Investigating the role of Paf1 in transcription by RNA polymerase I
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PROVIDER: PRJNA1188147 | ENA |
REPOSITORIES: ENA
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Project description:Polymerase-associated factor 1 complex (Paf1C) has been identified to promote transcription by Pols I and II and is conserved across eukaryotes. This transcription factor has been well-studied for Pol II, but its role in transcription by Pol I has not been fully characterized. Therefore, the goal of this study was to use a high-resolution technique, native elongating transcript sequencing (NET-seq), to investigate the effect of the loss of two of the five Paf1C subunits (Paf1 and Cdc73) on Pol I occupancy in yeast. Most notably, we found that in both paf1D and cdc73D mutants, there was a significant reduction in Pol I occupancy at the 5’ end of the rDNA template as compared to WT yeast. Overall, our results suggest that Paf1C is an important transcription elongation factor for Pol I.
2024-11-24 | GSE282332 | GEO
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
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
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. Keywords: single channel nucleotide
2005-08-26 | GSE3200 | GEO
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
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.
2015-08-13 | GSE70408 | GEO
Project description:In this study, we found that the ARF tumor suppressor directly binds the PAF1 subunit to block the assembly of PAF1 complex (PAF1c) and its interaction with RNAPII, thereby dampening PAF1c-dependent transcription in vitro and in cells. To investigate whether ARF regulates RNAPII and PAF1c occupancy at putative target genes identified through RNA-seq, we performed ChIP-seq (RNAPII, PAF1, and CTR9) in ARF (shARF) vs. control (shNT) knockdown MEFs. Our data show that loss of ARF increases RNAPII, PAF1, and CTR9 occupancy on upregulated genes, consistent with ARF-mediated gene-specific repression. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for RNA polymerase subunit RPB3 and PAF1 complex subunits PAF1 and CTR9 in mouse embryonic fibroblasts.
2024-10-10 | GSE217765 | GEO
Project description:The coordinated transcription of genes involves RNA polymerase II enzymes (RNAPII), which pull DNA through their active sites. DNA lesions in transcribed strands block RNAPII elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway ensures the efficient removal of transcription-blocking DNA lesions, but this is not sufficient to overcome this arrest and resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions specifically in response to DNA damage. PAF1C is dispensable for TCR-mediated repair,but is essential to resume RNA synthesis after UV irradiation, suggesting an unexpected uncoupling between DNA repair and transcription restart. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation waves throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.
2021-02-28 | PXD016198 | Pride
Project description:Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro. The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide.
In an effort to explore regulatory roles for Elongin A, we performed IP-MS. We constructed a DLD1 cell line expressing Flag-tagged Elongin A at endogenous levels and performed anti-Flag immuno-purification of solubilized chromatin followed by analysis of binding partners by mass spectrometry in triplicates. We identified a large group of Elongin A-associated proteins. We confirmed a subset of possible interacting proteins by Co-IP and western blotting. Consistent with previous studies, we identified RNA Pol II subunits and proteins related to transcription elongation and RNA processing. Among the top hits, we identified nearly all subunits of the PAF1 complex, except for RTF1, which does not stably associate with mammalian PAF1. Concordantly, in our previous PAF1 proteomic study, Elongin A was also among the top hits. We also identified several subunits of the Integrator complex, a multi-subunit complex that has been shown to participate in enhancer RNA (eRNA) processing, a finding consistent with our conclusion that Elongin A localizes to potential enhancers. Collectively, our results suggest that Elongin A stably interacts with RNAPII and the transcription machinery on chromatin.
Taken together, our studies suggest that Elongin A associates with the transcription machinery at actively transcribed genomic regions and may be involved in the release of paused RNAPII. However, Elongin A does not appear to be critical for maintaining transcription elongation rates in vivo.
2020-08-07 | MSV000085926 | MassIVE
Project description:The polymerase associated factor 1 complex (Paf1C) is a multifunctional epigenetic regulator of RNA polymerase II (Pol II) transcription. Paf1C controls gene expression by stimulating the placement of co-transcriptional histone modifications, influencing nucleosome occupancy in coding regions, facilitating transcription termination, and regulating nuclear export of RNAs. In this study, we investigate the extent to which these functions of Paf1C combine to influence the Saccharomyces cerevisiae transcriptome. Using conditions that enrich for unstable transcripts, we show that deletion of PAF1 affects all classes of Pol II-transcribed RNAs including multiple classes of noncoding transcripts. Gene ontology analysis revealed that mRNAs encoding genes involved in iron and phosphate homeostasis were differentially affected by deletion of PAF1. We further investigated these two groups of mRNAs with the goal of identifying overarching mechanisms of up and down-regulation in cells lacking Paf1. Our results indicate that only a subset of the observed changes result from loss of Paf1C-promoted histone modifications. We also found that transcription of the FET4 gene is differentially regulated by Paf1 and an upstream CUT. Together these data highlight the complexity of the epigenetic regulation of Pol II transcription imposed by Paf1C and identify a role for Paf1C in promoting CUT transcription.
2019-06-11 | GSE122704 | GEO