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Post-Transcriptional Role for the Yeast Paf1-RNA Polymerase II Complex is Revealed by Identification of Primary Targets

ABSTRACT: To identify the direct targets of the Paf1/RNA polymerase II complex we compared expression profiles of isogenic wild type and paf1 and ctr9 mutant strains. We also created a Tet-regulated form of Paf1 and monitored expression patterns after shut off of Paf1. Samples were isolated at one hour intervals from 1 to 8 hours after shut off. Keywords: single channel nucleotide

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE3200 | GEO | 2005/08/26

SECONDARY ACCESSION(S): PRJNA92865

REPOSITORIES: GEO

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Transcription profiling of S. cerevisiae paf1 and ctr9 mutants to identify direct targets of the Paf1/RNA polymerase II complex

Project description:To identify the direct targets of the Paf1/RNA polymerase II complex we compared expression profiles of isogenic wild type and paf1 and ctr9 mutant strains. We also created a Tet-regulated form of Paf1 and monitored expression patterns after shut off of Paf1. Samples were isolated at one hour intervals from 1 to 8 hours after shut off. Experiment Overall Design: Transcripts were compared on Affymetrix microarrays using standard protocols.

2007-09-19 | E-GEOD-3200 | biostudies-arrayexpress

RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II

Project description:Release of promoter-proximal paused RNA polymerase II (Pol II) during early elongation is a critical step in transcriptional regulation in metazoan cells. Paused Pol II release is thought to require the kinase activity of cyclin-dependent kinase 9 (CDK9) for the phosphorylation of DRB sensitivity-inducing factor, negative elongation factor, and C-terminal domain (CTD) serine-2 of Pol II. We found that Pol II-associated factor 1 (PAF1) is a critical regulator of paused Pol II release, that positive transcription elongation factor b (P-TEFb) directly regulates the initial recruitment of PAF1 complex (PAF1C) to genes, and that the subsequent recruitment of CDK12 is dependent on PAF1C. These findings reveal cooperativity among P-TEFb, PAF1C, and CDK12 in pausing release and Pol II CTD phosphorylation. Comparison of the chromatin occupancy of [1] PAF1, CDC73, LEO1, CTR9, total Pol II, and CTD serine 2-phosphorylated Pol II by ChIP-seq in THP1 cells; [2] PAF1, Pol II, Pol II (ser-5p), CDK12, and CDK9 by ChIP-seq in control and PAF1 knockdown cells; [3] LEO1 and Pol II by ChIP-seq in control and flavopiridol treated THP1 cells.

2015-12-11 | E-GEOD-62171 | biostudies-arrayexpress

PAF1, a molecular regulator of promoter-proximal pausing by RNA Polymerase II

Project description:The control of promoter-proximal pausing and the release of RNA polymerase II (RNA Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify Pol II associated Factor 1 (PAF1) as a major regulator of promoter-proximal pausing. Knockdown of PAF1 leads to increased release of paused Pol II into gene bodies at thousands of genes. Genes with the highest levels of paused Pol II exhibit the largest redistribution of Pol II from the promoter-proximal region into the gene body in the absence of PAF1. PAF1 depletion results in increased nascent transcription and increased levels of phosphorylation of Pol II’s c-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase Super Elongation Complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing a novel function for PAF1 as a major regulator of pausing in metazoans. ChIP-seq of Pol II of different forms, SEC subunits, PAFc subunits and H2Bub in human cell lines targeted by PAF1 or scramble shRNA. ChIP-seq of total Pol II in HCT116 cells targeted by BRE1A or scramble shRNA. ChIP-seq of total Pol II in S2 cells targeted by Paf1 or LacZ RNAi. Total RNA-seq, nascent RNA-seq and GRO-seq in HCT116 cells targeted by PAF1 or scramble shRNA.

