Role of the pre-initiation complex in Mediator recruitment and dynamics

Abstract

The Mediator complex stimulates the cooperative assembly of a pre-initiation complex (PIC) and recruitment of RNA Polymerase II (Pol II) for gene activation. The core Mediator complex is organized into head, middle, and tail modules, and in budding yeast (Saccharomyces cerevisiae), Mediator recruitment has generally been ascribed to sequence-specific activators engaging the tail module triad of Med2-Med3-Med15 at upstream activating sequences (UASs). We show that yeast lacking Med2-Med3-Med15 are viable and that Mediator and PolII are recruited to promoters genome-wide in these cells, albeit at reduced levels. To test whether Mediator might alternatively be recruited via interactions with the PIC, we examined Mediator association genome-wide after depleting PIC components. We found that depletion of Taf1, Rpb3, and TBP profoundly affected Mediator association at active gene promoters, with TBP being critical for transit of Mediator from UAS to promoter, while Pol II and Taf1 stabilize Mediator association at proximal promoters.

Kin28 depletion increases association of TFIID subunits Taf1 and Taf4 with promoters in Saccharomyces cerevisiae

“…We found that Taf1 occupancy was essentially unchanged upon simultaneous depletion of Kin28 and TBP (Fig. 4A); marked effects on Mediator and Pol II association provide evidence for efficient depletion of both Kin28 and TBP [21], as does the loss of the artifactual, transcription-dependent signal at the 3' ends of coding sequences ( Fig. 4A) [16,26].…”

“…To test whether the increased association of Taf1 seen upon Kin28 depletion requires stable TBP occupancy, we performed ChIP-seq against Taf1 in kin28-tbp-AA yeast, in which rapamycin addition causes depletion of both Kin28 and TBP [21]. We found that Taf1 occupancy was essentially unchanged upon simultaneous depletion of Kin28 and TBP (Fig.…”

“…We recently reported investigations of the interdepencies among the PIC components TBP, Taf1, and Pol II for occupancy of promoter regions, using ChIP-seq before and after conditional depletion of these same components [21]. Examination of occupancy of PIC components at a subset of genes, dubbed "UAS genes", that exhibit Mediator peaks at upstream activating sequences (UASs) in wild type yeast, revealed that depletion of TBP resulted in nearly complete loss of Pol II occupancy, but little change in occupancy by Taf1.…”

“…Stabilization of Mediator at gene promoters was accomplished by inactivation or depletion of Kin28, a kinase that is a subunit of TFIIH that phosphorylates the carboxy-terminal domain of the largest subunit of Pol II, thereby facilitating promoter escape [15,18]. We recently showed that this stabilization depends on Pol II, as it is suppressed if Pol II is depleted using the anchor away technique [21]. The transient occupancy of promoter regions by Mediator during the transcription cycle raises the question as to whether other components of the transcription machinery are stably bound or, like Mediator, bind and rapidly dissociate upon Pol II promoter escape.…”

Independent Recruitment of the Mediator Tail Module to theHOPromoter Suggests Mediator Core Limits Coactivator Recruitment inSaccharomyces cerevisiae

“…The Tail module attaches to the core Mediator through the Med14 (Rgr1) scaffold subunit (Tsai et al 2014) and interacts with sequence-specific transcription factors that recruit the Mediator complex to the UAS or enhancers of genes (Bhoite et al 2001;Kagey et al 2010;Jeronimo and Robert 2014). The tail module is required for efficient recruitment of Mediator and Pol II to promoters (Knoll et al 2018).…”