Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

DSIF and RNA polymerase II CTD Phosphorylation Coordinate the Recruitment of Rpd3S to Actively Transcribed Genes

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. In order to examine the genome-wide localization of RNAPII and Spt6 in Saccharomyces cerevisiae, RNAPII and Spt6 along with associated DNA sequences were immunoprecipitated using anti-8WG16 and anti-HA antibodies, respectively. The RNAPII and Spt6 chromatin immunoprecipitation was performed in duplicate from WT cells as described below. The extracted DNA was hybridized to a DNA microarray containing an average of 4 probes per kilobase across the whole yeast genome. The combined datasets are available in the supplemental files of the related publication.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: François Robert

PROVIDER: E-GEOD-22636 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

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

Drouin Simon S Laramée Louise L Jacques Pierre-Étienne PÉ Forest Audrey A Bergeron Maxime M Robert François F

PLoS genetics 20101028 10

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, Rpd3 ...[more]

PMID: 21060864

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Project description:Transcript elongation by RNA polymerase II (RNAPII) is accompanied by conserved patterns of histone modification within transcribed regions, but it remains uncertain how these modifications influence, or are influenced by, properties of the elongation complex. Here we establish an intimate link between Cdk9, the kinase component of positive transcription elongation factor b (P-TEFb), and mono-ubiquitylation of histone H2B (H2Bub1), in the fission yeast Schizosaccharomyces pombe. Mutations that impair Cdk9 function reduce H2Bub1 levels in vivo. Conversely, mutations that prevent H2Bub1 decrease phosphorylation of elongation factor subunit Spt5, a sensitive and specific indicator of Cdk9 activity. Chromatin immunoprecipitation (ChIP) analysis suggests this is due to impaired Cdk9 recruitment to H2Bub1-deficient chromatin. P-TEFb and H2Bub1 pathways also interact genetically: mutation of the histone H2B ubiquitin-acceptor residue decreases the requirement for Cdk9 activity in vivo, and multiple cdk9 mutations suppress morphological defects of H2Bub1-deficient strains. Moreover, H2Bub1 loss causes redistribution of transcribing RNAPII on chromatin that is corrected by a hypomorphic cdk9 mutation. Therefore, whereas mutual dependence of Spt5 phosphorylation and H2Bub1 suggests positive feedback between P-TEFb and the ubiquitylation machinery, mutual suppression by cdk9 and H2Bub1-pathway mutations indicates an antagonistic relationship, whereby the activities must be balanced to properly regulate elongation. In order to study the genome-wide localization of H2Bub1 in Schizosaccharomyces pombe, H2Bub1, H2B-Flag as well as RNAPII (along with associated DNA sequences) were immunoprecipitated using repectively anti-H2Bub1, anti-Flag and anti-8WG16 antibodies. The ChIPs were performed in duplicate from WT cells as well as in the H2B-K119R mutant. The extracted DNA was hybridized to a DNA microarray containing an average of 4 probes per kilobase across the whole yeast genome. The combined datasets are available in the supplemental files of the related publication.

Project description:H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of H2A.Z eviction, coupled to its pervasive incorporation by mislocalized SWR-C, alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z removal is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription. We profiled H2A.Z occupancy in S. cerevisiae by ChIP-chip on tiling arrays in different mutants for chromatin remodelers and histone chaperones. In most experiments, H2A.Z ChIP samples (Cy5-labeled) were performed using a polyclonal antibody against H2A.Z and hybridized against H2B ChIP samples (Cy3-labeled), also performed using a polyclonal antibody. Temperature-sensitive mutants for the histone chaperones Spt16 and Spt6 (spt16-197 and spt6-1004 respectively) showed strong H2A.Z localization defects so the role of these factors in H2A.Z localization was further analyzed. H2A.Z ChIP-chip experiments in spt16-197 and spt6-1004 were repeated using different experimental designs [normalized against Input DNA or Mock (IgG) ChIP samples]. The contribution of Spt16 and Spt6 on H2A.Z localization was also confirmed by nuclear depletion of Spt16 and Spt6 using the anchor-away system. H2A.Z ChIP-chip experiments were also performed in sic1Δ and spt16-197/sic1Δ cells in order to rule out any G1-arrest artifact. H2A.Z ChIP-chip experiments were repeated using spike-in controls for normalization, revealing widespread H2A.Z occupancy in Spt16 and Spt6 mutants. In addition, the localization of the chromatin remodeler SWR-C was determined in wild type cells as well as in spt16-197 and spt6-1004 cells. Finally, we also profiled histone H4 occupancy by ChIP-chip in wild type, spt16-197, spt6-1004, spt16-197/htz1Δ and spt6-1004/htz1Δ cells. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).

Dataset Information

DSIF and RNA polymerase II CTD Phosphorylation Coordinate the Recruitment of Rpd3S to Actively Transcribed Genes

Publications

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

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