RNA Polymerase II binding profiles of HEK-293T cells exposed to hyperosmotic stress
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE152062 | GEO | 2021/01/04
REPOSITORIES: GEO
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Project description:RNA Polymerase II binding profiles of HEK-293T cells exposed to hyperosmotic stress
| PRJNA638169 | ENA
Project description:Stress-induced readthrough transcription results in the synthesis of thousands of downstream-of-gene (DoG) containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse-seq revealed that hyperosmotic stress induces widespread transcriptional repression. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP-seq confirmed that the stress-induced redistribution of RNA Polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While subunits of the cleavage and polyadenylation machinery remained Pol II-associated, Integrator complex subunits dissociated from Pol II under stress conditions. Depleting the catalytic subunit of the Integrator complex, Int11, using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription. This SuperSeries is composed of the SubSeries listed below.
2021-01-04 | GSE152063 | 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:Abstract Background: Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair has been described to quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. However, the precise mechanism by which RNA polymerase II (Pol II) transcription is affected following UV irradiation during the repair processes genome-wide is not well understood. Results: We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We find that about 90% of the promoters of expressed genes show reduced Pol II occupancy 2-4 hours following UVB irradiation, and that the presence of Pol II is restored to “normal”, or higher, levels 5-6 hours after irradiation. We also identified a smaller set of genes, where the presence of Pol II at the promoter regions does not decrease after UVB irradiation, but often increases throughout the entire transcription units. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH but not TBP follows the behavior of Pol II suggesting that at these genes TFIIH may be sequestered for DNA repair upon UVB treatment. Conclusions: Thus, our study reveals a global negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed genes following sublethal UVB irradiation, and a small subset of genes (including regulators of repair, cell growth and survival), where Pol II escapes this negative regulation. Following genome-wide RNA Polymerase II redistribution over time upon UVB irradiation
2014-07-25 | E-GEOD-52789 | biostudies-arrayexpress
Project description:Elongin is a hetero-trimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. We solved three structures of human Elongin bound to transcribing Pol II using cryo-EM assisted by crosslinking mass spectrometry. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-8 ELOC subunit heterodimer. ELOA contains an N-terminal ‘latch’ that binds between the end of the RPB1 bridge helix and the funnel helices, thereby inducing a conformational change near the Pol II active center. The latch is strictly required for the elongation-stimulatory activity of Elongin, but not for its binding to Pol II, indicating that Elongin functions by allosterically influencing the conformational mobility of the active center. Structural comparisons show that Elongin binding to Pol II is incompatible with association of super elongation complex, the PAF1 complex, and RTF1, which also contains a latch element that stimulates Pol II.
2023-10-11 | PXD045446 | Pride
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
Project description:RNA polymerase II (Pol II) was immunoprecipitated from Arabidopsis thaliana seedlings, using antibodies that specifically recognize: 1) C’ terminal Domain (CTD), 2) CTD Serine 5 Phosphorylation and 3) CTD Serine 2 Phosphorylation. Proteins that co-immunoprecipitate with different Pol II pools were analysed.
2018-10-17 | PXD006004 | Pride
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.
2015-12-11 | GSE62171 | GEO
Project description:RNA polymerase II drives mRNA gene expression, yet our understanding of global Pol II repression is limited. Using auxin-inducible degron, we degraded Pol II's RPB1 subunit in mESCs, resulting in global repression, yet certain genes exhibited increased RNA levels post-degradation. These genes are associated with GPCR ligand binding and are characterized by being less paused and comprising polycomb-bound short genes. RPB1 degradation globally increased KDM6B binding, which was insufficient to explain specific gene activation. In contrast, RPB2 degradation repressed nearly all genes, accompanied by decreased H3K9me3 and SUV39H1 occupancy. We observed a specific increase in serine 2 phosphorylated Pol II at Pol II degradation-upregulated genes, indicating their resistance to degradation. Additionally, a-amanitin or UV treatment resulted in RPB1 degradation and global gene repression, unveiling subsets of upregulated genes. Our findings highlight the activation of signaling genes as a potential mechanism for cells to counter global transcription shutdown during stress.
2024-05-28 | PXD048023 |