In Vitro Transcription Recycling Assay Identifies PAF1 as a Driver of RNA Pol II Recycling
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA552087 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR15070000.fastq.gz | Fastqsanger.gz | |||
| SRR15070001.fastq.gz | Fastqsanger.gz | |||
| SRR15070002.fastq.gz | Fastqsanger.gz | |||
| SRR15070003.fastq.gz | Fastqsanger.gz | |||
| SRR15070004.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 123 |
Similar Datasets
Project description:RNA Polymerase II transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of novel RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from RNA Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed in vivo PAF1 complex cycling through gene bodies with RNA Pol II and recycling back to the transcription start sites, while PAF1 silencing triggered defective RNA Pol II recycling. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify RNA Pol II recycling as a potential target in cancer and demonstrate the assay’s applicability to characterize RNA Pol II recycling in cells and tissues from other disease states.
2021-10-07 | GSE133655 | GEO
Project description:RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states.
| S-EPMC8566496 | biostudies-literature
Project description:Nucleolar ribosomal DNA (rDNA) repeats control ribosome manufacturing. rDNA harbors a ribosomal RNA (rRNA) gene and an intergenic spacer (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding the rRNA components of ribosomes. Pol II at the IGS induces rRNA production by preventing Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli. At the IGS, Pol II regulatory processes and whether Pol I function can be beneficial remain unknown. Here, we identify IGS Pol II regulators, uncovering nucleolar optimization via IGS Pol I. Compartment-enriched proximity-dependent biotin identification (compBioID) showed enrichment of the TATA-less promoter-binding TBPL1 and transcription regulator PAF1 with IGS Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I and its ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the interactome of nucleolar Pol II and show its control by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and operation.
2024-10-09 | GSE261857 | GEO
Project description:RNA Polymerase II (Pol II) transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. By combing our in vitro transcription assays with other experiments (e.g. in vivo dynamic ChIP-seq assays), we identified PAF1 complex components as drivers for transcription recycling, revealing a new layer in controlling Pol II-dependent transcription. Our findings also point to RNA Pol II transcription recycling as a mechanism that functions aberrantly in disease states such as cancer, and indicate the potential to target factors such as PAF1 that drive RNA Pol II recycling in cancer.
2021-11-03 | PXD027143 | Pride
Project description:RNA Polymerase II (Pol II) transcriptional recycling is an underappreciated mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. By combing our in vitro transcription assays with other experiments (e.g. in vivo dynamic ChIP-seq assays), we identified PAF1 complex components and phospho-MED1 as drivers for transcription recycling, revealing a new layer in controlling Pol II-dependent transcription. Our findings also point to RNA Pol II transcription recycling as a mechanism that functions aberrantly in disease states such as cancer, and indicate the potential to target factors such as PAF1 and phospho-MED1 that drive RNA Pol II recycling in cancer.
2021-11-03 | PXD017881 | Pride
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: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
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:Mammalian RNA polymerase II (Pol II) initiation, elongation, termination and reinitiation are well studied, but how Pol II dynamically recycles after the transcription cycle remains unclear. By establishing in vitro and in vivo transcription recycling systems, we find that human Mediator 1 (MED1), when phosphorylated at the mammal-specific threonine 1032 by cyclin-dependent kinase 9, dynamically travels with Pol II throughout the transcribed genes to drive Pol II recycling. Mechanistically, MED1 phosphorylation leads to an increase of recycled Pol II via the molecular bridge of MED31, enhancing mRNA output during the transcription recycling process. Importantly, MED1 phosphorylation increases during prostate cancer progression to the lethal phase, and pharmacological inhibition of CDK9 decreases prostate tumor growth through decreasing MED1 phosphorylation and Pol II recycling. Our findings reveal essential mechanisms underlying Pol II recycling and suggest a neglected, yet fundamental Pol II transcription process for therapeutic intervention.
2022-04-20 | GSE127894 | GEO
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