. Nr2F1 putative TSSs are indicated by angled arrows (P1, P2 and P3). The proximal nucleotide sequences are shown for the Nr2F1 promoter and IEE region, and the putative enhancer element is underlined. The GC content of the evaluated regions is illustrated in the background graphs, with CpG islands marked in gray. Note the local depletion of CpGs proximal to the Nr2F1 RefSeq promoter (P3). White arrows indicate genomic regions included in the Nr2F1 reporter constructs ( Fig. 2 B, 0.5 and 1.0 kb, respectively). The gray bars specify primary transcripts from the indicated TSSs. ( B ) ChIP analysis shows that Polycomb repressive marks (Suz12, H3K27me3 and Ring1B) decrease at the Hoxa5 promoter and RARE, but increase at the Nr2F1 promoter and IEE in F9 WT cells in response to a 24 h RA treatment (left, boxed). Similar patterns of Nr2F1 epigenetic changes were observed in RAR knockout cells (KO, right). ( C ) ChIP analysis shows that transcriptional permissive marks (H3K9/K14ac, H3K4me3 and polII-CTD) increase at the Hoxa5 promoter and RARE, and at the Nr2F1 promoter and IEE in response to a 24 h RA treatment (left). Similar patterns of Nr2F1 epigenetic changes were observed in RARα knockout cells, whereas H3K9/14ac levels did not increase in RARβ 2 and RARγ knockout cells (right). ( D ) ChIP analysis shows Histone 3 occupancy (H3) and the non-specific background signal (IgG). The ChIP signals are depicted relative to the total chromatin input in each ChIP. Statistical significance ( P

Techniques Used: Knock-Out, Construct, Chromatin Immunoprecipitation

Summary model of Nr2F1 and Hoxa5 epigenetic signatures in response to RA. Nr2F1 and Hoxa5 display different epigenetic signatures on RA treatment of stem cells. Nr2F1 is characterized by increased levels of PRC (Suz12 and Ring1B) and the associated H3K27me3 histone mark in response to RA. In contrast, the Hoxa5 epigenetic signature is characterized by dissociation of PRC (Suz12 and Ring1B) and reduction of the H3K27me3 histone mark. Note that the epigenetic signatures of Hoxa5 are similar in presence and absence of Suz12 after RA addition. In contrast, the epigenetic signature of Nr2F1 on RA treatment differs in the presence and absence of Suz12, thus potentially explaining the increased transcriptional activity on Suz12 knockdown. For both Nr2F1 and Hoxa5 , the transcriptional induction is marked by increased levels of H3K4me3, H3K9ac and H3K14ac permissive histone marks. The different marks are depicted as shapes whose sizes reflect the relative abundance in the specified condition. Histone H3 is depicted as a gray circle. PRC1 and PRC2 are represented by Ring1B and Suz12, respectively.
Figure Legend Snippet: Summary model of Nr2F1 and Hoxa5 epigenetic signatures in response to RA. Nr2F1 and Hoxa5 display different epigenetic signatures on RA treatment of stem cells. Nr2F1 is characterized by increased levels of PRC (Suz12 and Ring1B) and the associated H3K27me3 histone mark in response to RA. In contrast, the Hoxa5 epigenetic signature is characterized by dissociation of PRC (Suz12 and Ring1B) and reduction of the H3K27me3 histone mark. Note that the epigenetic signatures of Hoxa5 are similar in presence and absence of Suz12 after RA addition. In contrast, the epigenetic signature of Nr2F1 on RA treatment differs in the presence and absence of Suz12, thus potentially explaining the increased transcriptional activity on Suz12 knockdown. For both Nr2F1 and Hoxa5 , the transcriptional induction is marked by increased levels of H3K4me3, H3K9ac and H3K14ac permissive histone marks. The different marks are depicted as shapes whose sizes reflect the relative abundance in the specified condition. Histone H3 is depicted as a gray circle. PRC1 and PRC2 are represented by Ring1B and Suz12, respectively.

Techniques Used: Activity Assay

36) Product Images from "Dynamically and epigenetically coordinated GATA/ETS/SOX transcription factor expression is indispensable for endothelial cell differentiation"

Article Title: Dynamically and epigenetically coordinated GATA/ETS/SOX transcription factor expression is indispensable for endothelial cell differentiation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx159

