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The Pol II Pre-Initiation Complex (PIC) Influences Mediator Binding but Not Promoter-Enhancer Looping [RNA-seq]

ABSTRACT: Knowledge of how Mediator and TFIID crosstalk contributes to promoter-enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genomewide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II pre-initiation complex (PIC) at the promoter, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by Promoter Capture Hi-C (PCHi-C). Degradation of MED4 had a minor effect of Mediator integrity but led to a consistent 2-fold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but no effect on the other PIC components. PCHi-C revealed no consistent effect of Med4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.

ORGANISM(S): Mus musculus

PROVIDER: GSE178849 | GEO | 2021/07/22

REPOSITORIES: GEO

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The Pol II Pre-Initiation Complex (PIC) Influences Mediator Binding but Not Promoter-Enhancer Looping [promoter_capture_Hi-C]

Project description:Knowledge of how Mediator and TFIID crosstalk contributes to promoter-enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genomewide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II pre-initiation complex (PIC) at the promoter, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by Promoter Capture Hi-C (PCHi-C). Degradation of MED4 had a minor effect of Mediator integrity but led to a consistent 2-fold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but no effect on the other PIC components. PCHi-C revealed no consistent effect of Med4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.

2021-07-22 | GSE178851 | GEO

The Pol II Pre-Initiation Complex (PIC) Influences Mediator Binding but Not Promoter-Enhancer Looping [nascent RNA-seq]

2021-07-22 | GSE178850 | GEO

The Pol II Pre-Initiation Complex (PIC) Influences Mediator Binding but Not Promoter-Enhancer Looping [ChIP-seq]

2021-07-22 | GSE178848 | GEO

Evidence that Mediator is essential for Pol II transcription but not a required component of the preinitiation complex in vivo

Project description:Mediator, a transcriptional co-activator complex, is recruited to enhancers by DNA-binding activators via its tail module, and it interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC) via its head module. Although Mediator has been described as a general transcription factor, it is unclear if it is essential for Pol II transcription and/or is a required component of the PIC in vivo. We comprehensively analyze this issue by measuring Pol II transcription upon rapid depletion of individual or multiple Mediator subunits from the nucleus. Depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in Pol II transcription. Transcriptional effects are stronger on SAGA-dependent genes as opposed to TFIID-dependent genes. Thus, Mediator modules that associate either with the enhancer or with the core promoter confer partial transcriptional activity. Furthermore, Pol II transcription can occur when Mediator is not detected at core promoters. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in Pol II transcription that is roughly comparable to the effect observed upon anchor-away-mediated depletion of Pol II or TBP. These results provide strong evidence that Mediator is essential for Pol II transcription in vivo, but that it is not a required component of the PIC.

2017-07-13 | GSE93190 | 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

The Pol II prenitiation complex (PIC) influences Mediator binding but not promoter-enhancer looping

Project description:The Pol II prenitiation complex (PIC) influences Mediator binding but not promoter-enhancer looping

| PRJNA741241 | ENA

TAF4 promotes pre-initiation complex formation and HNF4A occupancy of regulatory elements required to activation post-natal gene expression programme in hepatocytes (ChIP-seq)

Project description:The nuclear receptor HNF4A regulates embryonic and post-natal hepatocyte gene expression. Using hepatocyte-specific inactivation in mice, we show that the TAF4 subunit of TFIID acts as a cofactor for HNF4A in vivo and that HNF4A interacts directly with the TAF4-TAF12 heterodimer in vitro. In vivo, TAF4 is required to maintain HNF4A-directed embryonic gene expression at post-natal stages and for HNF4A-directed activation of post-natal gene expression. TAF4 promotes HNF4A occupancy of functional cis-regulatory elements located adjacent to the transcription start sites of post-natal expressed genes and for pre-initiation complex formation required for their expression. Promoter-proximal HNF4A-TFIID interactions are therefore required for pre-initiation complex formation and stable HNF4A occupancy of regulatory elements as two concomitant mutually dependent processes. Examination of PIC, H3k4me3, Ctcf and Hnf4a occupancy in wild-type and Taf4-/- livers by deep sequencing

2014-09-25 | E-GEOD-57807 | biostudies-arrayexpress

NET-prism enables RNA polymerase-dedicated transcriptional interrogation at nucleotide resolution

Project description:The advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. To better map transcription genome-wide at base pair resolution and with transcription/elongation factor dependency we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). NET-prism introduces an immunoprecipitation to capture RNA Pol II – associated proteins, which reveals the interaction of these proteins with active RNA Pol II. Application of NET-prism on different Pol II variants (Pol II S2ph, Pol II S5ph), elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals diverse Pol II signals, at a single nucleotide resolution, with regards to directionality and intensity over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity.

2019-07-02 | GSE107257 | GEO

An integrated SAGA and TFIID PIC assembly pathway selective for poised and induced promoters [ChIP-Exo]

Project description:Genome-wide, little is understood about how proteins organize at inducible promoters before and after in-duction, and to what extent inducible and constitutive architectures depend on cofactors. We report that se-quence-specific transcription factors and their tethered cofactors (e.g., SAGA, Mediator, TUP, NuA4, SWI/SNF, RPD3-L) are already bound to promoters prior to induction (“poised”), rather than recruited upon induction, whereas induction recruits the pre-initiation complex (PIC). Through depletion and/or deletion ex-periments we show that SAGA does not function at constitutive promoters, although a SAGA-independent Gcn5 does acetylate +1 nucleosomes there. At poised promoters, SAGA catalyzes +1 nucleosome acetylation but not PIC assembly. At induced promoters, SAGA catalyzes acetylation, deubiquitylation, and PIC assembly. Surprisingly, SAGA mediates induction by creating a PIC that allows TFIID to stably associate, rather than creating a TFIID-independent PIC, as is generally thought. These findings suggest that inducible systems, where present, evolved on top of constitutive systems.

2022-10-27 | GSE212654 | GEO

An integrated SAGA and TFIID PIC assembly pathway selective for poised and induced promoters [RNA-Seq]

2022-10-27 | GSE212653 | GEO

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