ChIP-chip of histone variants and modifications in S. pombe WT and various mutants
Ontology highlight
ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
SUBMITTER: François Robert
PROVIDER: E-GEOD-31071 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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.
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Project description:Spt6 is a highly conserved factor that carries out important functions in transcription and chromatin structure. To gain new insights into Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant and found that the level of nucleosomes is greatly reduced accross some but not all coding regions. In addition, genome-wide location analyses of RNA polymerase II showed that the nucleosome loss in the spt6 mutant occurs over highly-transcribed genes. Unexpectedly, the effects of the spt6 mutation on nucleosome levels did not correlate with its effects on mRNA levels, suggesting that Spt6 plays distinct roles in controlling chromatin structure across coding regions and in transcriptional regulation. We studied one case of transcriptional regulation by Stp6, at the CHA1 gene, and showed that regulation likely occurs by Spt6 controlling the position of the +1 nucleosome. These results, along with previous studies, suggest that Spt6 regulates transcription by controlling chromatin structure over regulatory regions, and its effects on nucleosome levels over coding regions likely serve and independent function. 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.
2010-12-01 | E-GEOD-21787 | biostudies-arrayexpress
Project description:Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C-terminus that recognizes Pol II CTD peptides phosphorylated on Ser2, Ser5 or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' end of genes, where phosphorylated Ser2 reaches its maximum level. Additionally, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' end of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo. We examined the genome-wide distribution (using ChIP-chip) of Spt6. Spt6 occupancy was also assayed in mutants for CTD Serine 2 and Serine 5 kinases and in mutants for histone deacetylases. ChIPs were performed with a Myc-tagged version of Spt6. Most ChIPs (in Cy5) were hybridyzed against a control ChIP sample from an isogenic non-tagged strain (in Cy3). In the ChIP experiments with the spt6-202del mutant, non immunoprecipitated DNA (input) was used as the control. In addition to Spt6 ChIPs, the project includes RNAPII (Rpb3) ChIP-chip datasets, where an anti-Rpb3 antibody was used to ChIP RNAPII and non immunoprecipitated DNA (input) was used as the control. 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).
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Project description:Determination of RNAPII, Rrd1-Myc, RNAPII-Ser2-P, RNAPII-Ser5-P abundance between wildtype and rrd1 deletion strains under rapamycin or control treatments.Rapamycin is an anticancer agent and immunosuppressant that acts by inhibiting the TOR signaling pathway. In yeast, rapamycin mediates a profound transcriptional response for which the RRD1 gene is required. To further investigate this connection, we performed genome-wide association studies of RNA polymerase II (RNAPII) and Rrd1 in response to rapamycin and demonstrate that Rrd1 colocalizes with RNAPII on actively transcribed genes and both are recruited to rapamycin responsive genes. Strikingly, when Rrd1 is lacking, RNAPII remains inappropriately associated to ribosomal genes and fails to be recruited to rapamycin responsive genes. This occurs independently of TATA box binding protein recruitment. Furthermore, Rrd1 modulates the phosphorylation status of RNAPII CTD and additional evidence suggests that Rrd1 is involved in various transcriptional stress responses. We propose that Rrd1 is a novel transcription elongation factor that fine-tunes the transcriptional stress response of RNAPII.
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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).
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