Identification of the master transcription factor for EC differentiation. ( A ) Venn diagram depicting the overlap of expression cluster 2,3 and 4 in Figure 2B and histone class 1 in Figure 3C . The numbers indicate the overlapped or unique genes count. The common 15 genes are classified by GO term (right panel). ( B ) Determination of enriched transcription factor motifs calculated from the enhancer regions of HUVECs database. The MODIC method ( 32 ) was used for identification of enriched sequences and the size of the character reflects the degree of enrichment. E -value and P -value indicate the probability that de novo enriched sequences obtained from FAIRE-seq are matched to the shown ‘Web logo’ and known consensus motifs by chance, respectively. ( C ) UCSC mm9 genome browser view around Gata2, Fli1, Sox7 and Sox18 genes. ChIP-seq tags were calculated by MACS and are shown in IGV. H3K4me3 peaks were shown in red, and H3K27me3 were in blue.
Figure Legend Snippet: Identification of the master transcription factor for EC differentiation. ( A ) Venn diagram depicting the overlap of expression cluster 2,3 and 4 in Figure 2B and histone class 1 in Figure 3C . The numbers indicate the overlapped or unique genes count. The common 15 genes are classified by GO term (right panel). ( B ) Determination of enriched transcription factor motifs calculated from the enhancer regions of HUVECs database. The MODIC method ( 32 ) was used for identification of enriched sequences and the size of the character reflects the degree of enrichment. E -value and P -value indicate the probability that de novo enriched sequences obtained from FAIRE-seq are matched to the shown ‘Web logo’ and known consensus motifs by chance, respectively. ( C ) UCSC mm9 genome browser view around Gata2, Fli1, Sox7 and Sox18 genes. ChIP-seq tags were calculated by MACS and are shown in IGV. H3K4me3 peaks were shown in red, and H3K27me3 were in blue.

Techniques Used: Expressing, Chromatin Immunoprecipitation, Magnetic Cell Separation

Global and dynamic histone code analysis on the EC differentiation. ( A ) Enrichment of the H3K4me3 modification around the promoters from 200 EC specific genes in Flk-1 positive mesoderm cells for 0 (green line), 6 (yellow line) and 48 h (blue line). VEGF treatment (left) or no treatment (right panel). The average histone code profiles are shown with −5 to +5 kbp from the transcription start sites (TSS). ( B ) Enrichment of the H3K27me3 modification around the promoters from 200 EC specific genes (left) and non-EC specific genes (right panel) for 0 (green line), 6 (yellow line) and 48 h (blue line) VEGF treatment. The average histone code profiles are shown with −5 to +5 kbp from the TSS. ( C ) Heat map representation of the VEGF responsive genes. Genes were classified based on the histone modification patterns using the algorithm ‘HOPACH’. Colour intensity: red (H3K4me3 mark) and blue (H3K27me3 mark) indicate higher and white indicate lower, shown relative to median. ( D ) UCSC mm9 genome browser view around the Etv2 and Erg genes. ChIP-seq tags were calculated by MACS and shown in Integrative Genomics Viewer (IGV). H3K4me3 peaks were shown in red, and H3K27me3 were in blue. ( E ) Functional annotations for each class are shown. The enrichment scores of each group from DAVID are shown in the bar graphs.
Figure Legend Snippet: Global and dynamic histone code analysis on the EC differentiation. ( A ) Enrichment of the H3K4me3 modification around the promoters from 200 EC specific genes in Flk-1 positive mesoderm cells for 0 (green line), 6 (yellow line) and 48 h (blue line). VEGF treatment (left) or no treatment (right panel). The average histone code profiles are shown with −5 to +5 kbp from the transcription start sites (TSS). ( B ) Enrichment of the H3K27me3 modification around the promoters from 200 EC specific genes (left) and non-EC specific genes (right panel) for 0 (green line), 6 (yellow line) and 48 h (blue line) VEGF treatment. The average histone code profiles are shown with −5 to +5 kbp from the TSS. ( C ) Heat map representation of the VEGF responsive genes. Genes were classified based on the histone modification patterns using the algorithm ‘HOPACH’. Colour intensity: red (H3K4me3 mark) and blue (H3K27me3 mark) indicate higher and white indicate lower, shown relative to median. ( D ) UCSC mm9 genome browser view around the Etv2 and Erg genes. ChIP-seq tags were calculated by MACS and shown in Integrative Genomics Viewer (IGV). H3K4me3 peaks were shown in red, and H3K27me3 were in blue. ( E ) Functional annotations for each class are shown. The enrichment scores of each group from DAVID are shown in the bar graphs.

