Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1089218 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28380354_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR28380355_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR28380356_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR28380357_subreads.fastq.gz | Fastqsanger.gz | |||
| SRR28380358_subreads.fastq.gz | Fastqsanger.gz |
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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.
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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.
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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.
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Project description:Chromatin places fundamental physical constraints on transcription (Gamarra and Narlikar, 2021). The PAF1 complex (PAF1C), a hexamer of PAF1, LEO1, CTR9, SKI8, CDC73 and RTF1, plays a critical role in transcription with incompletely understood mechanisms. During transcriptional elongation, PAF1C is one of the positive elongation factors in complex with RNA polymerase II (Pol II) to facilitate elongation through chromatin (Vos et al., 2018), but it is unclear yet if negative elongation factors are needed concurrently to restrain elongation. Here we show that besides decreasing elongation rate, LEO1 knockout in human K562 cells increases transcriptional readthrough and cellular level of C-terminal domain (CTD) phosphorylated Pol II while increases and decreases transcriptional output of several hundred genes, respectively. Mechanistic analyses taking proteomic, functional genomic and biochemical approaches discovered that PAF1C regulates transcriptional termination in part through recruiting PNUTS-PP1γ complex and facilitates Pol II transcriptional re-initiation through recruiting TOX4-PP1α complex. Moreover, Paf1 conditional knockout in mice severely blocks T cell development, increases cellular level of CTD phosphorylated Pol II, mainly decreases Pol II occupancy and transcriptional output in double positive T cells, and importantly, the regulation of re-initiation by PAF1C through TOX4-PP1α complex is conserved between mouse and human. Our results also suggest that PNUTS-PP1γ and TOX4-PP1α bind PAF1C to restrain Pol II elongation through chromatin. These findings not only establish PAF1C as a critical regulator of transcriptional termination and re-initiation besides elongation but also advanced current understanding of elongation.
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