EnChAMP-seq genomic localization and footprinting of RNA Pol II and NELF-A
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE311646 | GEO | 2026/01/06
REPOSITORIES: GEO
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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).
2012-10-25 | E-GEOD-23542 | 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 | GSE23543 | 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 | GSE23542 | 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
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:Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is not fully understood. Here we identify a novel activity of the co-regulator and histone acetyltransferase p300/CBP in positioning promoter-proximal paused Pol II. We find that CBP inhibition impedes transcription through the +1 nucleosome, causing âdribblingâ of Pol II from the canonical pause site genome-wide. We further discovered that promoters strongly occupied by Drosophila CBP and GAGA-factor have high levels of paused Pol II, a unique chromatin signature and strong expression regardless of cell type. Interestingly, CBP activity is rate-limiting for Pol II recruitment to these highly-paused promoters but for transit into elongation at other genes. Thus, we uncover a key role for CBP during transcription in directly controlling different rate-limiting steps depending on promoter features. Examination of transcriptional regulation with and without CBP inhibition for 10 minutes in Drosophila S2 cells. Two biological replicates for each condition.
2016-08-01 | E-GEOD-81649 | biostudies-arrayexpress
Project description:Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is not fully understood. Here we identify a novel activity of the co-regulator and histone acetyltransferase p300/CBP in positioning promoter-proximal paused Pol II. We find that CBP inhibition impedes transcription through the +1 nucleosome, causing “dribbling” of Pol II from the canonical pause site genome-wide. We further discovered that promoters strongly occupied by Drosophila CBP and GAGA-factor have high levels of paused Pol II, a unique chromatin signature and strong expression regardless of cell type. Interestingly, CBP activity is rate-limiting for Pol II recruitment to these highly-paused promoters but for transit into elongation at other genes. Thus, we uncover a key role for CBP during transcription in directly controlling different rate-limiting steps depending on promoter features.
2016-08-01 | GSE81649 | GEO
Project description:Transcription is a multi-stage process that coordinates several steps within the transcription cycle including chromatin reorganization, RNA polymerase II recruitment, initiation, promoter clearance and elongation. Recent advances have identified the super elongation complex (SEC), containing the eleven nineteen lysine rich leukemia protein (ELL), as a key regulator of transcriptional elongation. We show here that ELL plays a diverse and kinetically distinct role prior to its assembly into the SEC by stabilizing Pol II recruitment/initiation and entry into the pause site. Loss of ELL destabilizes the PIC complexes and results in disruption of early elongation and promoter proximal chromatin structure prior to recruitment of AFF4 and other SEC components. These changes result in significantly reduced transcriptional activation of rapidly induced genes. Thus, ELL plays an early and essential role during rapid high amplitude gene expression that is required for both Pol II pause site entry and release.
2011-12-09 | GSE34104 | GEO
Project description:Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome’s primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide, nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally-engaged RNA polymerases in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ~40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II’s entry into elongation. Furthermore, “bivalent” ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb Group Complexes PRC 1 and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5’ proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.
2011-04-01 | GSE27037 | GEO
Project description:Transcription is a multi-stage process that coordinates several steps within the transcription cycle including chromatin reorganization, RNA polymerase II recruitment, initiation, promoter clearance and elongation. Recent advances have identified the super elongation complex (SEC), containing the eleven nineteen lysine rich leukemia protein (ELL), as a key regulator of transcriptional elongation. We show here that ELL plays a diverse and kinetically distinct role prior to its assembly into the SEC by stabilizing Pol II recruitment/initiation and entry into the pause site. Loss of ELL destabilizes the PIC complexes and results in disruption of early elongation and promoter proximal chromatin structure prior to recruitment of AFF4 and other SEC components. These changes result in significantly reduced transcriptional activation of rapidly induced genes. Thus, ELL plays an early and essential role during rapid high amplitude gene expression that is required for both Pol II pause site entry and release. Total RNA for unstimulated and for simulated with EGF (50 ng/mL) for 1hr from wild type and stably induced ELL KD in HEK293 cells were isolated and purified and submitted to LMT-Affymetrix microarray facility to obtain the raw data. Affymetrix Human Exon ST1.0 platform was used for the microarray analysis. Raw data in CEL files were preprocessed using Affymetrix Expression Console software.
2011-12-09 | E-GEOD-34104 | biostudies-arrayexpress