Techniques Used: Modification, Chromatin Immunoprecipitation, Magnetic Cell Separation, Functional Assay

37) Product Images from "The methyltransferase EZH2 is not required for mammary cancer development, although high EZH2 and low H3K27me3 correlate with poor prognosis of ER-positive breast cancers"

Article Title: The methyltransferase EZH2 is not required for mammary cancer development, although high EZH2 and low H3K27me3 correlate with poor prognosis of ER-positive breast cancers

Journal: Molecular carcinogenesis

doi: 10.1002/mc.22188

Kaplan-Meier survival curves based on abundance of H3K27me3 and EZH2. Overall survival plots stratified by high or low expression of H3K27me3 and EZH2 in all cases (A, B, C). Overall survival plots stratified by high or low expression of H3K27me3 and
Figure Legend Snippet: Kaplan-Meier survival curves based on abundance of H3K27me3 and EZH2. Overall survival plots stratified by high or low expression of H3K27me3 and EZH2 in all cases (A, B, C). Overall survival plots stratified by high or low expression of H3K27me3 and

Techniques Used: Expressing

Correlation of H3K27me3 and EZH2 levels with clinicopathological parameters
Figure Legend Snippet: Correlation of H3K27me3 and EZH2 levels with clinicopathological parameters

Techniques Used:

Survival analysis based on H3K27me3 and EZH2 levels
Figure Legend Snippet: Survival analysis based on H3K27me3 and EZH2 levels

Techniques Used:

Immunohistochemical staining of breast tumors for H3K27me3, EZH2 and UTX. Shown are representative examples of low and high expression, respectively.
Figure Legend Snippet: Immunohistochemical staining of breast tumors for H3K27me3, EZH2 and UTX. Shown are representative examples of low and high expression, respectively.

Techniques Used: Immunohistochemistry, Staining, Expressing

38) Product Images from "The methyltransferase EZH2 is not required for mammary cancer development, although high EZH2 and low H3K27me3 correlate with poor prognosis of ER-positive breast cancers"

Article Title: The methyltransferase EZH2 is not required for mammary cancer development, although high EZH2 and low H3K27me3 correlate with poor prognosis of ER-positive breast cancers

Journal: Molecular carcinogenesis

doi: 10.1002/mc.22188

Kaplan-Meier survival curves based on abundance of H3K27me3 and EZH2. Overall survival plots stratified by high or low expression of H3K27me3 and EZH2 in all cases (A, B, C). Overall survival plots stratified by high or low expression of H3K27me3 and
Figure Legend Snippet: Kaplan-Meier survival curves based on abundance of H3K27me3 and EZH2. Overall survival plots stratified by high or low expression of H3K27me3 and EZH2 in all cases (A, B, C). Overall survival plots stratified by high or low expression of H3K27me3 and

Techniques Used: Expressing

Correlation of H3K27me3 and EZH2 levels with clinicopathological parameters
Figure Legend Snippet: Correlation of H3K27me3 and EZH2 levels with clinicopathological parameters

Techniques Used:

Survival analysis based on H3K27me3 and EZH2 levels
Figure Legend Snippet: Survival analysis based on H3K27me3 and EZH2 levels

Techniques Used:

Immunohistochemical staining of breast tumors for H3K27me3, EZH2 and UTX. Shown are representative examples of low and high expression, respectively.
Figure Legend Snippet: Immunohistochemical staining of breast tumors for H3K27me3, EZH2 and UTX. Shown are representative examples of low and high expression, respectively.

Techniques Used: Immunohistochemistry, Staining, Expressing

39) Product Images from "The methyltransferase EZH2 is not required for mammary cancer development, although high EZH2 and low H3K27me3 correlate with poor prognosis of ER-positive breast cancers"

Article Title: The methyltransferase EZH2 is not required for mammary cancer development, although high EZH2 and low H3K27me3 correlate with poor prognosis of ER-positive breast cancers

Journal: Molecular carcinogenesis

doi: 10.1002/mc.22188

Kaplan-Meier survival curves based on abundance of H3K27me3 and EZH2. Overall survival plots stratified by high or low expression of H3K27me3 and EZH2 in all cases (A, B, C). Overall survival plots stratified by high or low expression of H3K27me3 and
Figure Legend Snippet: Kaplan-Meier survival curves based on abundance of H3K27me3 and EZH2. Overall survival plots stratified by high or low expression of H3K27me3 and EZH2 in all cases (A, B, C). Overall survival plots stratified by high or low expression of H3K27me3 and

Techniques Used: Expressing

Correlation of H3K27me3 and EZH2 levels with clinicopathological parameters
Figure Legend Snippet: Correlation of H3K27me3 and EZH2 levels with clinicopathological parameters

Techniques Used:

Survival analysis based on H3K27me3 and EZH2 levels
Figure Legend Snippet: Survival analysis based on H3K27me3 and EZH2 levels

Techniques Used:

Immunohistochemical staining of breast tumors for H3K27me3, EZH2 and UTX. Shown are representative examples of low and high expression, respectively.
Figure Legend Snippet: Immunohistochemical staining of breast tumors for H3K27me3, EZH2 and UTX. Shown are representative examples of low and high expression, respectively.

