Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters.

ABSTRACT: Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.

SUBMITTER: Krebs AR

PROVIDER: S-EPMC5548954 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Genome-wide single molecule footprinting reveals high RNA polymerase II turnover at paused promoters

Project description:Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA Polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here we harnessed single molecule footprinting to quantify distinct steps of initiation in vivo throughout the drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpected high turnover of polymerases at paused promoters--an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat-shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single molecule resolution and refines concepts of transcriptional pausing.

2017-06-23 | GSE77369 | GEO

Dynamic turnover of paused Pol II complexes at human promoters.

Project description:Paused RNA polymerase II (Pol II) that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused Pol II is a stable or dynamic target remains unresolved. We report that most 5' paused Pol II throughout the genome is turned over within 2 min. This process is revealed under hypertonic conditions that prevent Pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation, suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel preinitiated state of Pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that Pol II occupancy near 5' ends is governed by a cycle of ongoing assembly of preinitiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of Pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause is by escape from premature termination.

| S-EPMC6120720 | biostudies-literature

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.

| S-EPMC8729149 | biostudies-literature

Live-cell analysis of endogenous GFP-RPB1 uncovers rapid turnover of initiating and promoter-paused RNA Polymerase II.

Project description:Initiation and promoter-proximal pausing are key regulatory steps of RNA Polymerase II (Pol II) transcription. To study the in vivo dynamics of endogenous Pol II during these steps, we generated fully functional GFP-RPB1 knockin cells. GFP-RPB1 photobleaching combined with computational modeling revealed four kinetically distinct Pol II fractions and showed that on average 7% of Pol II are freely diffusing, while 10% are chromatin-bound for 2.4 seconds during initiation, and 23% are promoter-paused for only 42 seconds. This unexpectedly high turnover of Pol II at promoters is most likely caused by premature termination of initiating and promoter-paused Pol II and is in sharp contrast to the 23 minutes that elongating Pol II resides on chromatin. Our live-cell-imaging approach provides insights into Pol II dynamics and suggests that the continuous release and reinitiation of promoter-bound Pol II is an important component of transcriptional regulation.

| S-EPMC5948963 | biostudies-literature

Genome-wide reductions in active chromatin, paused RNA Polymerase II and nucleosome turnover during heat shock

Project description:Heat shock rapidly induces expression of a small set of genes while globally repressing transcription, making it an attractive system for studying alterations in the chromatin landscape that accompany changes in gene regulation. We have characterized these changes using low-salt extraction of intact micrococcal nuclease (MNase)-treated Drosophila S2 cell nuclei to determine the active nucleosomal and subnucleosomal chromatin landscapes. The low-salt-soluble fraction corresponds to classical "active" chromatin and includes distinct size fractions of MNase-protected particles that can be precisely mapped by paired-end sequencing. After heat shock, the distribution of low-salt-soluble nucleosomes showed an overall reduction over gene bodies, consistent with down-regulation of transcription. No global changes were detected in the subnucleosomal landscape upstream of transcriptional start sites, however, we observed a genome-wide reduction of paused RNA Polymerase II from the active chromatin fraction. Furthermore, nucleosome turnover decreased within gene bodies in a pattern similar to that observed when transcription elongation was artificially inhibited. These observations suggest that reduced Pol II affinity and processivity is the dominant nuclear mechanism for genome-wide repression during heat shock. Our ability to precisely map both nucleosomal and subnucleosomal particles directly from classical active chromatin extracts to assay changes in the chromatin landscape provides a simple general strategy for epigenome characterization. High-throughput sequencing (Illumina HiSeq 2000) We have characterized changes to the active nucleosomal and subnucleosomal landscape during the heat shock response in Drosophila cells by genome-wide profiling of low-salt extracted micrococcal nuclease-treated nuclei, paused RNA Polymerase II and CATCH-IT nucleosome turnover.

2011-11-15 | E-GEOD-30755 | biostudies-arrayexpress

Heat shock reduces stalled RNA polymerase II and nucleosome turnover genome-wide.

