Widespread backtracking by RNA pol II is a major effector of gene activation, 5’ pause release, termination and transcription elongation rate
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ABSTRACT:
PROVIDER: PRJNA491943 | ENA |
REPOSITORIES: ENA
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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.
2018-11-30 | GSE120201 | GEO
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.
2023-05-27 | GSE205545 | GEO
Project description: Not available
| S-EPMC6320282 | biostudies-literature
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.
2020-10-06 | GSE150844 | GEO
Project description:Transcription termination was analyzed by anti RNA pol II ChIP-seq in isogenic human HEK293 cell lines that inducibly express a-amanitin resistant mutants of the RNA polymerase II large subunit with slow and fast elongation rates and in lines that inducbily over-express WT or an active site mutant of the RNA exonuclease "torpedo" Xrn2. Transcription termination zones were mapped by anti-pol II ChIP-seq under conditions where transcription elongation rate was increased or decreased by point mutations in the large subunit of the enzyme. Termination was also assayed under conditions where Xrn2 exonuclease activity was inhibited by over-expression of an active site mutant (D235A).
2015-11-19 | E-GEOD-72800 | biostudies-arrayexpress
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. Refer to individual Series
2014-10-07 | E-GEOD-59833 | biostudies-arrayexpress
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.
2014-10-07 | E-GEOD-59828 | biostudies-arrayexpress
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. Examination of differential gene expression after overexpression of Cdk9-DN at 4 time points of somatic cell reprogramming
2014-10-07 | E-GEOD-59813 | biostudies-arrayexpress
Project description:Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through inhibition of pause release, we determine that pause-release rates are highly variable and most promoter-proximal paused RNA Polymerase II molecules prematurely terminate (~80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with induction of promoter-proximal termination under hyperosmotic stress but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.
2021-09-13 | GSE166202 | GEO
Project description:Distal enhancers characterized by H3K4me1 mark play critical roles in developmental and transcriptional programs. However, potential roles of specific distal regulatory elements in regulating RNA Polymerase II (Pol II) promoter-proximal pause release remain poorly investigated. Here we report that a unique cohort of jumonji C domain-containing protein 6 (JMJD6) and bromodomain-containing protein 4 (Brd4) co-bound distal enhancers, termed anti-pause enhancers (A-PEs), regulate promoter-proximal pause release of a large subset of transcription units via long-range interactions. Brd4-dependent JMJD6 recruitment on A-PEs mediates erasure of H4R3me2(s), which is directly read by 7SK snRNA, and decapping/demethylation of 7SK snRNA, ensuring the dismissal of the 7SKsnRNA/HEXIM inhibitory complex. The interactions of both JMJD6 and Brd4 with the P-TEFb complex permit its activation and pause release of regulated coding genes. The functions of JMJD6/ Brd4-associated dual histone and RNA demethylase activity on anti-pause enhancers have intriguing implications for these proteins in development, homeostasis and disease. All Gro-seq(s) were designed to reveal the transcriptional targets of JMJD6 and Brd4, and assess the role of JMJD6 and Brd4 in Pol II promoter-proximal pause release. All ChIP-seq(s) were designed to understand the unique features, associated molecular mechanisms and functions of the anti-pause enhancers (A-PEs) discovered in the current study.
2014-01-14 | E-GEOD-51633 | biostudies-arrayexpress