2015-08-13 | E-GEOD-70408 | biostudies-arrayexpress

Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1

Project description:Pleiotropic transcription regulator RNA polymerase II (Pol II)-associated factor 1 (PAF1) governs multiple transcriptional steps and the deposition of several epigenetic marks. However, it remains unclear how ultimate transcriptional outcome is determined by PAF1 and whether it relates to PAF1-controlled epigenetic marks. We utilize rapid degradation systems and reveal direct PAF1 functions in governing pausing partially by recruiting Integrator-PP2A (INTAC), in addition to ensuring elongation. Following acute PAF1 degradation, destabilized polymerase undergoes early termination or effective release, which presumably relies on skewed balance between INTAC and P-TEFb resulting in hyperphosphorylated substrates including SPT5. Impaired Pol II progression during elongation, along with altered pause release frequency, determines the final transcriptional outputs. Moreover, PAF1 degradation causes a cumulative decline in histone modifications. These epigenetic alterations in chromatin likely further influence the production of transcripts from PAF1 target genes.

2022-01-30 | GSE192848 | GEO

PAF1, a molecular regulator of promoter-proximal pausing by RNA Polymerase II

2015-08-13 | GSE70408 | GEO

Paf1 complex factors, Leo1 and Paf1, promote local histone turnover to modulate chromatin states in fission yeast [ncRNA-Seq]

Project description:Maintenance of open and repressed chromatin states is crucial for regulation of gene expression. To study the genes involved in maintaining chromatin states we generated a random mutant library using the Hermes transposon mutagenesis system in fission yeast Schizosacchromyces pombe. The silencing of reporter genes inserted in the euchromatic region adjacent to the heterochromatic mating type locus was monitored. We identified Leo1-Paf1, a subcomplex of the RNA Polymerase II Associated Factor 1 Complex (Paf1C), required to prevent spreading of heterochromatin into euchromatin. Through high-resolution genome-wide ChIP (ChIP-exo) we mapped the heterochromatin mark H3K9me2 in leo1∆ cells. Loss of Leo1-Paf1 led to increased heterochromatin stability at several facultative heterochromatin loci. The RNAi machinery is the major pathway for heterochromatin formation in S. pombe. However, small RNA sequencing showed that heterochromatin assembly in leo1∆ cells was RNAi-independent. By examining histone turnover rate in leo1∆ cells, we showed that deletion of Leo1 decreased nucleosome turnover, which led to heterochromatin spreading. Our data revealed that Leo1-Paf1 promotes chromatin state fluctuations by enhancing histone turnover.

2015-10-18 | GSE66940 | GEO

Paf1 complex factors, Leo1 and Paf1, promote local histone turnover to modulate chromatin states in fission yeast [ChIP-exo]

2015-10-18 | GSE66939 | GEO

PAF1 regulation of promoter-proximal pause release via enhancer activation

Project description:Gene expression in metazoans is regulated by RNA Polymerase II (Pol II) promoter-proximal pausing and its release. Previously, we identified that Pol II-associated factor 1 (PAF1) modulates the release of paused Pol II into productive elongation. Here, we find that PAF1 occupies transcriptional enhancers and restrains hyperactivation of a subset of these enhancers. Enhancer activation as the result of Paf1 loss releases Pol II from paused promoters of nearby PAF1 target genes. Knockout of PAF1-regulated enhancers attenuates the release of paused Pol II on PAF1 target genes without major interference in the establishment of pausing at their cognate promoters. Thus, a subset of enhancers can primarily modulate gene expression by controlling the release of paused Pol II in a PAF1-dependent manner.

2017-09-01 | GSE97527 | GEO

Effects of innate immune stimuli on PAF1 knockout cells

Project description:PAF1 knockout and ncgRNA control cells were treated with multiple innate immune stimuli for 3 hours to mimic various microbial stresses.

2022-04-10 | E-MTAB-11620 | biostudies-arrayexpress

Characterization of Paf1 complex occupancy in mouse embryonic stem cells under control conditions or Phf5a knockdown

Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed for genome-wide localization of Paf1, Leo1 and Cdc73 subunits of the Paf1 complex in mouse ESCs under conditions of shControl and shPhf5a knockdown. These results revealed that downregualtion of Phf5a results in the significant decrease of Paf1 complex binding to its targets in ESCs.

2016-08-31 | GSE73445 | GEO

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