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Genome-wide map of RNA polymerase II, NELF, and DSIF in HeLa cells

ABSTRACT: Most metazoan promoters have RNA polymerase II (Pol II) paused slightly downstream of the transcription start site by NELF and DSIF. This pausing keeps these promoters available for rapid induction by P-TEFb, whose activity causes NELF to dissociate and Pol II and DSIF to elongate on the gene. ChIP-Seq data was generated for Pol II, NELF, and DSIF in HeLa cells and used to look at pausing downstream of unannotated promoters. 3x ChIP-Seq (Pol II, NELF, DSIF) in HeLa cells

ORGANISM(S): Homo sapiens

SUBMITTER: Kyle Nilson

PROVIDER: E-GEOD-53008 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein (snRNP) activates hexamethylene bisacetamide-inducible protein (HEXIM1) transcription.

Liu Pingyang P Xiang Yanhui Y Fujinaga Koh K Bartholomeeusen Koen K Nilson Kyle A KA Price David H DH Peterlin B Matija BM

The Journal of biological chemistry 20140210 14

By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, add ...[more]

PMID: 24515107

Similar Datasets

Genome-wide map of RNA polymerase II, NELF, and DSIF in HeLa cells

Project description:Most metazoan promoters have RNA polymerase II (Pol II) paused slightly downstream of the transcription start site by NELF and DSIF. This pausing keeps these promoters available for rapid induction by P-TEFb, whose activity causes NELF to dissociate and Pol II and DSIF to elongate on the gene. ChIP-Seq data was generated for Pol II, NELF, and DSIF in HeLa cells and used to look at pausing downstream of unannotated promoters.

2014-02-01 | GSE53008 | GEO

SPT6 functions in transcriptional pause/release via PAF1C recruitment

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute 5 protein-depletion approach, prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Mechanistically, loss of SPT6 results in loss of PAF1 complex (PAF1C) from RNA Pol II, leading to NELF-bound RNA Pol II release into the gene bodies. Furthermore, SPT6 and/or PAF1 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through the recruitment of PAF1C during the early elongation.

2022-08-09 | PXD033771 | Pride

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

The negative elongation factor NELF promotes active transcription of Drosophila ecdysone-dependent genes

Project description:The current work describes molecular mechanism used by the Negative elongation complex (NELF) to control the Pol II pause at genes whose transcription is induced by 20-hydroxyecdysone (20E). According to our data NELF complex is recruited to the promoters and enhancers of 20E-dependent genes. Its presence at the regulatory sites of 20E-dependent genes correlates with the described interaction of its NELF-A subunit with EcR receptor. The NELF complex, consisting of at least three subunits, is formed at the 20E-dependent promoters and participates in their active transcription and maintenance of their repressed state. NELF depletion causes a significant decrease in transcription induced by 20E. We associate this effect with the disruption of Pol II elongation complexes formation. A considerable reduction in the promoter-bound level of Spt5 subunit of DSIF was observed at the 20E-dependent genes upon NELF depletion. We presume that participation in stabilization of the Pol II-DSIF complex can be an important function of NELF with a big impact on transcription of its target genes. Part of our study was to directly link NELF to regulation of 20E-dependent genes in development. We showed presence of NELF at the promoters of 20E-dependent genes during their active transcription both in embryogenesis and metamorphosis. We also demonstrate that 20E-dependent promoters remain in a Pol II paused state at the larva stage, where they are temporarily repressed.

2021-01-10 | GSE156847 | GEO

Role of RNA polymerase II "pausing" in Drosophila development

Project description:According to previous studies, during Drosophila embryogenesis, RNA polymerase II is recruited to promoters at developmental stages preceding the stages of active transcription of genes. This work is aimed at exploring whether this mechanism is used during Drosophila metamorphosis. We performed ChIP-Seq analysis using antibodies to various modifications of RNA polymerase II (total, Pol II CTD Ser5P and Pol II CTD Ser2P), as well as to subunits of NELF, DSIF, PAF complexes and Brd4/Fs(1)h that control transcription elongation. We found that like in mid-embryogenesis during metamorphosis, promoters bind RNA polymerase II in the "paused" state preparing for activation at later stages of development. During mid-embryogenesis, RNA polymerase II in "pause" is phosphorylated at Ser5 and Ser2 of Rpb1 CTD and binds NELF, DSIF, and PAF complexes, but not Brd4/Fs(1)h. During metamorphosis, the "paused" RNA polymerase II complex includes Brd4/Fs(1)h in addition to NELF, DSIF, and PAF. The RNA polymerase II in this complex is phosphorylated at Ser5 at Rpb1 CTD, but not at Ser2.

2022-09-10 | GSE210971 | GEO

Functional association of Gdown1 with RNA Polymerase II in Human

Project description:Most human genes are loaded with promoter proximally paused RNA polymerase II (RNAP II) molecules that are poised for release into productive elongation by P-TEFb. Gdown1, a protein that renders RNAP II responsive to mediator, is involved in RNAP II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. ChIP-Seq data demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb, and mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function. 7 ChIP-Seq data for Pol II and Gdown1 in human HeLa cell. See associated publication.

2012-01-13 | E-GEOD-32442 | biostudies-arrayexpress

Regulation of Mammalian Cell Growth and Survival By an RNA Polymerase II-Pausing Factor (ChIP-seq)

Project description:Many mammalian genes are occupied by paused RNA polymerase II (pol II) at promoter-proximal regions on both sides of transcription start sites (TSSs). However, the consequences of pol II pausing on gene expression and cell biology are not fully understood. Here we report that genetic ablation of the b subunit of mouse negative elongation factor (Nelf-b), a key pol II-pausing factor, results in slower progression at multiple cell cycle stages and increased apoptosis. Consistently, a whole-genome analysis indicates that growth and cell death-related genes are highly enriched among the direct target genes of Nelf-b. In particular, Nelf-b deletion increases pol II density in the promoter-distal region of stress response genes and their overall expression levels in the absence of any external stress signals. In addition, our work also reveals a previously unappreciated role of Nelf-b role in curbing TSS-upstream transcription of many mammalian genes. We suggest that Nelf-mediated pol II pausing balances the cellular needs for growth/survival and stress response by preventing excessive basal transcription of stress-induced genes. Examination of Nelf-b bindings sites on chromosomes in mouse embryonic fibroblasts

2011-08-17 | E-GEOD-24113 | biostudies-arrayexpress

SPT6 functions in transcriptional pause-release via PAF1C recruitment [RNA-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

2022-08-09 | GSE202189 | GEO

SPT6 functions in transcriptional pause-release via PAF1C recruitment [PRO-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

2022-08-09 | GSE202187 | GEO

SPT6 functions in transcriptional pause-release via PAF1C recruitment [ChIP-seq]

Project description:In vitro studies identified various factors including P-TEFb, SEC, SPT6, PAF1, DSIF, and NELF functioning at different stages of transcription elongation driven by RNA polymerase II (RNA Pol II). What remains unclear is how these factors cooperatively regulate pause/release and productive elongation in the context of living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in release of paused RNA Pol II. Short genes demonstrate a prominent release and a subsequent increase in mature transcripts, whereas long genes fail to yield mature transcripts due to a loss of processivity. Unexpectedly, the recruitment of PAF1 complex (PAF1C) to RNA Pol II fails upon SPT6 depletion, leading to the release of NELF-bound RNA Pol II into the gene bodies. Furthermore, SPT6 depletion impairs heat shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause-release through PAF1C recruitment and NELF removal during the early elongation.

2022-08-09 | GSE202184 | GEO

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