Mediator orchestrates PIC assembly
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Project description:In this study, we generate genomic maps of Mediator, Pol II, TBP and TFIIH, by ChIP coupled to next generation sequencing technology (ChIP-seq), in wild type (WT) strains and med17-ts mutants from Saccharomyces cerevisiae. Some of the data, concerning WT strains are also deposited at ArrayExpress under accession number E-MTAB-1595 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-1595). There are 2 series of experiment: 1- WT (see E-MTAB-1595) and mutants med17-98, med17-444, and med17-670 (this submission) 2- WT and mutant med17-444 (this submission).
2015-09-03 | E-MTAB-2961 | biostudies-arrayexpress
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 | GSE212653 | GEO
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
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
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 | GSE178849 | GEO
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 | GSE178848 | GEO
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 | GSE178850 | GEO
Project description:Pol II(G) is a novel form of RNA polymerase II that contains a tightly associated Gdown1 polypeptide. Whereas Pol II suffices for robust activator-dependent transcription in the absence of Mediator, Pol II(G) is highly dependent upon Mediator in a biochemically defined in vitro system. However, the mechanism(s) whereby Gdown1 alters the coactivator-dependence of Pol II is unknown. Here we show that Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II in vitro, resulting in inhibition of a critical function of TFIIF in facilitating PIC assembly. The apparent inability of Pol II(G) to associate with TFIIF suggests that Gdown1 must be removed from Pol II for transcription initiation. However, Mediator can actually help Pol II(G) bind to the promoter prior to subsequent Mediator functions. Complementary cell-based analyses reveal that Pol II(G) is recruited to promoter regions of subsets of actively transcribed genes, where Pol II(G) appears to modulate transcription. Our findings indicate that differential regulation of subsets of genes by Pol II and Pol II(G) occurs through the regulation of TFIIF and consequent alterations in Mediator requirements. Genome-wide localization of PolII and Gdown1 in human IMR90 fibroblast cells using ChIP-seq
2012-01-13 | E-GEOD-33128 | biostudies-arrayexpress
Project description:Transcriptional profiling of the melanoma SK-mel-103 comparing control untreated cells with cells treated with 1ug/ml of polyinosine-polycytidylic acid (pIC) or 1ug/ml of pIC with polyethyleneimine (PEI) for 4 or 10 hours. Keywords: Treatment Four-condition experiment, SK-Mel-103 untreated vs. SK-Mel-103 treated with pIC/pIC+PEI at 4 and 10 hours. One replicate per array.
2010-04-08 | E-GEOD-14445 | biostudies-arrayexpress
Project description:CBP/p300 is one group of histone acetyltransferases, which catalyze the acetylation of histone lysine in order to loosen the chromatin structure and promote transcription. The multiple-subunits complex MEDIATOR is important for the transcription initiation through participating in the assembly of transcriptional pre-initiation complex (PIC). Our study identified that MEDIATOR interacts with HAC1 and HAC5, and participate in the regulation of transcription coordinately, thereby regulating the development of Arabidopsis. Besides, MEDIATOR, HAC1, and HAC5 may be organized together by the LLPS to regulate transcription.
2021-01-01 | GSE162458 | GEO