Techniques Used: Immunohistochemistry, Staining, Expressing

40) Product Images from "The phytochemical 3,3′-Diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells"

Article Title: The phytochemical 3,3′-Diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells

Journal: The Journal of nutritional biochemistry

doi: 10.1016/j.jnutbio.2017.05.005

DIM stimulates a repressive chromatin code in PSA regulatory regions. A) Schematic of AREs in the promoter and enhancer regions of PSA . Grey bars indicate positions of amplicons analyzed relative to transcriptional start site. ChIPs and qPCR assays were used to evaluate H3K4me3, H3K9me3 and H3K27me3 levels at PSA regulatory regions in LNCaP cells treated with 15 μM DIM for 48h. B) promoter (−120 bp), C) ARE-promoter (−170 bp) and D) ARE-enhancer regions of PSA gene. ChIP-data are expressed as % input compared to DMSO vehicle control. IgG (DMSO+DIM) is an average of two ChIP assays from each treatment, using Normal-IgG as negative control. Data represents three biological replicates. Graphs depict mean + standard deviation. Significance was determined by Student’s t-Test with *p
Figure Legend Snippet: DIM stimulates a repressive chromatin code in PSA regulatory regions. A) Schematic of AREs in the promoter and enhancer regions of PSA . Grey bars indicate positions of amplicons analyzed relative to transcriptional start site. ChIPs and qPCR assays were used to evaluate H3K4me3, H3K9me3 and H3K27me3 levels at PSA regulatory regions in LNCaP cells treated with 15 μM DIM for 48h. B) promoter (−120 bp), C) ARE-promoter (−170 bp) and D) ARE-enhancer regions of PSA gene. ChIP-data are expressed as % input compared to DMSO vehicle control. IgG (DMSO+DIM) is an average of two ChIP assays from each treatment, using Normal-IgG as negative control. Data represents three biological replicates. Graphs depict mean + standard deviation. Significance was determined by Student’s t-Test with *p

Techniques Used: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Standard Deviation

Increase in repressive chromatin mark H3K27me3 at the promoter region of DNA repair AR-target genes in response to DIM. LNCaP cells were treated with 15 μM DIM or DMSO as a vehicle control for 48h. H3K27me3 and H3K4me3 levels were assessed by ChIP in the promoters of A) PARP1 , B) MRE11 and C) DNA-PK . H3K9me3 levels following DIM treatment were also assessed in the DNA-PK promoter. IgG (DMSO+DIM) is an average of two ChIP assays from each treatment, using Normal-IgG as negative control ChIP-data are expressed as % input relative to DMSO vehicle control. Data represents three biological replicates. Graphs depict mean + standard deviation. Significance was determined by Student’s t-Test with *p
Figure Legend Snippet: Increase in repressive chromatin mark H3K27me3 at the promoter region of DNA repair AR-target genes in response to DIM. LNCaP cells were treated with 15 μM DIM or DMSO as a vehicle control for 48h. H3K27me3 and H3K4me3 levels were assessed by ChIP in the promoters of A) PARP1 , B) MRE11 and C) DNA-PK . H3K9me3 levels following DIM treatment were also assessed in the DNA-PK promoter. IgG (DMSO+DIM) is an average of two ChIP assays from each treatment, using Normal-IgG as negative control ChIP-data are expressed as % input relative to DMSO vehicle control. Data represents three biological replicates. Graphs depict mean + standard deviation. Significance was determined by Student’s t-Test with *p

Techniques Used: Chromatin Immunoprecipitation, Negative Control, Standard Deviation

Related Articles

other:

Article Title: ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity
Article Snippet: Consistent with previous studies, we found that H3K27me3 is highly enriched in genic regions in both ES cells and G1ME cells, with 41 and 45% of total reads falling within a gene and 3 kb promoter region with these regions constituting < 2% of the genome.

Article Title: miR-125b-1 is repressed by histone modifications in breast cancer cell lines
Article Snippet: However, in this study, we were interested in evaluating the relationship between miR -125b -1 repression and repressive histone modifications such as H3K9me3 and H3K27me3.

Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae
Article Snippet: Genes enriched in either H3K27ac or H3K27me3, were assigned to multiple different KEGG pathways.

Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae
Article Snippet: We find that the presence of H3K27ac coincides with actively transcribed genes, whereas H3K27me3 is mostly associated with clusters of repressed genes that show low levels of transcription.

Article Title: ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity
Article Snippet: Hon et al. ( ) confirmed a strong association between H3K27me3 and transcriptional repression, but they did not identify genes that carry H3K27me3 specifically in the promoter region.

Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae
Article Snippet: Results described here for the mosquito A. gambiae agree with these observations and suggest a close association between the presence of H3K27ac and H3K27me3, and the transcriptional state of genes.

Article Title: Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays
Article Snippet: Enriched GO terms with gene list among expressed and non-expressed genes marked by H3K4me3, H3K27me3 or both marks. (XLS) Click here for additional data file.

