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Stably-paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor Triptolide

ABSTRACT: Transcription by RNA Polymerase II (Pol II) in metazoan is regulated in several steps, including preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional RNA-processing and termination. Genome-wide studies have demonstrated that the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes involved in developmental and stimulus-responsive pathways. However, a mechanistic understanding of the paused Pol II states at promoters is limited. For example, at a global level, it’s unclear to what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid cycles of initiation and termination. Here we used the small molecule Triptolide (TPL), an XPB/TFIIH inhibitor, to block transcriptional initiation followed by measuring Pol II occupancy by ChIP-seq. This inhibition of initiation enables us to investigate different states of paused Pol II. Specifically, our global analysis reveals that most genes with paused Pol II, as defined by pausing index, show significant clearance of Pol II during the period of TPL treatment. Our study further identifies a group of genes with unexpectedly stably-paused Pol II, with unchanged Pol II occupancy even after one hour of inhibition of initiation. This group of genes constitutes a small portion of all paused genes defined by the conventional criterion of pausing index. These findings could pave the way for evaluating the contribution of different elongation/pausing factors on different states of Pol II pausing in developmental and other stimulus-responsive pathways.

ORGANISM(S): Homo sapiens

PROVIDER: GSE63314 | GEO | 2015/01/05

SECONDARY ACCESSION(S): PRJNA267258

REPOSITORIES: GEO

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Stably-paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor Triptolide

Project description:Transcription by RNA Polymerase II (Pol II) in metazoan is regulated in several steps, including preinitiation complex (PIC) formation, initiation, Pol II escape, productive elongation, cotranscriptional RNA-processing and termination. Genome-wide studies have demonstrated that the phenomenon of promoter-bound Pol II pausing is widespread, especially for genes involved in developmental and stimulus-responsive pathways. However, a mechanistic understanding of the paused Pol II states at promoters is limited. For example, at a global level, it’s unclear to what extent the engaged paused Pol II is stably tethered to the promoter or undergoes rapid cycles of initiation and termination. Here we used the small molecule Triptolide (TPL), an XPB/TFIIH inhibitor, to block transcriptional initiation followed by measuring Pol II occupancy by ChIP-seq. This inhibition of initiation enables us to investigate different states of paused Pol II. Specifically, our global analysis reveals that most genes with paused Pol II, as defined by pausing index, show significant clearance of Pol II during the period of TPL treatment. Our study further identifies a group of genes with unexpectedly stably-paused Pol II, with unchanged Pol II occupancy even after one hour of inhibition of initiation. This group of genes constitutes a small portion of all paused genes defined by the conventional criterion of pausing index. These findings could pave the way for evaluating the contribution of different elongation/pausing factors on different states of Pol II pausing in developmental and other stimulus-responsive pathways. ChIP-Seq of total/Ser5P Pol II in HCT116 cells treated with DMSO or TPL in serum starvation/activation or normal conditions. Nascent RNA-seq in HCT116 cells treated with DMSO or TPL in starved condition.

2015-01-05 | E-GEOD-63314 | biostudies-arrayexpress

RNA Polymerase II Pausing Can Be Retained or Acquired During Activation of Genes Involved in the Epithelial to Mesenchymal Transition

Project description:Promoter-proximal RNA polymerase II (Pol II) pausing is implicated in the regulation of gene transcription. However, the mechanisms of pausing including its dynamics during transcriptional responses remain to be fully understood. We performed global analysis of short capped RNAs and Pol II Chromatin Immunoprecipitation sequencing in MCF-7 breast cancer cells to map Pol II pausing across the genome, and used permanganate footprinting to specifically follow pausing during transcriptional activation of several genes involved in the Epithelial to Mesenchymal Transition (EMT). We find that the gene for EMT master regulator Snail (SNAI1), but not Slug (SNAI2), shows evidence of Pol II pausing before activation. Transcriptional activation of the paused SNAI1 gene is accompanied by a further increase in Pol II pausing signal whereas activation of non-paused SNAI2 gene results in the acquisition of a typical pausing signature. The increase in pausing signal reflects increased transcription initiation without changes in Pol II pausing. Activation of the heat shock HSP70 gene involves pausing release that speeds up Pol II turnover, but does not change pausing location. We suggest that Pol II pausing is retained during transcriptional activation and can further undergo regulated release in a signal-specific manner. Untreated MCF-7 cells were analyzed for the distribution of Pol II using ChIP-sequencing with Anti-Pol II N-20 antibody (two independent biological replicates, A, B), and for the distribution of paused RNA polymerase II by sequencing of short capped RNAs (scRNAs) prepared from nuclei (three independent biological replicates, 1-3). All samples were sequenced on a MiSeq instrument in paired-end format

2015-03-27 | E-GEOD-67041 | biostudies-arrayexpress

TFIID enables RNA polymerase II promoter-proximal pausing

Project description:RNA polymerase II (pol II) transcribes all protein-coding and many non-coding RNAs in the human genome. Pol II transcription initiation is governed by the Pre-Initiation Complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, pol II, and Mediator. After initiation, pol II enzymes typically pause after transcribing less than 100 bases, and paused polymerases represent a common regulatory intermediate. Accordingly, paused pol II has been implicated in enhancer function, development and homeostasis, and diseases ranging from cancer to viral pathogenesis. Precisely how pol II promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes such as NELF and DSIF increase pausing whereas the activity of CDK9 (P-TEFb complex) correlates with pause release. To address specific mechanistic questions about pol II pausing and its regulation, we reconstituted human pol II promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pol II pausing in vitro, whereas P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pol II pausing, suggesting that pausing is an inherent property of the PIC. In agreement, pol II pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP); moreover, pausing was dependent upon TFIID subunits TAF1 and TAF2. TAF1/2 bind genomic DNA downstream of the pol II initiation site, invoking a “complex interaction” model for pausing. Consistent with this model, PRO-Seq experiments revealed increased transcription upon acute depletion (t=60 min) of TAF1 and TAF2 in human cells, and pol II pausing was disrupted at thousands of genes. Similar results were obtained in TAF1-depleted Drosophila S2 cells. Collectively, these data establish the general transcription factor TFIID as a genome-wide regulator of pol II promoter-proximal pausing.

2020-05-28 | GSE132764 | GEO

Paused RNA polymerase II inhibits new transcriptional initiation

Project description:RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation. This pause is a key component of metazoan gene expression regulation. Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression. This requires multiple rounds of transcription and thus cannot rely solely on pause release. But it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription. Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation. We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.

2017-04-17 | GSE85741 | GEO

Paused RNA polymerase II inhibits new transcriptional initiation [ChIP-Seq]

2017-04-17 | GSE85740 | GEO

Paused RNA polymerase II inhibits new transcriptional initiation [ChIP-nexus spike-in]

2017-04-17 | GSE85739 | GEO

Paused RNA polymerase II inhibits new transcriptional initiation [ChIP-nexus]

2017-04-17 | GSE85738 | GEO

RNA Polymerase II Pausing Can Be Retained or Acquired During Activation of Genes Involved in the Epithelial to Mesenchymal Transition

2015-03-27 | GSE67041 | GEO

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release

Project description:Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.

2024-05-31 | PXD037842 | Pride

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

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