Schizosaccharomyces pombe
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ABSTRACT:
PROVIDER: PRJNA144845 | ENA |
REPOSITORIES: ENA
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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.
2012-12-01 | E-GEOD-31070 | biostudies-arrayexpress
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.
2012-12-01 | GSE31070 | GEO
Project description:Histone H2B mono-ubiquitylation (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. How Cdk9 activity and H2Bub1 cooperate to regulate the expression of individual genes remains unclear. Here we show Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of distinct gene subsets; ablation of both pathways derepresses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both are enriched in coding regions, and H2Bub1 levels are directly proportional to those of phospho-Spt5. Cdk9-dependence of antisense suppression correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Combined reduction of Cdk9 activity and loss of H2Bub1 prevent recruitment of the histone deacetylase Clr6-CII to transcribed genes, and lead to decreased histone occupancy and increased histone acetylation within gene coding regions. These results uncover new pathways linking regulators of RNAPII transcription elongation to suppression of aberrant antisense transcription, and demonstrate novel interactions between co-transcriptional histone modification pathways.
2020-05-21 | GSE115682 | GEO
Project description:Transcription by RNA polymerase II is regulated by epigenetic modifications to the chromatin template. The mono-ubiquitylation of H2B is established during transcription elongation and broadly impacts chromatin architecture and gene expression. The Polymerase Associated Factor 1 complex (Paf1C) is required for H2B ubiquitylation through an unknown mechanism. Here, we find that a 66-amino acid histone modification domain (HMD) within the Rtf1 subunit of Paf1C promotes H2B ubiquitylation in cells lacking all Paf1C members and present the crystal structure of this domain. Using site-specific in vivo crosslinking, we show that Rtf1 directly interacts with the ubiquitin conjugase Rad6 through a conserved surface on the HMD. Through ChIP-exo analysis, we observe that enrichment of Paf1C correlates with H2B ubiquitylation, and that the HMD, Rad6 and Bre1 localize to H2B. Finally, we demonstrate that the HMD directly stimulates H2B ubiquitylation in a reconstituted system, arguing that Paf1C functions as a cofactor for Rad6-Bre1 mediated catalysis.
2016-11-07 | GSE83348 | GEO
Project description:Chromatin modifications play a pivotal role in cell fate decision. In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis when cells starve. The methylation of H3K4 by Set1-COMPASS and consequent promoter nucleosome deacetylation was shown to repress ste11 induction and cell differentiation but the regulatory steps remain poorly understood. A genetic screen highlighted H2B ubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2B-ub1 at the promoter where it represses expression, and over the transcribed region where it sustains transcription of ste11. By promoting H3K4 methylation at the promoter, H2B-ub1 initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR formation leads to high expression. Therefore, H2B-ub1 represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene. Samples from mononucleosomal DNA from S. pombe strains h-972 and h-972 rsc1::kanR were sequenced (Illumina NextSeq 500 platform) using the pair-end read protocol
2016-07-22 | E-GEOD-80524 | biostudies-arrayexpress
Project description:Chromatin modifications play a pivotal role in cell fate decision. In fission yeast, the ste11 gene encodes the master regulator initiating the switch from vegetative growth to gametogenesis when cells starve. The methylation of H3K4 by Set1-COMPASS and consequent promoter nucleosome deacetylation was shown to repress ste11 induction and cell differentiation but the regulatory steps remain poorly understood. A genetic screen highlighted H2B ubiquitylation and the RSC remodeling complex as activators of ste11 expression. Mechanistic analyses revealed more complex, opposite roles of H2B-ub1 at the promoter where it represses expression, and over the transcribed region where it sustains transcription of ste11. By promoting H3K4 methylation at the promoter, H2B-ub1 initiates the deacetylation process, which decreases chromatin remodeling by RSC. Upon induction, this process is reversed and efficient NDR formation leads to high expression. Therefore, H2B-ub1 represses gametogenesis by opposing the recruitment of RSC at the promoter of the master regulator ste11 gene.
2016-07-22 | GSE80524 | GEO
Project description:Faithful propagation of functionally distinct chromatin states is crucial for maintaining cellular identity, and its breakdown can lead to diseases such as cancer. Whereas mechanisms that sustain repressed states have been intensely studied, regulatory circuits that protect active chromatin from inactivating signals are not well understood. Here we discover a self-reinforcing feedback loop that preserves the transcription-competent state of RNA polymerase II transcribed genes. We found that the chromatin reader Pdp3 recruits the histone acetyltransferase Mst2 to H3K36me3 marked chromatin. Thereby, Mst2 binds to all transcriptionally active regions genome-wide. Besides acetylating histone H3K14, Mst2 also acetylates Brl1, a component of the histone H2B ubiquitin ligase complex. Brl1 acetylation results in increased histone H2B ubiquitination, which together with H3K14ac positively feeds back on transcription and prevents assembly of ectopic heterochromatin. Our work uncovers a molecular pathway that secures epigenome integrity and highlights the importance of opposing feedback loops for the partitioning of chromatin into transcriptionally active and inactive states.
2017-06-28 | PXD005714 | Pride
Project description:CDK9 is the kinase subunit of P-TEFb that enables RNA polymerase (Pol) II to transit from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to the lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9’s activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb’s loss of activity, only the simultaneous inhibition of CDK9 and MYC can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy. ChIP-seq of Pol II in HeLa cells before or after i-CDk9 treatment
2015-06-17 | E-GEOD-60953 | biostudies-arrayexpress
Project description:CDK9 is the kinase subunit of P-TEFb that enables RNA polymerase (Pol) II to transit from promoter-proximal pausing to productive elongation. Although considerable interest exists in CDK9 as a therapeutic target, little progress has been made due to the lack of highly selective inhibitors. Here, we describe the development of i-CDK9 as such an inhibitor that potently suppresses CDK9 phosphorylation of substrates and causes genome-wide Pol II pausing. While most genes experience reduced expression, MYC and other primary response genes increase expression upon sustained i-CDK9 treatment. Essential for this increase, the bromodomain protein BRD4 captures P-TEFb from 7SK snRNP to deliver to target genes and also enhances CDK9’s activity and resistance to inhibition. Because the i-CDK9-induced MYC expression and binding to P-TEFb compensate for P-TEFb’s loss of activity, only the simultaneous inhibition of CDK9 and MYC can efficiently induce growth arrest and apoptosis of cancer cells, suggesting the potential of a combinatorial treatment strategy. We used microarrays to examine the global impact on gene expression by imhibiting CDK9 at different time durations. HeLa cell lines treated with CDK9 inhibitor at different time points
2015-06-17 | E-GEOD-60952 | biostudies-arrayexpress