Article Title: ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity
Article Snippet: They have enrichment for H3K27me3 across the entire gene that can extend into the flanking regions and have little to no RNApol-II, H3K36me3 or H3K4me3.

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    Millipore h3k27me3
    H3K4me3 and <t>H3K27me3</t> enrichment profiles in ZF4 cells. (A) Distinct H3K4me3 and H3K27me3 enrichment profiles on indicated genomic regions. Genomic positioning is indicated by nucleotide number of the first (5′) and last (3′) probe in the tiled region. Gene names or accession numbers as well as their genomic position are shown in blue. (B) 2-D scatter plot of averaged MaxSixty values for H3K4me3 vs. H3K27me3 log 2 signal intensities. Data points (all points being shown in gray) were colored to visualize classification according to peak calling highlighting H3K4me3-enriched promoters (purple; N = 6315), H3K27me3-enriched promoters (green; N = 1079) and H3K4me3/K27me3-co-enriched promoters (blue; N = 2120). Red line is the regression line through all data points. (C) Venn diagram analysis of H3K4me3 and H3K27me3 genes.
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    H3K4me3 and H3K27me3 enrichment profiles in ZF4 cells. (A) Distinct H3K4me3 and H3K27me3 enrichment profiles on indicated genomic regions. Genomic positioning is indicated by nucleotide number of the first (5′) and last (3′) probe in the tiled region. Gene names or accession numbers as well as their genomic position are shown in blue. (B) 2-D scatter plot of averaged MaxSixty values for H3K4me3 vs. H3K27me3 log 2 signal intensities. Data points (all points being shown in gray) were colored to visualize classification according to peak calling highlighting H3K4me3-enriched promoters (purple; N = 6315), H3K27me3-enriched promoters (green; N = 1079) and H3K4me3/K27me3-co-enriched promoters (blue; N = 2120). Red line is the regression line through all data points. (C) Venn diagram analysis of H3K4me3 and H3K27me3 genes.

    Journal: PLoS ONE

    Article Title: Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays

    doi: 10.1371/journal.pone.0015651

    Figure Lengend Snippet: H3K4me3 and H3K27me3 enrichment profiles in ZF4 cells. (A) Distinct H3K4me3 and H3K27me3 enrichment profiles on indicated genomic regions. Genomic positioning is indicated by nucleotide number of the first (5′) and last (3′) probe in the tiled region. Gene names or accession numbers as well as their genomic position are shown in blue. (B) 2-D scatter plot of averaged MaxSixty values for H3K4me3 vs. H3K27me3 log 2 signal intensities. Data points (all points being shown in gray) were colored to visualize classification according to peak calling highlighting H3K4me3-enriched promoters (purple; N = 6315), H3K27me3-enriched promoters (green; N = 1079) and H3K4me3/K27me3-co-enriched promoters (blue; N = 2120). Red line is the regression line through all data points. (C) Venn diagram analysis of H3K4me3 and H3K27me3 genes.

    Article Snippet: Enriched GO terms with gene list among expressed and non-expressed genes marked by H3K4me3, H3K27me3 or both marks. (XLS) Click here for additional data file.

    Techniques:

    Distribution of H3K4me3 and H3K27me3 on promoters. Metagene analysis of the distribution of H3K4me3 and H3K27me3 occupancy on (A) H3K4me3-only, (B) H3K27me3-only and (C) H3K4me3/K27me3 tiled regions, relative to the TSS (red vertical bar). (D) Sequential ChIP analysis of H3K4me3 and H3K27me3 co-enrichment on the sox3 , sox2 promoters and on bactin1 , downstream of the coding region. Panels on the left show results from the first ChIP using antibodies indicated on the x-axis. The graph on the right shows results of the re-ChIP experiment as indicated on the x-axis.

    Journal: PLoS ONE

    Article Title: Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays

    doi: 10.1371/journal.pone.0015651

    Figure Lengend Snippet: Distribution of H3K4me3 and H3K27me3 on promoters. Metagene analysis of the distribution of H3K4me3 and H3K27me3 occupancy on (A) H3K4me3-only, (B) H3K27me3-only and (C) H3K4me3/K27me3 tiled regions, relative to the TSS (red vertical bar). (D) Sequential ChIP analysis of H3K4me3 and H3K27me3 co-enrichment on the sox3 , sox2 promoters and on bactin1 , downstream of the coding region. Panels on the left show results from the first ChIP using antibodies indicated on the x-axis. The graph on the right shows results of the re-ChIP experiment as indicated on the x-axis.

    Article Snippet: Enriched GO terms with gene list among expressed and non-expressed genes marked by H3K4me3, H3K27me3 or both marks. (XLS) Click here for additional data file.

