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Architectural Mediator subunits are differentially essential for global transcription in yeast (ChIP-seq)

ABSTRACT: Mediator is a modular complex required for RNA polymerase II transcription in vivo. We show that Mediator destabilization via depletion of the main complex scaffold Med14 or the head module scaffold Med17 is lethal and results in global transcriptional downregulation, though Med17 destruction has a markedly greater negative effect. Depletion of Med14 or Med17 impairs pre-initiation complex (PIC) formation and/or stability to similar degrees, suggesting that the differential transcriptional effects of head module destabilization and overall dissociation of the complex are not due to different extents of defective PIC formation. Co-depletion of Med14 and Med17 reduced transcription and TFIIB promoter occupancy to similar degrees as Med17 loss alone, suggesting that the independent head module can weakly stimulate transcription in vivo, though not at a level that maintains viability. Collectively, our data suggest that the head is the major functional module of Mediator with respect to PIC function in vivo.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE145494 | GEO | 2020/03/05

REPOSITORIES: GEO

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Connection of core and and tail Mediator modules restrains transcription from TFIID-dependent promoters [ChEC-seq]

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The Cdk8 kinase module regulates Mediator-RNA polymerase II interaction

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Architectural Mediator subunits are differentially essential for global transcription in yeast (RNA-seq/4tU-seq ; Med14-Med17-doubleAID)

Project description:This project aims to understand the function of Mediator and the interplay in transcription in S. cerevisiae. Thus we analyzed total and newly synthesized RNA levels in a conditional Med14 and Med17 depletion strain. For this purpose we used an auxin inducible degron system system to specifically induce the degradation Med14 and Med17 by addition of auxin into the culture medium. We measured transcript levels after 30 min auxin or DMSO (control) treatment induction of degradation of Med14 and Med17 by the addition of auxin (30 min) or with the carrier DMSO (control) followed by a 6 min labelling of newly synthesized RNA with 4-thiouracil (4tU). In parallel wiltype S.pombe cells were labelled with 4tU for 6min during midlog growth phase. Prior to RNA extraction (Ribopure Yeast Kit, Ambion) our labelled S.cerevisiae cells of interest were mixed with labelled S.pombe cells as a spike to allow normalization against an exogenous reference (ratio 3 S .cerevisiae for 1 S.pombe cell. From the total RNA we purified the 4tU labelled fraction of RNAs containing 4tU. Both the total RNA and the purified newly synthesized RNAs will then be subject to RNA-sequencing after ribosomal RNA depletion. In summary, we will provide 8 samples for this project containing RNA from both S.cerevisiae and S.pombe. The sample read counts were normalized with size factors computed by the median-of-ratios method proposed by Anders and Huber (Anders et al. 2010) on S. pombe genes to make these counts comparable between samples.

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