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DSIF and RNA polymerase II CTD phosphorylation coordinate the recruitment of Rpd3S to actively transcribed genes.

Drouin S, Laramée L, Jacques PÉ, Forest A, Bergeron M, Robert F - PLoS Genet. (2010)

Bottom Line: However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive.Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes.Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada.

ABSTRACT
Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP-Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.

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Set2-dependent H3K36 methylation is required for the function of Rpd3S.(A,B) SOM clustering of H4K5ac enrichment (A) and RNAPII occupancy (B) on ORFs of all genes in WT cells, along with the difference calculated between the enrichment observed in various mutants and WT cells.
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pgen-1001173-g003: Set2-dependent H3K36 methylation is required for the function of Rpd3S.(A,B) SOM clustering of H4K5ac enrichment (A) and RNAPII occupancy (B) on ORFs of all genes in WT cells, along with the difference calculated between the enrichment observed in various mutants and WT cells.

Mentions: Since we—quite surprisingly—observed, however, that Rpd3S binding to genes is mostly independent of histone methylation by Set2, we decided to test whether the activity of Rpd3S requires methylation of H3K36 by Set2. To do so, we looked at H4K5 acetylation (H4K5ac) by ChIP-chip. We used H4K5 acetylation to score for Rpd3S activity because it was shown previously to be a robust read out for Rpd3 activity in ChIP-chip assays [41]. Similar to other groups [34], [35], [38], [40], we observed decreased acetylation on promoters in set2Δ, H3K36A or Rpd3S mutants (Figure S3). Histone acetylation is also dramatically affected across ORFs in these mutants. As shown in Figure 3A, we observed a loss of acetylation for normally highly acetylated ORFs, and a gain in acetylation for ORFs that exhibit low levels in the wild type. Because Set2 and Rpd3S are both known to prevent cryptic initiation within ORFs, we repeated the same analyses on RNAPII ChIP-chip results, and found a similar pattern to that observed for H4K5ac, namely that ORFs with high RNAPII enrichment show decreased RNAPII levels in the absence of Set2 or Rpd3S, whereas ORFs with low RNAPII tend to display higher levels of polymerase (Figure 3B). These results clearly show that the activity of the Rpd3S complex requires methylation of H3K36 by Set2.


DSIF and RNA polymerase II CTD phosphorylation coordinate the recruitment of Rpd3S to actively transcribed genes.

Drouin S, Laramée L, Jacques PÉ, Forest A, Bergeron M, Robert F - PLoS Genet. (2010)

Set2-dependent H3K36 methylation is required for the function of Rpd3S.(A,B) SOM clustering of H4K5ac enrichment (A) and RNAPII occupancy (B) on ORFs of all genes in WT cells, along with the difference calculated between the enrichment observed in various mutants and WT cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965751&req=5

pgen-1001173-g003: Set2-dependent H3K36 methylation is required for the function of Rpd3S.(A,B) SOM clustering of H4K5ac enrichment (A) and RNAPII occupancy (B) on ORFs of all genes in WT cells, along with the difference calculated between the enrichment observed in various mutants and WT cells.
Mentions: Since we—quite surprisingly—observed, however, that Rpd3S binding to genes is mostly independent of histone methylation by Set2, we decided to test whether the activity of Rpd3S requires methylation of H3K36 by Set2. To do so, we looked at H4K5 acetylation (H4K5ac) by ChIP-chip. We used H4K5 acetylation to score for Rpd3S activity because it was shown previously to be a robust read out for Rpd3 activity in ChIP-chip assays [41]. Similar to other groups [34], [35], [38], [40], we observed decreased acetylation on promoters in set2Δ, H3K36A or Rpd3S mutants (Figure S3). Histone acetylation is also dramatically affected across ORFs in these mutants. As shown in Figure 3A, we observed a loss of acetylation for normally highly acetylated ORFs, and a gain in acetylation for ORFs that exhibit low levels in the wild type. Because Set2 and Rpd3S are both known to prevent cryptic initiation within ORFs, we repeated the same analyses on RNAPII ChIP-chip results, and found a similar pattern to that observed for H4K5ac, namely that ORFs with high RNAPII enrichment show decreased RNAPII levels in the absence of Set2 or Rpd3S, whereas ORFs with low RNAPII tend to display higher levels of polymerase (Figure 3B). These results clearly show that the activity of the Rpd3S complex requires methylation of H3K36 by Set2.

Bottom Line: However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive.Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes.Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.

View Article: PubMed Central - PubMed

Affiliation: Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada.

ABSTRACT
Histone deacetylase Rpd3 is part of two distinct complexes: the large (Rpd3L) and small (Rpd3S) complexes. While Rpd3L targets specific promoters for gene repression, Rpd3S is recruited to ORFs to deacetylate histones in the wake of RNA polymerase II, to prevent cryptic initiation within genes. Methylation of histone H3 at lysine 36 by the Set2 methyltransferase is thought to mediate the recruitment of Rpd3S. Here, we confirm by ChIP-Chip that Rpd3S binds active ORFs. Surprisingly, however, Rpd3S is not recruited to all active genes, and its recruitment is Set2-independent. However, Rpd3S complexes recruited in the absence of H3K36 methylation appear to be inactive. Finally, we present evidence implicating the yeast DSIF complex (Spt4/5) and RNA polymerase II phosphorylation by Kin28 and Ctk1 in the recruitment of Rpd3S to active genes. Taken together, our data support a model where Set2-dependent histone H3 methylation is required for the activation of Rpd3S following its recruitment to the RNA polymerase II C-terminal domain.

Show MeSH
Related in: MedlinePlus