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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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Landscape of ASH2 binding and histone methylation profiles in the wing imaginal disc. (A) UCSC Genome Browser overview of the ChIP-Seq reads across two regions of chromosome 3R: from the top, the input sample, ASH2, H3K4me3, H3K27me3, H3K36me3 and RefSeq genes. The height of the peaks represents the number of reads obtained for each mark in each region by ChIP-Seq. The enriched regions (targets) are shown in brown boxes below the corresponding sample. (B) Venn diagrams showing the intersection between ASH2, H3K4me3 and H3K27me3 (left) or H3K36me3 (right). (C) Projection of ASH2, H3K4me3, H3K36me3 and H3K27me3 over the TSS and the idealized gene.
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Figure 1: Landscape of ASH2 binding and histone methylation profiles in the wing imaginal disc. (A) UCSC Genome Browser overview of the ChIP-Seq reads across two regions of chromosome 3R: from the top, the input sample, ASH2, H3K4me3, H3K27me3, H3K36me3 and RefSeq genes. The height of the peaks represents the number of reads obtained for each mark in each region by ChIP-Seq. The enriched regions (targets) are shown in brown boxes below the corresponding sample. (B) Venn diagrams showing the intersection between ASH2, H3K4me3 and H3K27me3 (left) or H3K36me3 (right). (C) Projection of ASH2, H3K4me3, H3K36me3 and H3K27me3 over the TSS and the idealized gene.

Mentions: ASH2 occupancy was mapped using chromatin isolated from third instar larva wing imaginal discs, obtaining 8009 target genes (Figure 1 and Supplementary Table S1). GO analysis revealed a significant enrichment in development and morphogenesis categories (Supplementary Figure S1). We also determined the genomic distribution of H3K4me3 and H3K36me3, as specific marks of positive transcriptional regulation and H3K27me3 as a negative mark (Supplementary Table S1). We identified 5730 target genes for H3K4me3, 4919 for H3K36me3 and 2999 for H3K27me3. Figure 1A shows an example of ASH2 binding between two peaks of H3K4me3 associated with katanin-60 and Mms19 genes, which display opposite transcriptional orientation. H3K36me3 extends over the gene region of both expressed genes. By contrast, in an example of a gene silenced in the wing disc, H3K27me3 is spread throughout the Deformed (Dfd) locus. As anticipated, there was extensive overlap between ASH2 occupancy and the H3K4me3 and H3K36me3 marks, but not with H3K27me3 (Figure 1B). A subset of 441 genes has both activating and silencing marks, and among those, 423 contain ASH2 binding sites. Genes from several pathways known to be expressed differentially in the wing disc are included in this group (Supplementary Figure S1), suggesting that the two marks are present in various cell types according to their transcriptional state. Thus, we can predict that uncharacterized genes of this group display heterogeneous expression patterns in the wing tissue.Figure 1.


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)

Landscape of ASH2 binding and histone methylation profiles in the wing imaginal disc. (A) UCSC Genome Browser overview of the ChIP-Seq reads across two regions of chromosome 3R: from the top, the input sample, ASH2, H3K4me3, H3K27me3, H3K36me3 and RefSeq genes. The height of the peaks represents the number of reads obtained for each mark in each region by ChIP-Seq. The enriched regions (targets) are shown in brown boxes below the corresponding sample. (B) Venn diagrams showing the intersection between ASH2, H3K4me3 and H3K27me3 (left) or H3K36me3 (right). (C) Projection of ASH2, H3K4me3, H3K36me3 and H3K27me3 over the TSS and the idealized gene.
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Related In: Results  -  Collection

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Figure 1: Landscape of ASH2 binding and histone methylation profiles in the wing imaginal disc. (A) UCSC Genome Browser overview of the ChIP-Seq reads across two regions of chromosome 3R: from the top, the input sample, ASH2, H3K4me3, H3K27me3, H3K36me3 and RefSeq genes. The height of the peaks represents the number of reads obtained for each mark in each region by ChIP-Seq. The enriched regions (targets) are shown in brown boxes below the corresponding sample. (B) Venn diagrams showing the intersection between ASH2, H3K4me3 and H3K27me3 (left) or H3K36me3 (right). (C) Projection of ASH2, H3K4me3, H3K36me3 and H3K27me3 over the TSS and the idealized gene.
Mentions: ASH2 occupancy was mapped using chromatin isolated from third instar larva wing imaginal discs, obtaining 8009 target genes (Figure 1 and Supplementary Table S1). GO analysis revealed a significant enrichment in development and morphogenesis categories (Supplementary Figure S1). We also determined the genomic distribution of H3K4me3 and H3K36me3, as specific marks of positive transcriptional regulation and H3K27me3 as a negative mark (Supplementary Table S1). We identified 5730 target genes for H3K4me3, 4919 for H3K36me3 and 2999 for H3K27me3. Figure 1A shows an example of ASH2 binding between two peaks of H3K4me3 associated with katanin-60 and Mms19 genes, which display opposite transcriptional orientation. H3K36me3 extends over the gene region of both expressed genes. By contrast, in an example of a gene silenced in the wing disc, H3K27me3 is spread throughout the Deformed (Dfd) locus. As anticipated, there was extensive overlap between ASH2 occupancy and the H3K4me3 and H3K36me3 marks, but not with H3K27me3 (Figure 1B). A subset of 441 genes has both activating and silencing marks, and among those, 423 contain ASH2 binding sites. Genes from several pathways known to be expressed differentially in the wing disc are included in this group (Supplementary Figure S1), suggesting that the two marks are present in various cell types according to their transcriptional state. Thus, we can predict that uncharacterized genes of this group display heterogeneous expression patterns in the wing tissue.Figure 1.

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.

Show MeSH