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Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing.

Pérez-Lluch S, Blanco E, Carbonell A, Raha D, Snyder M, Serras F, Corominas M - Nucleic Acids Res. (2011)

Bottom Line: We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription.Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies.Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica i Institut de Biomedicina (IBUB), Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain.

ABSTRACT
An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.

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Correlation between gene expression levels, ASH2, histone marks and RNA Polymerase II occupancy. (A) Ranking of genes according to their expression levels in the wing imaginal disc. Genes on the Affymetrix array (left) are classified as highly expressed (dark red), expressed (orange) or silenced (blue); target genes of ASH2, histone modifications, PolIIS5P and PolIIS2P are distributed relative to their expression levels (right). (B) Distribution of ChIP-Seq reads of genes belonging to each expression category in the Affymetrix array across the TSS and the idealized gene. Highly expressed genes are shown in red, expressed genes in orange and silenced genes in blue.
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Figure 2: Correlation between gene expression levels, ASH2, histone marks and RNA Polymerase II occupancy. (A) Ranking of genes according to their expression levels in the wing imaginal disc. Genes on the Affymetrix array (left) are classified as highly expressed (dark red), expressed (orange) or silenced (blue); target genes of ASH2, histone modifications, PolIIS5P and PolIIS2P are distributed relative to their expression levels (right). (B) Distribution of ChIP-Seq reads of genes belonging to each expression category in the Affymetrix array across the TSS and the idealized gene. Highly expressed genes are shown in red, expressed genes in orange and silenced genes in blue.

Mentions: To uncover the relationship between ASH2 and transcription, we took advantage of previously published data on the wing disc transcriptome (14). We classified the genes into three categories according to their expression level: silenced, expressed and highly expressed genes (Figure 2A). We also performed ChIP-Seq analysis using specific antibodies against two modified forms of RNA Polymerase II: serine 5 phosphorylated (PolIIS5P, as a mark of the stalled polymerase at the TSS) and serine 2 phosphorylated (PolIIS2P, as the elongating mark) (Supplementary Table S1). We found 1080 genes containing only PolIIS5P mark (putatively stalled genes), 1452 genes showing only the elongating PolIIS2P mark and 1817 genes with both. As expected, PolIIS5P, like ASH2, peaks around the TSS, and the elongating polymerase (PolIIS2P) is present in actively transcribed regions, coinciding with H3K36me3 (Figure 2B). We uncovered a positive association between gene expression and number of ChIP-Seq reads as previously reported (40). The correlation between the expression level and the ChIP-Seq data (Spearman’s rank correlation coefficient, see ‘Material and Methods’ section) confirmed that the set of expressed genes is clearly enriched in ASH2 (correlation coefficient 0.88), H3K4me3 (0.93), PolIIS5P (0.95), PolIIS2P (0.95) and H3K36me3 (0.93) targets. In contrast, H3K27me3 (–0.84) is primarily associated with silenced genes (Figure 2A).Figure 2.


Genome-wide chromatin occupancy analysis reveals a role for ASH2 in transcriptional pausing.

Pérez-Lluch S, Blanco E, Carbonell A, Raha D, Snyder M, Serras F, Corominas M - Nucleic Acids Res. (2011)

Correlation between gene expression levels, ASH2, histone marks and RNA Polymerase II occupancy. (A) Ranking of genes according to their expression levels in the wing imaginal disc. Genes on the Affymetrix array (left) are classified as highly expressed (dark red), expressed (orange) or silenced (blue); target genes of ASH2, histone modifications, PolIIS5P and PolIIS2P are distributed relative to their expression levels (right). (B) Distribution of ChIP-Seq reads of genes belonging to each expression category in the Affymetrix array across the TSS and the idealized gene. Highly expressed genes are shown in red, expressed genes in orange and silenced genes in blue.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 2: Correlation between gene expression levels, ASH2, histone marks and RNA Polymerase II occupancy. (A) Ranking of genes according to their expression levels in the wing imaginal disc. Genes on the Affymetrix array (left) are classified as highly expressed (dark red), expressed (orange) or silenced (blue); target genes of ASH2, histone modifications, PolIIS5P and PolIIS2P are distributed relative to their expression levels (right). (B) Distribution of ChIP-Seq reads of genes belonging to each expression category in the Affymetrix array across the TSS and the idealized gene. Highly expressed genes are shown in red, expressed genes in orange and silenced genes in blue.
Mentions: To uncover the relationship between ASH2 and transcription, we took advantage of previously published data on the wing disc transcriptome (14). We classified the genes into three categories according to their expression level: silenced, expressed and highly expressed genes (Figure 2A). We also performed ChIP-Seq analysis using specific antibodies against two modified forms of RNA Polymerase II: serine 5 phosphorylated (PolIIS5P, as a mark of the stalled polymerase at the TSS) and serine 2 phosphorylated (PolIIS2P, as the elongating mark) (Supplementary Table S1). We found 1080 genes containing only PolIIS5P mark (putatively stalled genes), 1452 genes showing only the elongating PolIIS2P mark and 1817 genes with both. As expected, PolIIS5P, like ASH2, peaks around the TSS, and the elongating polymerase (PolIIS2P) is present in actively transcribed regions, coinciding with H3K36me3 (Figure 2B). We uncovered a positive association between gene expression and number of ChIP-Seq reads as previously reported (40). The correlation between the expression level and the ChIP-Seq data (Spearman’s rank correlation coefficient, see ‘Material and Methods’ section) confirmed that the set of expressed genes is clearly enriched in ASH2 (correlation coefficient 0.88), H3K4me3 (0.93), PolIIS5P (0.95), PolIIS2P (0.95) and H3K36me3 (0.93) targets. In contrast, H3K27me3 (–0.84) is primarily associated with silenced genes (Figure 2A).Figure 2.

Bottom Line: We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription.Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies.Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants.

View Article: PubMed Central - PubMed

Affiliation: Departament de Genètica i Institut de Biomedicina (IBUB), Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain.

ABSTRACT
An important mechanism for gene regulation involves chromatin changes via histone modification. One such modification is histone H3 lysine 4 trimethylation (H3K4me3), which requires histone methyltranferase complexes (HMT) containing the trithorax-group (trxG) protein ASH2. Mutations in ash2 cause a variety of pattern formation defects in the Drosophila wing. We have identified genome-wide binding of ASH2 in wing imaginal discs using chromatin immunoprecipitation combined with sequencing (ChIP-Seq). Our results show that genes with functions in development and transcriptional regulation are activated by ASH2 via H3K4 trimethylation in nearby nucleosomes. We have characterized the occupancy of phosphorylated forms of RNA Polymerase II and histone marks associated with activation and repression of transcription. ASH2 occupancy correlates with phosphorylated forms of RNA Polymerase II and histone activating marks in expressed genes. Additionally, RNA Polymerase II phosphorylation on serine 5 and H3K4me3 are reduced in ash2 mutants in comparison to wild-type flies. Finally, we have identified specific motifs associated with ASH2 binding in genes that are differentially expressed in ash2 mutants. Our data suggest that recruitment of the ASH2-containing HMT complexes is context specific and points to a function of ASH2 and H3K4me3 in transcriptional pausing control.

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