    Techniques: Chromatin Immunoprecipitation

    Reproducibility of zebrafish ChIP-chip experiments. (A) Two-dimensional scatter plots of MaxSixty values for H3K4me3 and H3K27me3 log 2 signal intensities detected in each of two ChIP-chip replicates from ZF4 cells. Correlation coefficient (R) and regression line are shown. (B) H3K4me3 and H3K27me3 profiles detected by ChIP-chip in two independent replicates through 310 kb of zebrafish chromosome 10. Data are expressed as log 2 ChIP/input ratios. Position of methylation peaks are shown as blue horizontal bars. Tracks representing primary transcripts and tiled regions are also shown. Primary transcripts included in the region are as follows: 1) sin2 ; 2) NM_001003421; 3) NM_200663; 4) NM_001008616; 5) ENSDART00000081978; 6) ripply3 , 7) ENSDART00000081992; 8) dyrk1aa ; 9) ENSDART00000058411; 10) ENSDART00000088605; 11) NM_001037708; 12) hyou1 ; 13) hist2h2l ; 14) znf259 . Red bars in the H3K4me3 tracks indicate probes with out-of-scale signal intensity.

    Journal: PLoS ONE

    Article Title: Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays

    doi: 10.1371/journal.pone.0015651

    Figure Lengend Snippet: Reproducibility of zebrafish ChIP-chip experiments. (A) Two-dimensional scatter plots of MaxSixty values for H3K4me3 and H3K27me3 log 2 signal intensities detected in each of two ChIP-chip replicates from ZF4 cells. Correlation coefficient (R) and regression line are shown. (B) H3K4me3 and H3K27me3 profiles detected by ChIP-chip in two independent replicates through 310 kb of zebrafish chromosome 10. Data are expressed as log 2 ChIP/input ratios. Position of methylation peaks are shown as blue horizontal bars. Tracks representing primary transcripts and tiled regions are also shown. Primary transcripts included in the region are as follows: 1) sin2 ; 2) NM_001003421; 3) NM_200663; 4) NM_001008616; 5) ENSDART00000081978; 6) ripply3 , 7) ENSDART00000081992; 8) dyrk1aa ; 9) ENSDART00000058411; 10) ENSDART00000088605; 11) NM_001037708; 12) hyou1 ; 13) hist2h2l ; 14) znf259 . Red bars in the H3K4me3 tracks indicate probes with out-of-scale signal intensity.

    Article Snippet: Enriched GO terms with gene list among expressed and non-expressed genes marked by H3K4me3, H3K27me3 or both marks. (XLS) Click here for additional data file.

    Techniques: Chromatin Immunoprecipitation, Methylation

    Quantitative PCR validation of ChIP-chip data. (A) ChIP-on-chip profiles of H3K4me3 and H3K27me3 enrichment on indicated genes. Position of primary transcripts and TSS (arrow) are shown. (B) ChIP-qPCR analysis of H3K4me3 and H3K27me3 enrichment on the same genes as in (A) from separate duplicate ChIPs. ChIP DNA was not WGA amplified prior to PCR. Position of amplicons and primer sequences for each gene are shown in Table S3 . Note the correlation between ChIP- chip and ChIP-qPCR data.

    Journal: PLoS ONE

    Article Title: Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays

    doi: 10.1371/journal.pone.0015651

    Figure Lengend Snippet: Quantitative PCR validation of ChIP-chip data. (A) ChIP-on-chip profiles of H3K4me3 and H3K27me3 enrichment on indicated genes. Position of primary transcripts and TSS (arrow) are shown. (B) ChIP-qPCR analysis of H3K4me3 and H3K27me3 enrichment on the same genes as in (A) from separate duplicate ChIPs. ChIP DNA was not WGA amplified prior to PCR. Position of amplicons and primer sequences for each gene are shown in Table S3 . Note the correlation between ChIP- chip and ChIP-qPCR data.

    Article Snippet: Enriched GO terms with gene list among expressed and non-expressed genes marked by H3K4me3, H3K27me3 or both marks. (XLS) Click here for additional data file.

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Whole Genome Amplification, Amplification, Polymerase Chain Reaction

    Genes marked by H3K4me3 and/or H3K27me3 are linked to distinct functional GO terms. (A) GO term enrichment of genes containing H3K4me3, H3K27me3 or H3K4/K27me3 promoters in ZF4 cells. The twelve most significant GO terms are shown as a function of significance from bottom (highest significance) to top. (B) Representation of all enriched GO terms among H3K4/K27me3 genes. All enriched GO terms are listed in Table S1 . (C) H3K4me3 and H3K27me3 enrichment profiles on the developmentally regulated hoxc locus, expressed as log 2 ChIP/Input (y axis).

