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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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Characterization of ASH2 binding regions. (A) Motifs identified in the region around the TSS of 196 downregulated (left) and 137 upregulated genes (right) in ash2 mutants. The following information is shown for each motif: transcription factor, motif logo, distribution of sites around the TSS, total number of predictions and number of conserved sites in at least five Drosophila species (in parenthesis). (B) Venn diagrams showing the intersection between genes harbouring the characteristic motifs in ASH2 binding regions of downregulated and upregulated sets of genes. (C) Identification of modules constituted of two different motifs in ASH2 binding regions (maximum distance to define a module is 100 bp). A selection of four classes out of the full set of combinations is shown here.
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Figure 4: Characterization of ASH2 binding regions. (A) Motifs identified in the region around the TSS of 196 downregulated (left) and 137 upregulated genes (right) in ash2 mutants. The following information is shown for each motif: transcription factor, motif logo, distribution of sites around the TSS, total number of predictions and number of conserved sites in at least five Drosophila species (in parenthesis). (B) Venn diagrams showing the intersection between genes harbouring the characteristic motifs in ASH2 binding regions of downregulated and upregulated sets of genes. (C) Identification of modules constituted of two different motifs in ASH2 binding regions (maximum distance to define a module is 100 bp). A selection of four classes out of the full set of combinations is shown here.

Mentions: To address this possibility and to understand the sequence determinants of ASH2 binding, we proceeded to computationally characterize the regions around the TSS of upregulated and downregulated genes. Using motif discovery tools, we first identified multiple regulatory sites specifically present on each set of sequences. Complementary to this approach, we used TRANSFAC and JASPAR to scan these regions and enrich the initial collection of motifs. We next filtered out those predictions that were not confirmed by phylogenetic footprinting using the genomes of 12 Drosophilae by selecting only those sites conserved in D. pseudoobscura and at least four additional Drosophilids (enrichment calculated in comparison to the total number of conserved sites of each class in the D. melanogaster genome, see ‘Materials and Methods’ section). Roughly, 50% of ASH2-regulated genes presented at least one evolutionarily conserved motif (see Figure 4A). As anticipated, we found the GAGA motif, known to engage the GAGA transcription factor GAF, within the ASH2-binding regions of a significant subset of downregulated genes (58 genes, P-value <10−12). Recently published data from ChIP-on-chip analysis of GAF in Drosophila embryos (39) support our predictions, since 74% of GAGA predicted sites are located within GAF ChIP-on-chip regions. Interestingly, ASH2 binding regions are enriched in E2F-binding sites (42 genes, P-value <10−7) known to recruit E2F transcription factors. A different situation was observed in the set of upregulated genes, where we identified a non-canonical E-box (48 genes, P-value <10−10) and a DRE motif (39 genes, P-value <10−8), known to recruit the DNA replication-related element factor (DREF) (41). We also identified a common motif in both lists of genes (TGGTCACACTG) that is reportedly involved in the recruitment of Mnt/Max complexes (42). In fact, 18 putative Mnt/Max sites overlap with binding regions previously defined by DamID analysis (42) supporting our predictions. One novel motif was additionally identified in each group (Motifs 1 and 2 in Figure 4A). We believe that these novel sequences, together with the transcription factors, participate in ASH2 binding. Again, given the stringent protocol employed to identify these motifs, our results are likely to underestimate the actual number of binding sites. In order to decipher putative cis-regulatory modules underlying ASH2-binding regions, we depict the genes in both sets containing two or more motifs (Figure 4B). We next focused on those cases in which the binding motifs are located at a distance up to 100 bp, thus constituting a plausible regulatory unit. As shown in Figure 4C, we characterized several ASH2-binding regions that manifest specific preferences concerning local positioning and order between the components of each potential module.Figure 4.


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)

Characterization of ASH2 binding regions. (A) Motifs identified in the region around the TSS of 196 downregulated (left) and 137 upregulated genes (right) in ash2 mutants. The following information is shown for each motif: transcription factor, motif logo, distribution of sites around the TSS, total number of predictions and number of conserved sites in at least five Drosophila species (in parenthesis). (B) Venn diagrams showing the intersection between genes harbouring the characteristic motifs in ASH2 binding regions of downregulated and upregulated sets of genes. (C) Identification of modules constituted of two different motifs in ASH2 binding regions (maximum distance to define a module is 100 bp). A selection of four classes out of the full set of combinations is shown here.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Characterization of ASH2 binding regions. (A) Motifs identified in the region around the TSS of 196 downregulated (left) and 137 upregulated genes (right) in ash2 mutants. The following information is shown for each motif: transcription factor, motif logo, distribution of sites around the TSS, total number of predictions and number of conserved sites in at least five Drosophila species (in parenthesis). (B) Venn diagrams showing the intersection between genes harbouring the characteristic motifs in ASH2 binding regions of downregulated and upregulated sets of genes. (C) Identification of modules constituted of two different motifs in ASH2 binding regions (maximum distance to define a module is 100 bp). A selection of four classes out of the full set of combinations is shown here.
Mentions: To address this possibility and to understand the sequence determinants of ASH2 binding, we proceeded to computationally characterize the regions around the TSS of upregulated and downregulated genes. Using motif discovery tools, we first identified multiple regulatory sites specifically present on each set of sequences. Complementary to this approach, we used TRANSFAC and JASPAR to scan these regions and enrich the initial collection of motifs. We next filtered out those predictions that were not confirmed by phylogenetic footprinting using the genomes of 12 Drosophilae by selecting only those sites conserved in D. pseudoobscura and at least four additional Drosophilids (enrichment calculated in comparison to the total number of conserved sites of each class in the D. melanogaster genome, see ‘Materials and Methods’ section). Roughly, 50% of ASH2-regulated genes presented at least one evolutionarily conserved motif (see Figure 4A). As anticipated, we found the GAGA motif, known to engage the GAGA transcription factor GAF, within the ASH2-binding regions of a significant subset of downregulated genes (58 genes, P-value <10−12). Recently published data from ChIP-on-chip analysis of GAF in Drosophila embryos (39) support our predictions, since 74% of GAGA predicted sites are located within GAF ChIP-on-chip regions. Interestingly, ASH2 binding regions are enriched in E2F-binding sites (42 genes, P-value <10−7) known to recruit E2F transcription factors. A different situation was observed in the set of upregulated genes, where we identified a non-canonical E-box (48 genes, P-value <10−10) and a DRE motif (39 genes, P-value <10−8), known to recruit the DNA replication-related element factor (DREF) (41). We also identified a common motif in both lists of genes (TGGTCACACTG) that is reportedly involved in the recruitment of Mnt/Max complexes (42). In fact, 18 putative Mnt/Max sites overlap with binding regions previously defined by DamID analysis (42) supporting our predictions. One novel motif was additionally identified in each group (Motifs 1 and 2 in Figure 4A). We believe that these novel sequences, together with the transcription factors, participate in ASH2 binding. Again, given the stringent protocol employed to identify these motifs, our results are likely to underestimate the actual number of binding sites. In order to decipher putative cis-regulatory modules underlying ASH2-binding regions, we depict the genes in both sets containing two or more motifs (Figure 4B). We next focused on those cases in which the binding motifs are located at a distance up to 100 bp, thus constituting a plausible regulatory unit. As shown in Figure 4C, we characterized several ASH2-binding regions that manifest specific preferences concerning local positioning and order between the components of each potential module.Figure 4.

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