Mediator independently orchestrates multiple steps of preinitiation complex assemblyin vivo

Abstract

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

Functional interplay between Mediator and TFIIB in preinitiation complex assembly in relation to promoter architecture

“…These ChIP results indicated that a mutant in the Mediator middle module has a pronounced effect on the recruitment and/or stability of specific PIC components in vivo. Figure S4A shows an example of ChIP-seq density distributions for all proteins on a selected class II gene in wild-type strains after subtraction of the normalized control of an untagged strain as described previously (Eyboulet et al 2013(Eyboulet et al , 2015. As expected, a metagene analysis around the transcription start sites (TSSs) in wild-type strains showed that Pol II is distributed inside transcribed regions and that Mediator (Med15 and Med17 subunits) is located upstream of TSSs on regulatory regions (Supplemental Fig.…”

“…1E) and was analyzed by mass spectrometry as described previously ( Fig. 1F; Eyboulet et al 2015). All 20 Mediator subunits detectable in a wildtype strain by this approach were present in the Mediator complex purified from the med10 mutant grown at 30°C (Fig.…”

“…To detect the potential global effects of Mediator mutations on PIC component occupancy, we performed the normalization step relative to quantitative PCR (qPCR) data on a set of selected regions as described previously (Eyboulet et al 2015). Regression analysis of PIC component binding in med10-196 versus wild type was systematically performed.…”

“…Gene-specific effects of med10-196 mutation on PIC assembly in vivo Our previous analysis of the impact of conditional mutations in the Med17 Mediator subunit on genome-wide PIC assembly showed a global nature of the effects but also suggested that some effects could still be gene-specific (Eyboulet et al 2015). The high correlation coefficients obtained when comparing the genome-wide occupancy of the different PIC components between med10-196 and the wild type highlight the global effects of the mutation Figure 3.…”

“…It also stimulates the phosphorylation of the Pol II C-terminal domain (CTD) by TFIIH (Kim et al 1994;Jiang et al 1998). Mediator cooperates with GTFs to stabilize the PIC, in particular through interactions with TFIID, TFIIH, and Pol II (Johnson et al 2002;Esnault et al 2008;Soutourina et al 2011;Eyboulet et al 2015). A functional interplay between Mediator and TFIIB was also suggested based on in vitro experiments with human Mediator (Baek et al 2006).…”