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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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ASH2 direct targets and the transcriptome of ash2 mutants. (A) Gene Ontology (GO) term enrichment of ASH2 and H3K4me3 target genes identified as downregulated (left) and upregulated (right) in ash2 mutant versus wild-type wing discs. The number of genes in each category is shown within the bars. (B) Distribution of ASH2 (left) and H3K4me3 (right) reads over the transcriptional start site (TSS) of upregulated and downregulated genes in the mutant array. In the ASH2 plot, the black box depicts the preferred position of ASH2 over the gene. This region (–370 to +560) was aligned to identify enriched motifs within each list of genes.
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Figure 3: ASH2 direct targets and the transcriptome of ash2 mutants. (A) Gene Ontology (GO) term enrichment of ASH2 and H3K4me3 target genes identified as downregulated (left) and upregulated (right) in ash2 mutant versus wild-type wing discs. The number of genes in each category is shown within the bars. (B) Distribution of ASH2 (left) and H3K4me3 (right) reads over the transcriptional start site (TSS) of upregulated and downregulated genes in the mutant array. In the ASH2 plot, the black box depicts the preferred position of ASH2 over the gene. This region (–370 to +560) was aligned to identify enriched motifs within each list of genes.

Mentions: In light of our results, we reanalysed the expression data obtained previously in microarray analyses of ash2 mutant discs (14). By comparing wild-type with ash2I1 mutants, we identified 342 downregulated genes and 368 upregulated genes in wing imaginal discs (see ‘Materials and Methods’ section). A significant fraction of these differentially expressed genes are ASH2 target genes: 294 downregulated genes (85%) and 253 upregulated genes (69%). We next selected those genes that also present the H3K4me3 mark and found 196 ASH2 and H3K4me3 target genes among the downregulated genes and 137 among the upregulated ones. These genes display distinct features in terms of GO categories (Figure 3A). The downregulated set of genes is enriched in development and transcription categories, whereas the upregulated list is enriched in ribosomal and mitochondrial metabolism categories. Downregulated genes and upregulated genes also show significant differences (see ‘Materials and Methods’ section) in gene size (on average 14 068 and 6858 bp, respectively, P-value <10−5), number of exons (5.9 and 3.6 exons, P-value <10−13) and number of alternative forms as annotated in RefSeq (2.3 and 1.3 alternative transcripts, P-value <10−11). Moreover, genes showing a higher expression level in the mutant condition do not correspond to silenced genes in the wild-type disc. Instead, those genes were already expressed and only increased their values in the absence of ASH2. The projection of ASH2 and H3K4me3 reads over the TSS of downregulated and upregulated genes uncovers no differences in their occupancy. The difference in number of reads of H3K4me3 may reflect the number of cells presenting this activating mark in the wing disc (Figure 3B). Taken together, our data suggest that ASH2 action is dependent on interactions with other transcriptional regulators.Figure 3.


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)

ASH2 direct targets and the transcriptome of ash2 mutants. (A) Gene Ontology (GO) term enrichment of ASH2 and H3K4me3 target genes identified as downregulated (left) and upregulated (right) in ash2 mutant versus wild-type wing discs. The number of genes in each category is shown within the bars. (B) Distribution of ASH2 (left) and H3K4me3 (right) reads over the transcriptional start site (TSS) of upregulated and downregulated genes in the mutant array. In the ASH2 plot, the black box depicts the preferred position of ASH2 over the gene. This region (–370 to +560) was aligned to identify enriched motifs within each list of genes.
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Related In: Results  -  Collection

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Figure 3: ASH2 direct targets and the transcriptome of ash2 mutants. (A) Gene Ontology (GO) term enrichment of ASH2 and H3K4me3 target genes identified as downregulated (left) and upregulated (right) in ash2 mutant versus wild-type wing discs. The number of genes in each category is shown within the bars. (B) Distribution of ASH2 (left) and H3K4me3 (right) reads over the transcriptional start site (TSS) of upregulated and downregulated genes in the mutant array. In the ASH2 plot, the black box depicts the preferred position of ASH2 over the gene. This region (–370 to +560) was aligned to identify enriched motifs within each list of genes.
Mentions: In light of our results, we reanalysed the expression data obtained previously in microarray analyses of ash2 mutant discs (14). By comparing wild-type with ash2I1 mutants, we identified 342 downregulated genes and 368 upregulated genes in wing imaginal discs (see ‘Materials and Methods’ section). A significant fraction of these differentially expressed genes are ASH2 target genes: 294 downregulated genes (85%) and 253 upregulated genes (69%). We next selected those genes that also present the H3K4me3 mark and found 196 ASH2 and H3K4me3 target genes among the downregulated genes and 137 among the upregulated ones. These genes display distinct features in terms of GO categories (Figure 3A). The downregulated set of genes is enriched in development and transcription categories, whereas the upregulated list is enriched in ribosomal and mitochondrial metabolism categories. Downregulated genes and upregulated genes also show significant differences (see ‘Materials and Methods’ section) in gene size (on average 14 068 and 6858 bp, respectively, P-value <10−5), number of exons (5.9 and 3.6 exons, P-value <10−13) and number of alternative forms as annotated in RefSeq (2.3 and 1.3 alternative transcripts, P-value <10−11). Moreover, genes showing a higher expression level in the mutant condition do not correspond to silenced genes in the wild-type disc. Instead, those genes were already expressed and only increased their values in the absence of ASH2. The projection of ASH2 and H3K4me3 reads over the TSS of downregulated and upregulated genes uncovers no differences in their occupancy. The difference in number of reads of H3K4me3 may reflect the number of cells presenting this activating mark in the wing disc (Figure 3B). Taken together, our data suggest that ASH2 action is dependent on interactions with other transcriptional regulators.Figure 3.

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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