Genome-wide map of RNA polymerase II, NELF, and DSIF in HeLa cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE53008 | GEO | 2014/02/01
SECONDARY ACCESSION(S): PRJNA230673
REPOSITORIES: GEO
Similar Datasets
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. 3x ChIP-Seq (Pol II, NELF, DSIF) in HeLa cells
2014-02-01 | E-GEOD-53008 | biostudies-arrayexpress
Project description:Recent studies have found that promoter-proximal pausing occurs at most Pol2-regulated genes. DSIF and NELF function as negative elongation factors and promote Pol2 pausing. P-TEFb, whose enzymatic activity lies in the kinase Cdk9, positively regulates the transition into productive elongation by phosphorylating subunits in DSIF, NELF and Pol2. To gain insights into the interplay between these factors in regulating transcriptional pause release, we tested if knockdown of either pausing factor could bypass P-TEFb function. We find that P-TEFb function is still required for transcriptional elongation after DSIF or NELF knockdown. mES cells were infected with a shRNA hairpin to knockdown Spt5, Spt4, NelfE or control. The resulting cells were then treated with flavopiridol (1uM for 60 minutes). DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing. A sample of whole cell extract was sequenced and used as the background to determine enrichment. ChIP was performed using an antibody against total RNA Pol2 (Rpb1 N-terminus, Santa Cruz sc-899).
2011-01-19 | E-GEOD-26658 | biostudies-arrayexpress
Project description:Recent studies have found that promoter-proximal pausing occurs at most Pol2-regulated genes. DSIF and NELF function as negative elongation factors and promote Pol2 pausing. P-TEFb, whose enzymatic activity lies in the kinase Cdk9, positively regulates the transition into productive elongation by phosphorylating subunits in DSIF, NELF and Pol2. To gain insights into the interplay between these factors in regulating transcriptional pause release, we tested if knockdown of either pausing factor could bypass P-TEFb function. We find that P-TEFb function is still required for transcriptional elongation after DSIF or NELF knockdown.
2011-01-19 | GSE26658 | GEO
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
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
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
Project description:Negative elongation factor (NELF) is known to enforce promoter-proximal pausing of RNA polymerase II (Pol II), a pervasive phenomenon observed across multicellular genomes. However, the physiological impact of NELF on tissue homeostasis remains unclear. Here we show for the first time that whole-body conditional deletion of the B subunit of NELF (NELF-B) in adult mice results in cardiomyopathy and impaired response to cardiac stress. Tissue-specific knockout of NELF-B confirms its cell-autonomous function in cardiomyocytes. NELF directly supports transcription of those genes encoding rate-limiting enzymes in fatty acid oxidation and the tricarboxylic acid (TCA) cycle. NELF also shares extensively transcriptional target genes with peroxisome proliferator-activated receptors alpha (PPARalpha), a master regulator of energy metabolism in myocardium. Mechanistically, NELF helps stablize the transcription initation complex at the metabolism-related genes. Our findings strongly indicate that NELF is part of the PPARalpha-mediated transcription regulatory network that maintains metabolic homeostasis in cardiomyocytes. 3 Nelf-b f/f and 3 Nelf-b f/f; CreER female mice were injected with Tamoxifen at 8 wk old. Heart tissue were harvested at 20 wks old and used for RNA preparation.
2014-03-01 | E-GEOD-54372 | biostudies-arrayexpress
Project description:RNA polymerase II (Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, Pol II is rapidly released from pausing at heat shock induced genes, while most genes are paused and transcriptionally downregulated during the heat shock response. We find that both aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5’ cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from Pol II pause-release.
2020-03-09 | GSE144786 | GEO
Project description:Cdk9 is an essential transcriptional kinase that is conserved across distant eukaryotes, but we previously found that acute inhibition of Cdk9 causes different transcriptional phenotypes in NELF-containing higher eukaryotes (like mammals and Drosophila) vs. the NELF-lacking fission yeast S. pombe. NELF is known to participate in promoter-proximal pausing of RNA Polymerase II, and using NELF-depleted Drosophila cells, we find that this NELF-mediated pause is necessary to prevent gene body entry by Pol II in the absence of Cdk9. Without NELF, the abnormal gene body entry that occurs following Cdk9 inhibition is strikingly similar to that seen in S. pombe, and in either case, these Pol II complexes have elongation defects and are unable to complete gene transcription. These results show that NELF enforces an early checkpoint for Cdk9 and efficiently shuts down gene transcription in its absence. We propose this would have been critical for the emergence of gene regulatory strategies acting via Cdk9 to modulate transcription of specific genes.
2023-03-22 | GSE211397 | GEO
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