Evidence for Multiple Mediator Complexes in Yeast Independently Recruited by Activated Heat Shock Factor

Abstract

Mediator is an evolutionarily conserved coactivator complex essential for RNA polymerase II transcription. Although it has been generally assumed that in Saccharomyces cerevisiae, Mediator is a stable trimodular complex, its structural state in vivo remains unclear. Using the "anchor away" (AA) technique to conditionally deplete select subunits within Mediator and its reversibly associated Cdk8 kinase module (CKM), we provide evidence that Mediator's tail module is highly dynamic and that a subcomplex consisting of Med2, Med3, and Med15 can be independently recruited to the regulatory regions of heat shock factor 1 (Hsf1)-activated genes. Fluorescence microscopy of a scaffold subunit (Med14)-anchored strain confirmed parallel cytoplasmic sequestration of core subunits located outside the tail triad. In addition, and contrary to current models, we provide evidence that Hsf1 can recruit the CKM independently of core Mediator and that core Mediator has a role in regulating postinitiation events. Collectively, our results suggest that yeast Mediator is not monolithic but potentially has a dynamic complexity heretofore unappreciated. Multiple species, including CKM-Mediator, the 21-subunit core complex, the Med2-Med3-Med15 tail triad, and the foursubunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains.

Mediator Undergoes a Compositional Change during Transcriptional Activation

“…Our results are in apparent conflict with a very recent paper claiming that heat shock factor can recruit the kinase module independently of the rest of Mediator (Anandhakumar et al, 2016). First, on a genomic scale, association of the kinase module with enhancers is dependent on Kin28, which associates solely with the core promoter and hence should not directly affect independent association with the enhancer.…”

Heat Shock Factor 1 Drives Intergenic Association of Its Target Gene Loci Upon Heat Shock

“…Hence, Hsf1 may recruit a distinct set and/or quantity of coactivators and Pol IIassociated machinery. Mediator is a particularly attractive candidate, as it is abundant at activated HSP genes while nearly undetectable at comparably expressed CTT1 and RPL genes (Anandhakumar et al, 2016;Fan et al, 2006;Kim and Gross, 2013). The relative scarcity of Mediator at CTT1 and RPL may reflect its transient association with these genes despite their strong activation (Jeronimo and Robert, 2014;Wong et al, 2014).…”

“…One possibility is that Hsf1 recruits a distinct set and/or quantity of coactivators, in particular Mediator, which is prominently recruited to HSP genes (Anandhakumar et al, 2016;Fan et al, 2006;Kim and Gross, 2013). Notably, Mediator is nearly undetectable at comparably activated CTT1 and RPL genes (Fan et al, 2006); such relative scarcity may reflect its transient association with those loci (Jeronimo and Robert, 2014;Wong et al, 2014).…”

Med15B Regulates Acid Stress Response and Tolerance in Candida glabrata by Altering Membrane Lipid Composition

“…Med17 links transcription and DNA repair by facilitating the recruitment of the Rad2/XPG endonuclease to transcribed genes during exogenous genotoxic stress (18). The tail module subunits Med2, Med3, and Med15 can form a subcomplex and are involved in a heat shock response through recruitment by activated heat shock transcription factor 1 (Hsf1) to its target genes (19). By changing the Mediator subunit composition, the tail subcomplex affects the response to oxidative stress (20).…”