Widespread backtracking by RNA pol II is a major effector of gene activation, 5’ pause release, termination and transcription elongation rate
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA491943 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR16101039.fastq.gz | Fastqsanger.gz | |||
| SRR16101040.fastq.gz | Fastqsanger.gz | |||
| SRR7879130.fastq.gz | Fastqsanger.gz | |||
| SRR7879140.fastq.gz | Fastqsanger.gz | |||
| SRR7879150.fastq.gz | Fastqsanger.gz |
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Project description:In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. We identified backtracking sites using mNET-seq in human cells expressing dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3’ flanking regions where termination is enhanced by TFIISDN. Inhibition of the rescue from backtracked pol II by TFIISDN impaired the escape from 5’ pause sites, the completion of long transcripts, and activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half as measured by sequencing nascent transcripts pulse labeled with Bromouridine (Bru-seq) and by anti-pol II ChIP-seq at time points after release of a DRB block. These results suggest that rescue of backtracked pol II by TFIIS is a major stimulus of elongation.
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Project description:In Arabidopsis, RNA Polymerase II (Pol II) often pauses within a few hundred base pairs downstream of the polyadenylation site, reflecting efficient transcriptional termination, but how such pause is regulated remains largely elusive. Here, we studied Pol II dynamics at gene 3’ end by combining comprehensive experiments with mathematical modelling. We generated high-resolution Ser2P Pol II positioning data specifically enriched at gene 3’ end and defined a 3’ end pause index (3’PI). The position but not the extent of the 3’ end pause correlates with termination window size. The 3’PI is not decreased but even mildly increased in the termination deficient mutant xrn3, indicating 3’ end pause is a regulatory step early during the termination and before XRN3 mediated RNA decay that releases the Pol II. Unexpectedly, 3’PI is closely associated with gene exon numbers and co-transcriptional splicing efficiency. Multiple exons genes often display stronger 3’ end pauses and more efficient on-chromatin splicing than genes with few exons. Chemical inhibition of splicing strongly reduces the 3’PI and disrupts its correlation with exon numbers but does not globally impact 3’ end readthrough levels. These results were further confirmed by fitting Pol II positioning data with a mathematical model, which enables the estimation of parameters that define Pol II dynamics. Our work highlights that the number of exons via co-transcriptional splicing is a major determinant of Pol II pausing levels at gene 3’ end in plants.
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Project description:Efficient release of promoter-proximally paused Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated. Here, we report that Integrator subunit 8 (IntS8) is critical for transcription repression through its association with Protein Phosphatase 2A (PP2A). We find that Integrator-bound PP2A dephosphorylates the Pol II C-terminal domain and Spt5, and prevents the transition to productive elongation. Blocking PP2A association with Integrator thus stimulates pause release and gene activation. These results reveal a second catalytic function associated with Integrator-mediated transcription termination, and suggest a new model for the control of productive elongation involving active competition between transcriptional kinases and phosphatases.
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Project description:We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a and Dcp2 and the termination factor TTF2 co-immunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease torpedo that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2 and TTF2 localize near transcription start sites (TSSs) by ChIP-Seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors, Xrn2 and TTF2, shifted polymerase away from the TSS toward upstream and downstream distal positions. This re-distribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the torpedo mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated co-transcriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation. RNA pol II (GSE30895: GSM766171), Xrn2, TTF2 and Dcp1a were localized by ChIP-Seq in HeLa cells. RNA pol II was localized in control HEK293 cells and cells infected with lentiviruses expressing a scrambled control shRNA (scr), and shRNAs targeting the following proteins: Xrn2, TTF2, Xrn2+TTF2, Edc3, Dcp1a, and Dcp2.
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2014-10-07 | E-GEOD-59833 | biostudies-arrayexpress
Project description:Gene expression is regulated by controlling distinct steps of the transcriptional cycle, including initiation, pausing, elongation, and termination. Kinases phosphorylate RNA Polymerase II and associated factors to control transitions between these steps and act as central gene regulatory nodes. Similarly, phosphatases that dephosphorylate these components are emerging as important regulators of transcription, though their roles remain less well understood. Here we discover that the PNUTS-PP1 phosphatase complex plays an essential role in controlling transcription pause release in addition to its previously described function in transcription termination. Transcription pause release by the PNUTS complex is essential for almost all RNA Pol II-dependent gene transcription, relies on its PP1 phosphatase subunit, and controls the phosphorylation of factors required for pause release and elongation. Together, this reveals an essential new role for a phosphatase complex in transcription pause release and shows that the PNUTS complex is essential for RNA Poll II-dependent transcription.
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Project description:Reactivation of the pluripotency network during somatic cell reprogramming by exogenous transcription factors involves chromatin remodeling and the recruitment of RNA polymerase II (Pol II) to target loci. Here, we report that Pol II is engaged at pluripotency promoters in reprogramming but remains paused and inefficiently released. We also show that bromodomain-containing protein 4 (BRD4) stimulates productive transcriptional elongation of pluripotency genes by dissociating the pause release factor P-TEFb from an inactive complex containing HEXIM1. Consequently, BRD4 overexpression enhances reprogramming efficiency and HEXIM1 suppresses it, whereas Brd4 and Hexim1 knockdown do the opposite. We further demonstrate that the reprogramming factor KLF4 helps recruit P-TEFb to pluripotency promoters. Our work thus provides a mechanism for explaining the reactivation of pluripotency genes in reprogramming and unveils an unanticipated role for KLF4 in transcriptional pause release. Pol II ChIP-seq for MEFs, ESCs and bulk populations of OSKM reprogramming intermediates at two time points.
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Project description:In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3' flanking regions where termination is enhanced by TFIISDN, suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5' pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions.
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Project description:We report a function of human mRNA decapping factors in control of transcription by RNA polymerase II. Decapping proteins Edc3, Dcp1a and Dcp2 and the termination factor TTF2 co-immunoprecipitate with Xrn2, the nuclear 5'-3' exonuclease torpedo that facilitates transcription termination at the 3' ends of genes. Dcp1a, Xrn2 and TTF2 localize near transcription start sites (TSSs) by ChIP-Seq. At genes with 5' peaks of paused pol II, knockdown of decapping or termination factors, Xrn2 and TTF2, shifted polymerase away from the TSS toward upstream and downstream distal positions. This re-distribution of pol II is similar in magnitude to that caused by depletion of the elongation factor Spt5. We propose that coupled decapping of nascent transcripts and premature termination by the torpedo mechanism is a widespread mechanism that limits bidirectional pol II elongation. Regulated co-transcriptional decapping near promoter-proximal pause sites followed by premature termination could control productive pol II elongation.
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Project description:Gene expression is regulated by controlling distinct steps of the transcriptional cycle, including initiation, pausing, elongation, and termination. Kinases phosphorylate RNA Polymerase II and associated factors to control transitions between these steps and act as central gene regulatory nodes. Similarly, phosphatases that dephosphorylate these components are emerging as important regulators of transcription, though their roles remain less well understood. Here we discover that the mouse PNUTS-PP1 phosphatase complex plays an essential role in controlling transcription pause release in addition to its previously described function in transcription termination. Transcription pause release by the PNUTS complex is essential for almost all RNA Pol II-dependent gene transcription, relies on its PP1 phosphatase subunit, and controls the phosphorylation of factors required for pause release and elongation. Together, these findings reveal an essential new role for a phosphatase complex in transcription pause release and shows that the PNUTS complex is essential for RNA Pol II-dependent transcription.
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