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 four-subunit CKM, can be independently recruited by activated Hsf1 to its target genes in AA strains.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00005-16.

ACKNOWLEDGMENTS

We thank Kelly Tatchell for facilities, reagents, assistance with fluorescence microscopy, and helpful discussions; Denes Hnisz and Sunyoung Kim for critical reading of the manuscript; Richard Young, Steve Hahn, Mark Ptashne, and David Stillman for antibodies; Frank Holstege for yeast strains; and David Pincus and Kim Nasmyth for plasmids.

Y.W.M. and D.S.G. conceived the study. Y.W.M., A.S.K., J.A., S.C., and D.S.G. designed the experiments. J.A., Y.W.M., S.C., and A.S.K. performed the experiments. Y.W.M., J.A., A.S.K., S.C., and D.S.G. interpreted the data. D.S.G. wrote the paper.

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Funding

The funders of this study had no role in study design, data collection and interpretation, or the decision to submit the work for publication.