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Cell-type-specific RNA Pol II activity maps in intact-tissues: gateway to mammalian gene regulatory mechanisms in vivo [CUT&RUN]

ABSTRACT: Accessing ongoing RNA Pol II activity in specific cell types within intact-tissue is critical to reveal regulatory mechanisms of development. We developed PReCIS-seq, a method combining Cre-inducible GFP-tagging of endogenous Pol II with transcriptional run-on and GFP-immunoprecipitation, to map transcriptionally-engaged Pol II genome-wide in targeted cell types of mouse tissues. Applied to keratinocytes within intact-skin, PReCIS-seq demonstrates that transcriptionally activated functions of biological transitions generally employ both Pol II promoter-recruitment and promoter-proximal pause-release mechanisms. We uncover a global Pol II regulatory polarization that modulates cellular safeguarding versus lineage identity genes across embryonic development to adult homeostasis. This polarization is associated with distinct proximal-promoter structures distinguishing high-paused genes with restricted Pol II pause-release from low-paused genes undergoing rapid Pol II firing into productive elongation. PReCIS-seq also identifies active enhancers based on divergent transcription. This approach enables high-resolution, cell-type-specific analysis of Pol II dynamics in intact-tissues across mammalian development, homeostasis, and disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE306498 | GEO | 2025/08/27

REPOSITORIES: GEO

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Cell-type-specific RNA Pol II activity maps in intact-tissues: gateway to mammalian gene regulatory mechanisms in vivo [PReCIS_seq]

Project description:Accessing ongoing RNA Pol II activity in specific cell types within intact-tissue is critical to reveal regulatory mechanisms of development. We developed PReCIS-seq, a method combining Cre-inducible GFP-tagging of endogenous Pol II with transcriptional run-on and GFP-immunoprecipitation, to map transcriptionally-engaged Pol II genome-wide in targeted cell types of mouse tissues. Applied to keratinocytes within intact-skin, PReCIS-seq demonstrates that transcriptionally activated functions of biological transitions generally employ both Pol II promoter-recruitment and promoter-proximal pause-release mechanisms. We uncover a global Pol II regulatory polarization that modulates cellular safeguarding versus lineage identity genes across embryonic development to adult homeostasis. This polarization is associated with distinct proximal-promoter structures distinguishing high-paused genes with restricted Pol II pause-release from low-paused genes undergoing rapid Pol II firing into productive elongation. PReCIS-seq also identifies active enhancers based on divergent transcription. This approach enables high-resolution, cell-type-specific analysis of Pol II dynamics in intact-tissues across mammalian development, homeostasis, and disease.

2024-12-04 | GSE278911 | GEO

Cell-type-specific RNA Pol II activity maps in intact-tissues: gateway to mammalian gene regulatory mechanisms in vivo [RNA-seq]

2024-12-04 | GSE278913 | GEO

Cell-type-specific RNA Pol II activity maps in intact-tissues: gateway to mammalian gene regulatory mechanisms in vivo [ChIP-seq]

2024-12-04 | GSE278912 | GEO

Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-Seq

Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-seq data for Pol II and additional factors controlling pause release in mouse ES cells.

2010-04-02 | E-GEOD-20530 | biostudies-arrayexpress

Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-chip

Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-chip data for Pol II and additional factors controlling pause release in mouse ES cells.

2010-04-02 | E-GEOD-20529 | biostudies-arrayexpress

CBP regulates promoter-proximal RNA polymerase II

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 forÂ 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

NELF regulates a promoter-proximal step distinct from RNA Pol II pause-release

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

Stable pausing by RNA polymerase II provides an opportunity to target and integrate regulatory signals

Project description:Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 minutes at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions. This submission includes 13 raw data files. Four samples (chromatin, soluble, mock-treated, and Rrp40-depleted) are represented by two biological replicate raw data files, while five samples (cells, DMSO, FP, mock-treated + FP, and Rrp-40depleted + FP) are represented by single raw data files.

2013-10-31 | E-GEOD-49078 | biostudies-arrayexpress

Knockdown of Brd4 or SEC affects the HMBA-induced global Pol II pausing release

Project description:To test whether Brd4 and SEC co-regulate the release of promoter-proximally paused Pol II, we performed Pol II ChIP-Seq to analyze the effect of depletion of Brd4 or SEC on HMBA-induced pause release in HCT116 cells.

2019-01-12 | GSE76784 | GEO

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

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