Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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The RSC Complex localizes to coding sequences to regulate Pol II and histone occupancy (Agilent)

ABSTRACT: ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome-wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions. ChIP-chip experiments to measure Sth1, Rpb3 and H3 occupancy in WT and various mutants (histone acetyltransferase and Pol II CTD kinase mutants). The histone H3 and Rpb3 occupancy were also measured in cells upon Sth1 depletion. The WT and mutant strains were grown in Synthetic complete or YPD media to an O.D. 600 of 0.6-0.8. For inducing Gcn4, the cells grown in SC were treated with Sulfometuron methyl for 20-25 minutes and process for chromatin immunoprecipitation using antibodies again Myc, Rpb3 or histone H3.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Chhabi Govind

PROVIDER: E-GEOD-62519 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:RSC (remodels the structure of chromatin) is an essential ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae. The catalytic subunit of RSC, Sth1 uses its ATPase activity to slide or remove nucleosomes. RSC has been shown to regulate the width of the nucleosome-depleted regions (NDRs) by sliding the flanking nucleosomes away from NDRs. As such the nucleosomes encroach NDRs when RSC is depleted and leads to transcription initiation defects. In this study, we examined the effects of the catalytic-dead Sth1 on transcription and compared them to the effects observed during acute and rapid Sth1 depletion by auxin-induced degron strategy. We found that rapid depletion of Sth1 reduces recruitment of TBP and Pol II in highly transcribed genes, as would be expected considering its role in regulating chromatin structure at promoters. In contrast, cells harboring the catalytic-dead Sth1 exhibited a severe reduction in TBP binding, but surprisingly, also displayed a substantial accumulation in Pol II occupancies within coding regions. After depleting endogenous Sth1 in the catalytic dead mutant, we observed a further increase in Pol II occupancies, suggesting that the inactive Sth1 contributed to the observed accumulation of Pol II in coding regions. Notwithstanding the Pol II increase, the ORF occupancies of histone chaperones FACT and Spt6 were significantly reduced in the mutant. These results suggest a potential role for RSC in recruiting/retaining these chaperones in coding regions. Pol II accumulation despite substantial reductions in TBP, FACT, and Spt6 occupancies in the catalytic-dead mutant could be indicative of severe transcription elongation and termination defects. Such defects would be consistent with studies showing that RSC is recruited to coding regions in a transcription-dependent manner. Thus, these findings imply a role for RSC in transcription elongation and termination processes, in addition to its established role in transcription initiation.

Project description:Cmr1 (changed mutation rate 1) is a largely uncharacterized nuclear protein that has recently emerged in several global genetic interaction and protein localization studies. It clusters with proteins involved in DNA damage and replication stress response, suggesting a role in maintaining genome integrity. Under conditions of proteasome inhibition or replication stress, this protein localizes to distinct sub-nuclear foci termed as intranuclear quality control (INQ) compartments, which sequester proteins for their subsequent degradation. Interestingly, it also interacts with histones, chromatin remodelers and modifiers, as well as with proteins involved in transcription including subunits of RNA Pol I and Pol III, but not with those of Pol II. It is not known whether Cmr1 plays a role in regulating transcription of Pol II target genes. Here, we show that Cmr1 is recruited to the coding regions of transcribed genes of S. cerevisiae. Cmr1 occupancy correlates with the Pol II occupancy genome-wide, indicating that it is recruited to coding sequences in a transcription-dependent manner. Cmr1-enriched genes include Gcn4 targets and ribosomal protein genes. Furthermore, our results show that Cmr1 recruitment to coding sequences is stimulated by Pol II CTD kinase, Kin28, and the histone deacetylases, Rpd3 and Hos2. Finally, our genome-wide analyses implicate Cmr1 in regulating Pol II occupancy at transcribed coding sequences. However, it is dispensable for maintaining co-transcriptional histone occupancy and histone modification (acetylation and methylation). Collectively, our results show that Cmr1 facilitates transcription by directly engaging with transcribed coding regions. ChIp-chip experiments were perfomed to determine genome-wide distribution of Cmr1 in WT and gcn4Î cells (S. cerevisiae). Rpb3 occupancy in WT and cmr1Î cells was also determined to reveal the changes in Pol II occupancy in the absence of Cmr1. The WT and mutant strains were grown in Synthetic complete and cells were indcued for Gcn4 by treating with Sulfometuron methyl for 30 minutes and processed for chromatin immunoprecipitation using antibodies against Myc and Rpb3 (subunit of Pol II).

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The RSC Complex localizes to coding sequences to regulate Pol II and histone occupancy (Agilent)

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