    Journal: PLoS ONE

    Article Title: Tiling Histone H3 Lysine 4 and 27 Methylation in Zebrafish Using High-Density Microarrays

    doi: 10.1371/journal.pone.0015651

    Figure Lengend Snippet: Genes marked by H3K4me3 and/or H3K27me3 are linked to distinct functional GO terms. (A) GO term enrichment of genes containing H3K4me3, H3K27me3 or H3K4/K27me3 promoters in ZF4 cells. The twelve most significant GO terms are shown as a function of significance from bottom (highest significance) to top. (B) Representation of all enriched GO terms among H3K4/K27me3 genes. All enriched GO terms are listed in Table S1 . (C) H3K4me3 and H3K27me3 enrichment profiles on the developmentally regulated hoxc locus, expressed as log 2 ChIP/Input (y axis).

    Article Snippet: Enriched GO terms with gene list among expressed and non-expressed genes marked by H3K4me3, H3K27me3 or both marks. (XLS) Click here for additional data file.

    Techniques: Functional Assay, Chromatin Immunoprecipitation

    H3K9me3 and H3K27me3 are enriched on miR-125b-1 promoters in MDA-MB-231 and MCF7 breast cancer cells, respectively. To determine which histone modification was involved in miR-125b-1 repression, we evaluated H3K9me3 ( a ) and H3K27me3 ( b ) expression in the promoter regions by chromatin immunoprecipitation

    Journal: SpringerPlus

    Article Title: miR-125b-1 is repressed by histone modifications in breast cancer cell lines

    doi: 10.1186/s40064-016-2475-z

    Figure Lengend Snippet: H3K9me3 and H3K27me3 are enriched on miR-125b-1 promoters in MDA-MB-231 and MCF7 breast cancer cells, respectively. To determine which histone modification was involved in miR-125b-1 repression, we evaluated H3K9me3 ( a ) and H3K27me3 ( b ) expression in the promoter regions by chromatin immunoprecipitation

    Article Snippet: However, in this study, we were interested in evaluating the relationship between miR -125b -1 repression and repressive histone modifications such as H3K9me3 and H3K27me3.

    Techniques: Multiple Displacement Amplification, Modification, Expressing, Chromatin Immunoprecipitation

    An inhibitor of EZH2 reactivates miR-125b-1 in MCF7 breast cancer cells. To determine the dose of GSK126 that reduces H3K27me3 levels, we treated the breast cell lines with different GSK126 concentrations and performed Western blot analysis ( a ). b Densitometric analysis of triplicate Western blots. We chose the 1000-nM dose to evaluate pri-miR-125b-1 ( c ) and mature miR-125b expression levels ( d )

    Journal: SpringerPlus

    Article Title: miR-125b-1 is repressed by histone modifications in breast cancer cell lines

    doi: 10.1186/s40064-016-2475-z

    Figure Lengend Snippet: An inhibitor of EZH2 reactivates miR-125b-1 in MCF7 breast cancer cells. To determine the dose of GSK126 that reduces H3K27me3 levels, we treated the breast cell lines with different GSK126 concentrations and performed Western blot analysis ( a ). b Densitometric analysis of triplicate Western blots. We chose the 1000-nM dose to evaluate pri-miR-125b-1 ( c ) and mature miR-125b expression levels ( d )

    Article Snippet: However, in this study, we were interested in evaluating the relationship between miR -125b -1 repression and repressive histone modifications such as H3K9me3 and H3K27me3.

    Techniques: Western Blot, Expressing

    Elevated levels of miR-125b-1 affect the expression levels of BAK1. The target BAK1 was selected based on an evaluation of H3K27me3 enrichment in MCF7 cells by ENCODE ( a ). Next, we evaluated BAK1 expression levels by qRT-PCR ( b ). Finally, we determined protein levels by Western blotting ( c , d ). n = 3 *p > 0.05

    Journal: SpringerPlus

    Article Title: miR-125b-1 is repressed by histone modifications in breast cancer cell lines

    doi: 10.1186/s40064-016-2475-z

    Figure Lengend Snippet: Elevated levels of miR-125b-1 affect the expression levels of BAK1. The target BAK1 was selected based on an evaluation of H3K27me3 enrichment in MCF7 cells by ENCODE ( a ). Next, we evaluated BAK1 expression levels by qRT-PCR ( b ). Finally, we determined protein levels by Western blotting ( c , d ). n = 3 *p > 0.05

    Article Snippet: However, in this study, we were interested in evaluating the relationship between miR -125b -1 repression and repressive histone modifications such as H3K9me3 and H3K27me3.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Genome-wide distribution of histone modifications . Distribution of (A) H3K27me3 and (B) H3K27ac with respect to gene features in A. gambiae midguts. The enrichment or depletion is shown relative to chromatin input. The regions in the map comprise gene bodies flanked by a segment of 200 bp at the 5′ end of TSSs and TTSs. Average profiles across gene regions ±200 bp for each histone modification are shown on top. (C) Heatmap of RNA-seq data showing the level gene expression, as read count, profiled along the region. In all heatmaps (A–C) genes were organized into 3 clusters based on their level of H3K27me3. For H3K27ac, cluster genes are ordered by descent but independent to H3K27me3. The color bars indicate the range of intensities based on ChIP enrichment, from red to blues for higher to low enrichment values. Boxplots showing the mean enrichment of H3K27ac and H3K27me3 by cluster (D) , and mean level of gene expression by gene cluster (E) . Significant pairwise comparisons are indicated by asterisks (see text).