Project description:Heat shock rapidly induces expression of a subset of genes while globally repressing transcription, making it an attractive system to study alterations in the chromatin landscape that accompany changes in gene regulation. We characterized these changes in Drosophila cells by profiling classical low-salt-soluble chromatin, RNA polymerase II (Pol II), and nucleosome turnover dynamics at single-base-pair resolution. With heat shock, low-salt-soluble chromatin and stalled Pol II levels were found to decrease within gene bodies, but no overall changes were detected at transcriptional start sites. Strikingly, nucleosome turnover decreased genome-wide within gene bodies upon heat shock in a pattern similar to that observed with inhibition of Pol II elongation, especially at genes involved in the heat-shock response. Relatively high levels of nucleosome turnover were also observed throughout the bodies of genes with paused Pol II. These observations suggest that down-regulation of transcription during heat shock involves reduced nucleosome mobility and that this process has evolved to promote heat-shock gene regulation. Our ability to precisely map both nucleosomal and subnucleosomal particles directly from low-salt-soluble chromatin extracts to assay changes in the chromatin landscape provides a simple general strategy for epigenome characterization.

| S-EPMC3222904 | biostudies-literature

JMJD5 couples with CDK9 to release the paused RNA polymerase II.

Project description:More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

| S-EPMC7443887 | biostudies-literature

Dynamic turnover of paused pol II complexes at human promoters

Project description:Paused RNA polymerase II that piles up near most human promoters is the target of mechanisms that control entry into productive elongation. Whether paused pol II is a stable or a dynamic target remains unresolved. We report that most 5’-paused pol II throughout the genome is turned over within 2 minutes. This process is revealed under hypertonic conditions that prevent pol II recruitment to promoters. This turnover requires cell viability but is not prevented by inhibiting transcription elongation suggesting that it is mediated at the level of termination. When initiation was prevented by triptolide during recovery from high salt, a novel pre-initiated state of pol II lacking the pausing factor Spt5 accumulated at transcription start sites. We propose that pol II occupancy near 5’ ends is governed by a cycle of ongoing assembly of pre-initiated complexes that transition to pause sites followed by eviction from the DNA template. This model suggests that mechanisms regulating the transition to productive elongation at pause sites operate on a dynamic population of pol II that is turning over at rates far higher than previously suspected. We suggest that a plausible alternative to elongation control via escape from a stable pause, is by escape from premature termination.

2018-09-14 | GSE117006 | GEO

Genome-wide reductions in active chromatin, paused RNA Polymerase II and nucleosome turnover during heat shock

2011-11-15 | GSE30755 | GEO

Multiple P-TEFbs cooperatively regulate the release of promoter-proximally paused RNA polymerase II.

Project description:The association of DSIF and NELF with initiated RNA Polymerase II (Pol II) is the general mechanism for inducing promoter-proximal pausing of Pol II. However, it remains largely unclear how the paused Pol II is released in response to stimulation. Here, we show that the release of the paused Pol II is cooperatively regulated by multiple P-TEFbs which are recruited by bromodomain-containing protein Brd4 and super elongation complex (SEC) via different recruitment mechanisms. Upon stimulation, Brd4 recruits P-TEFb to Spt5/DSIF via a recruitment pathway consisting of Med1, Med23 and Tat-SF1, whereas SEC recruits P-TEFb to NELF-A and NELF-E via Paf1c and Med26, respectively. P-TEFb-mediated phosphorylation of Spt5, NELF-A and NELF-E results in the dissociation of NELF from Pol II, thereby transiting transcription from pausing to elongation. Additionally, we demonstrate that P-TEFb-mediated Ser2 phosphorylation of Pol II is dispensable for pause release. Therefore, our studies reveal a co-regulatory mechanism of Brd4 and SEC in modulating the transcriptional pause release by recruiting multiple P-TEFbs via a Mediator- and Paf1c-coordinated recruitment network.

| S-EPMC5001612 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data