Paf1C regulates RNA polymerase II progression by modulating elongation rate
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ABSTRACT:
PROVIDER: PRJNA477543 | ENA |
REPOSITORIES: ENA
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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
Project description: Not available
| S-EPMC6642404 | biostudies-literature
Project description:MYC family proteins are oncogenic transcription factors that can globally affect the function of RNA Polymerase II (RNAPII). The ability of MYC proteins to promote transcription elongation depends on their ubiquitination, but the underlying mechanism and its biological relevance are unknown. Here we show that MYC and the Polymerase II associated factor, PAF1c, interact directly and their function is mutually dependent, since the specific binding of MYC to active promoters depends on PAF1c and, conversely, PAF1c is required for MYC-dependent pause release. Upon binding, PAF1c is rapidly transferred from MYC onto RNAPII and this transfer is driven by the HUWE1 ubiquitin ligase. Both MYC and HUWE1 globally control histone H2B ubiquitylation, which promotes transcriptional elongation and alters chromatin structure for double-strand break repair. Consistently, MYC suppresses the accumulation of double-strand breaks at promoters in response to topoisomerase II inhibition. While depletion of PAF1c has only minor effects on MYC-dependent gene expression, MYC induces rampant transcription-dependent DNA damage in PAF1c-depleted cells. We propose that the HUWE1-dependent transfer of PAF1c from MYC onto RNAPII is critical for absorbing the topological stress accompanied with deregulated and oncogenic transcription.
2021-02-15 | PXD023242 | Pride
Project description:The coordinated transcription of genes involves the regulated release of RNA polymerase II (RNAPII) from promoter-proximal sites into active elongation. DNA lesions in transcribed strands block elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but this is not sufficient to 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 in response to DNA damage. PAF1C is dispensable for TCR-mediated repair, but is essential for recovery of RNA synthesis after UV irradiation, suggesting an uncoupling between DNA repair and transcription recovery. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation 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.
2021-01-15 | GSE140930 | GEO
Project description:The PAF complex (Paf1C) has been shown to regulate chromatin modifications, gene transcription, and PolII elongation. Here, we provide the first genome-wide analysis of chromatin occupancy by the entire PAF complex in mammalian cells. We show that Paf1C is recruited not only to promoters and gene bodies, but also to regions downstream of cleavage/polyadenylation (pA) sites at 3â ends, a profile that sharply contrasted with the yeast complex. Remarkably, our studies identified novel, subunit-specific links between Paf1C and regulation of alternative cleavage and polyadenylation (APA) and upstream antisense transcription. Moreover, we found that depletion of Paf1C subunits also resulted in the accumulation of RNA polymerase II (PolII) over gene bodies, which coincided with APA. Depletion of specific Paf1C subunits leads to global loss of histone H2B ubiquitylation, but surprisingly, there is little impact of Paf1C depletion on other histone modifications, including the tri-methylation of histone H3 on lysines 4 and 36 (H3K4me3 and H3K36me3), previously associated with this complex. Our results provide surprising differences with yeast, while unifying observations that link Paf1C with PolII elongation and RNA processing, and suggest that Paf1C could play a role in protecting transcripts from premature cleavage by preventing PolII accumulation at TSS-proximal pA sites. ChIP-seq, RNA-seq and 3'READS of Paf1C factors in mouse C2C12 myoblast cells
2016-01-15 | E-GEOD-72574 | biostudies-arrayexpress
Project description:Recent studies reveal a striking phenomenon that RNA Polymerase II (Pol II) appears to travel on gene body in an accelerated fashion, but the mechanism has remained unknown. We performed synchronized transcription coupled with deep sequencing, observing an inverse relationship between initial rate and acceleration in different cell types. We directly tested several correlative events and detected a positive contribution of the splicing commitment factor SRSF2 to Pol II acceleration, suggesting a functional benefit of co-transcriptional pre-mRNA splicing in transcription elongation. Unexpectedly, we found that perturbation of Pol II Ser2 phosphorylation had little impact on Pol II elongation or acceleration. While H3K79me2 has been positively correlated with Pol II elongation, we showed that reduction of this histone modification event actually accelerated Pol II elongation. Together, these data suggest a combined effect of gradual gain-of-competence and gradual lost-of-epigenetic barriers as the mechanism for accelerated Pol II elongation. DRB time-course releasing assay under functional perturbation
2015-12-31 | E-GEOD-66021 | biostudies-arrayexpress
Project description:Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA processing machineries, have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. Although H1s have a minor effect on global gene expression levels, they are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation rate of RNA polymerase II. Thus, H1 variants determine splicing fate by modulating the RNA polymerase II elongation rate.
2023-11-07 | GSE228239 | GEO
Project description:Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA processing machineries, have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. Although H1s have a minor effect on global gene expression levels, they are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation rate of RNA polymerase II. Thus, H1 variants determine splicing fate by modulating the RNA polymerase II elongation rate.
2023-11-07 | GSE228238 | GEO
Project description:Histones shape chromatin structure and the epigenetic landscape. H1, the most diverse histone in the human genome, has 11 variants. Due to high structural similarity between the H1s, their unique functions in transferring information from the chromatin to mRNA processing machineries, have remained elusive. Here, we generated human cell lines lacking up to five H1 subtypes, allowing us to characterize the genomic binding profiles of six H1 variants. Most H1s bind to specific sites, and binding depends on multiple factors including GC content. The highly expressed H1.2 has a high affinity for exons, whereas H1.3 binds intronic sequences. Although H1s have a minor effect on global gene expression levels, they are major splicing regulators, especially of exon skipping and intron retention events, through their effects on the elongation rate of RNA polymerase II. Thus, H1 variants determine splicing fate by modulating the RNA polymerase II elongation rate.
2023-11-07 | GSE228793 | GEO
Project description:To study the impact of the RNA polymerase II (Pol II) elongation rate on gene expression, we used CRISPR-Cas9 genome editing in S. pombe to generate a "slow" Pol II mutant with decreased elongation rate. Although the mutation is well tolerated as far as cell growth is concerned, transcriptomic analyses revealed that the slow mutant tends to terminate transcription prematurely. We distinguished two mechanisms by which premature termination affects gene expression in the slow mutant: It either (1) shortens 3'UTR, or (2) derepresses protein coding genes by prematurely terminating upstream interfering RNAs. Strikingly, the genes affected by these mechanisms are enriched for genes involved in phosphate uptake and purine synthesis, two processes essential for the maintenance of the nucleotide pool of the cell. Together with evidences that nucleotides are conditional for Pol II processive elongation, our results suggest that Pol II elongation rate acts as both sensor and effector in response to nucleotide depletion.
2020-05-29 | GSE140698 | GEO