    Journal: Frontiers in Genetics

    Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae

    doi: 10.3389/fgene.2014.00277

    Figure Lengend Snippet: Genome-wide distribution of histone modifications . Distribution of (A) H3K27me3 and (B) H3K27ac with respect to gene features in A. gambiae midguts. The enrichment or depletion is shown relative to chromatin input. The regions in the map comprise gene bodies flanked by a segment of 200 bp at the 5′ end of TSSs and TTSs. Average profiles across gene regions ±200 bp for each histone modification are shown on top. (C) Heatmap of RNA-seq data showing the level gene expression, as read count, profiled along the region. In all heatmaps (A–C) genes were organized into 3 clusters based on their level of H3K27me3. For H3K27ac, cluster genes are ordered by descent but independent to H3K27me3. The color bars indicate the range of intensities based on ChIP enrichment, from red to blues for higher to low enrichment values. Boxplots showing the mean enrichment of H3K27ac and H3K27me3 by cluster (D) , and mean level of gene expression by gene cluster (E) . Significant pairwise comparisons are indicated by asterisks (see text).

    Article Snippet: Genes enriched in either H3K27ac or H3K27me3, were assigned to multiple different KEGG pathways.

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

    Functional analysis of A. gambiae genes . The graphs show GO terms significantly associated with genes that show significant enrichment or depletion of H3K27ac and H3K27me3 at high levels (see Figure 2 ). Bars corresponds to number of sequences associated with each GO term. In the case of genes marked with H3K27ac from cluster 1 due to the number of records only most significant GO terms are shown (see Table S2 for the complete list).

    Journal: Frontiers in Genetics

    Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae

    doi: 10.3389/fgene.2014.00277

    Figure Lengend Snippet: Functional analysis of A. gambiae genes . The graphs show GO terms significantly associated with genes that show significant enrichment or depletion of H3K27ac and H3K27me3 at high levels (see Figure 2 ). Bars corresponds to number of sequences associated with each GO term. In the case of genes marked with H3K27ac from cluster 1 due to the number of records only most significant GO terms are shown (see Table S2 for the complete list).

    Article Snippet: Genes enriched in either H3K27ac or H3K27me3, were assigned to multiple different KEGG pathways.

    Techniques: Functional Assay

    Histone modification levels in selected genes. (A) Enrichment profile of histone PTMs in two Hox genes of A. gambiae (Antennapedia homeotic proteins, AGAP004659-AGAP004660). These are classic examples of Drosophila developmental genes where H3K27me3 is highly enriched and distributed in large domains that encompass both genic and intergenic regions in cells where the Antennapedia gene is silenced. Examples of various histone modification profiles, based on the level of enrichment or depletion in H3K27ac or H3K27me3, in two candidate genes: (B) Rel2 (AGAP006747, GO:0005515), and (C) Dscam (AGAP007092, GO: GO:0005515) (Tables S2, S3). The scale of the tracks is proportional to the number of sequencing reads for each histone modification. Gene expression in terms number of RNA reads mapped to each gene is also displayed.

    Journal: Frontiers in Genetics

    Article Title: Insights into the epigenomic landscape of the human malaria vector Anopheles gambiae

    doi: 10.3389/fgene.2014.00277

    Figure Lengend Snippet: Histone modification levels in selected genes. (A) Enrichment profile of histone PTMs in two Hox genes of A. gambiae (Antennapedia homeotic proteins, AGAP004659-AGAP004660). These are classic examples of Drosophila developmental genes where H3K27me3 is highly enriched and distributed in large domains that encompass both genic and intergenic regions in cells where the Antennapedia gene is silenced. Examples of various histone modification profiles, based on the level of enrichment or depletion in H3K27ac or H3K27me3, in two candidate genes: (B) Rel2 (AGAP006747, GO:0005515), and (C) Dscam (AGAP007092, GO: GO:0005515) (Tables S2, S3). The scale of the tracks is proportional to the number of sequencing reads for each histone modification. Gene expression in terms number of RNA reads mapped to each gene is also displayed.

    Article Snippet: Genes enriched in either H3K27ac or H3K27me3, were assigned to multiple different KEGG pathways.

    Techniques: Modification, Sequencing, Expressing