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Establishment of a novel in vitro transcription assay

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Hela Cell

DISEASE(S): Prostate Adenocarcinoma,Malignant Neoplasm Of Ovary

SUBMITTER: Zhong Chen

LAB HEAD: Qianben Wang

PROVIDER: PXD027143 | Pride | 2021-11-03

REPOSITORIES: Pride

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Establishment of a novel in vitro transcription assay

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

In Vitro Transcription Recycling Assay Identifies PAF1 as a Driver of RNA Pol II Recycling

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

SPT6 functions in transcriptional pause/release via PAF1C recruitment

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.

2022-08-09 | PXD033771 | Pride

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

In Vitro Transcription Recycling Assay Identifies PAF1 as a Driver of RNA Pol II Recycling

Project description:In Vitro Transcription Recycling Assay Identifies PAF1 as a Driver of RNA Pol II Recycling

| PRJNA552087 | ENA

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

CDK12 and Integrator-PP2A complex Modulates LEO1 Phosphorylation for Processive Transcription Elongation

Project description:Cyclin-dependent kinase 12 (CDK12) interacts with Cyclin K to form a functional nuclear kinase that promotes processive transcription elongation through phosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD). To gain a broader understanding of CDK12 cellular function, we used chemical-genetic and phosphoproteomic screening to identify a landscape of nuclear human CDK12 substrates, including regulators of transcription, chromatin organization, and RNA splicing. We further validated LEO1, a subunit of the PAF1 complex (PAF1C), as a bona fide cellular substrate of CDK12. Acute depletion of LEO1, or substituting LEO1 phosphorylation sites with alanine, attenuated PAF1C association with elongating Pol II and impaired processive transcription elongation. We also found that LEO1 interacts with, and is dephosphorylated by, the Integrator-PP2A complex (INTAC) and that INTAC promotes the association of PAF1C with Pol II. Together, this study reveals a previously unknown role for CDK12 and INTAC in regulating LEO1 phosphorylation for transcriptional regulation, providing important insights into gene transcription and its regulation.

2023-06-08 | PXD038558 | Pride

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

Paf1C regulates RNA polymerase II progression by modulating elongation rate

Project description:Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter proximal regions and within the first 20-30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20-30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.

2019-06-28 | GSE116169 | GEO

Structural basis of human transcription-DNA repair coupling

Project description:Transcription-coupled DNA repair (TCR) efficiently removes bulky DNA lesions from transcribed parts of the genome and constitutes a major defence against DNA damage by UV irradiation. TCR begins when RNA polymerase II (Pol II) stalls at a DNA lesion and recruits the Cockayne syndrome protein CsB, the E3 ubiquitin ligase complex CRL4CsA, and the UV-stimulated scaffold protein A (UVSSA). Here we provide five high-resolution structures of Pol II transcription complexes with bound TCR factors and complementary biochemical data. Together with published work, our results converge on a model for the molecular mechanism of transcription-DNA repair coupling. In this model, Pol II stalling triggers the formation of an alternative transcription elongation complex that we call ECact. In ECact, CsB has replaced the elongation factor DSIF, binds the PAF1 complex, and repositions upstream DNA such that it contacts the elongation factor SPT6. The CsB translocase now pulls on the upstream DNA template, thereby pushing Pol II forward. If the lesion cannot be bypassed, Pol II gets stably stalled. CRL4CsA spans over the Pol II clamp to reach the Pol II jaw and direct ubiquitination of RPB1 residue lysine-1268. This triggers the recruitment of TFIIH to UVSSA, which is located at downstream DNA, and enables nucleotide excision repair. Finally, large-scale conformational changes in CRL4CsA enable ubiquitination of CsB and the release of TCR factors, thereby allowing Pol II to resume elongation and continue transcription over repaired DNA.

2022-02-17 | PXD025328 | Pride

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