Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE42117 | GEO | 2013/02/01
SECONDARY ACCESSION(S): PRJNA179188
REPOSITORIES: GEO
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Project description:Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a Precision nuclear Run-On and sequencing assay (PRO-seq) to map the genome-wide distribution of transcriptionally-engaged Pol II at base-pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' poly-adenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing . We test this ‘Complex Interaction’ model with insertional mutagenesis of the Drosophila Hsp70 core promoter. Identification of RNA polymerase active sites in Drosophila S2 cell line using PRO-seq method. Identification of transcription initiation sites in Drosophila S2 cell line using PRO-cap method. Identification of changes in RNA polymerase active sites on transgenic Hsp70 promoters upon disruption of DNA sequence elements in 3 transgenic fly lines using PRO-seq method.
2013-02-01 | E-GEOD-42117 | biostudies-arrayexpress
Project description:RNA Polymerase II (Pol II) carries out transcription of both protein-coding and non-coding genes. Whereas Pol II initiation at protein-coding genes has been studied in detail, Pol II initiation at non-coding genes such as small nuclear RNA (snRNA) genes is not understood at the structural level. Here we study Pol II initiation at snRNA gene promoters and show that the snRNA-activating protein complex (SNAPc) enables DNA opening and transcription initiation independent of TFIIE and TFIIH in vitro. We then resolve cryo-EM structures of the SNAPc-containing Pol II preinitiation complex (PIC) assembled on U1 and U5 snRNA promoters. The core of SNAPc binds two turns of DNA and recognizes the snRNA promoter-specific proximal sequence element (PSE) located upstream of the TATA box-binding protein TBP. Two extensions of SNAPc called wing-1 and wing-2 bind TFIIA and TFIIB, respectively, explaining how SNAPc directs Pol II to snRNA promoters. Comparison of structures of closed and open promoter complexes elucidates TFIIH-independent DNA opening. These results provide the structural basis of Pol II initiation at non-coding RNA gene promoters.
2023-03-11 | PXD033638 | Pride
Project description:Control of RNA transcription is critical for the development and homeostasis of all organisms, and can occur at multiple steps of the transcription cycle, including RNA polymerase II (Pol II) recruitment, initiation, promoter-proximal pausing, and elongation. That Pol II accumulates on many promoters in metazoans implies that steps other than Pol II recruitment are rate-limiting and regulated 1-6. By integrating genome-wide Pol II chromatin immunoprecipition (ChIP) and Global Run-On (GRO) genomic data sets from Drosophila cells, we examined critical features of Pol II near promoters. The accumulation of promoter-proximal polymerase is widespread, occurring on 70% of active genes; and unlike elongating Pol II within the body of genes, promoter Pol II are held paused by factors like NELF, unable to transcribe unless nuclei are treated with strong detergent. Notably, we find that the vast majority of promoter-proximal Pol II detected by ChIP are paused, thereby identifying the biochemical nature of this rate-limiting step in transcription. Finally, we demonstrate that Drosophila promoters do not have the upstream divergent Pol II that is seen so broadly and prominently on mammalian promoters. We postulate this is a consequence of Drosophila’s extensive use of directional core promoter sequence elements, which contrasts with mammals’ lack of directional elements and prevalence of CpG island core promoters. In support of this idea, we show that the fraction of mammalian promoters containing a TATA box core element is dramatically depleted of upstream divergent transcription. ChIP-seq data set for Pol II (rpb3) (2 replicates).
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Project description:RNA polymerase II (Pol II) pausing is a general regulatory step in transcription, yet the stability of paused Pol II varies widely between genes. Although paused Pol II stability correlates with core promoter elements, the contribution of individual sequences remains unclear, in part because no rapid assay is available for measuring the changes in Pol II pausing as a result of altered promoter sequences. Here, we overcome this hurdle by showing that ChIP-nexus captures the endogenous Pol II pausing on transfected plasmids. Using this reporter-ChIP-nexus assay in Drosophila cells, we show that the pausing stability is influenced by downstream promoter sequences, but that the strongest contribution to Pol II pausing comes from the initiator sequence, in which a single nucleotide, a G at the +2 position, is critical for stable Pol II pausing. These results establish reporter-ChIP-nexus as a valuable tool to analyze Pol II pausing.
2019-04-05 | GSE116244 | GEO
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
Project description:Control of RNA transcription is critical for the development and homeostasis of all organisms, and can occur at multiple steps of the transcription cycle, including RNA polymerase II (Pol II) recruitment, initiation, promoter-proximal pausing, and elongation. That Pol II accumulates on many promoters in metazoans implies that steps other than Pol II recruitment are rate-limiting and regulated 1-6. By integrating genome-wide Pol II chromatin immunoprecipition (ChIP) and Global Run-On (GRO) genomic data sets from Drosophila cells, we examined critical features of Pol II near promoters. The accumulation of promoter-proximal polymerase is widespread, occurring on 70% of active genes; and unlike elongating Pol II within the body of genes, promoter Pol II are held paused by factors like NELF, unable to transcribe unless nuclei are treated with strong detergent. Notably, we find that the vast majority of promoter-proximal Pol II detected by ChIP are paused, thereby identifying the biochemical nature of this rate-limiting step in transcription. Finally, we demonstrate that Drosophila promoters do not have the upstream divergent Pol II that is seen so broadly and prominently on mammalian promoters. We postulate this is a consequence of Drosophila’s extensive use of directional core promoter sequence elements, which contrasts with mammals’ lack of directional elements and prevalence of CpG island core promoters. In support of this idea, we show that the fraction of mammalian promoters containing a TATA box core element is dramatically depleted of upstream divergent transcription.
2012-10-25 | GSE23542 | GEO
Project description:Control of RNA transcription is critical for the development and homeostasis of all organisms, and can occur at multiple steps of the transcription cycle, including RNA polymerase II (Pol II) recruitment, initiation, promoter-proximal pausing, and elongation. That Pol II accumulates on many promoters in metazoans implies that steps other than Pol II recruitment are rate-limiting and regulated 1-6. By integrating genome-wide Pol II chromatin immunoprecipition (ChIP) and Global Run-On (GRO) genomic data sets from Drosophila cells, we examined critical features of Pol II near promoters. The accumulation of promoter-proximal polymerase is widespread, occurring on 70% of active genes; and unlike elongating Pol II within the body of genes, promoter Pol II are held paused by factors like NELF, unable to transcribe unless nuclei are treated with strong detergent. Notably, we find that the vast majority of promoter-proximal Pol II detected by ChIP are paused, thereby identifying the biochemical nature of this rate-limiting step in transcription. Finally, we demonstrate that Drosophila promoters do not have the upstream divergent Pol II that is seen so broadly and prominently on mammalian promoters. We postulate this is a consequence of Drosophila’s extensive use of directional core promoter sequence elements, which contrasts with mammals’ lack of directional elements and prevalence of CpG island core promoters. In support of this idea, we show that the fraction of mammalian promoters containing a TATA box core element is dramatically depleted of upstream divergent transcription.
2012-10-25 | GSE23543 | GEO
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.
2015-01-05 | GSE63314 | GEO
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2021-02-01 | GSE130798 | GEO
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