Global regulation of transcription and the Hog1-mediated osmotic stress response in Saccharomyces cerevisiae

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At single genes many aspects of transcriptional regulation are understood in mechanistic detail. However, mechanisms that may influence gene regulation on a global level are yet unexplored. Within this research plan, I am proposing two projects to investigate mechanisms of transcriptional regulation on a global level in the yeast Saccharomyces cerevisiae.The first project aims at elucidating the mechanistic basis of transcriptional regulation during the osmotic stress response. After induction of osmotic stress the transcriptome undergoes a rapid reprogramming, dramatically shifting RNA polymerase (Pol) II occupancy from housekeeping to osmotic stress response genes, the mechanistic basis of which is unknown. The osmotic stress response is mediated by the high osmolarity glycerol (HOG) pathway and its mitogen activated protein kinase Hog1 that directly binds to Pol II. I will test the hypothesis that one or more osmostress polymerase complexes comprising Pol II, Hog1 and possibly other factors form the mechanistic basis of the osmostress-induced transcriptional reprogramming. I will establish an artificial Hog1 activation system 1) to define the sufficiency and requirement of Hog1, 2) to characterize the components, and 3) to quantify the fractional amounts of the osmostress polymerase complex without interference of high osmolarity or other signalling pathways. Further functional investigations will then either lead to a mechanistic explanation within the proposed hypothesis or support the alternative of classical gene-specific transcriptional regulation mediating the transcriptional stress response.The second project will explore the hypothesis of global control of transcription rates based on nuclear Pol II levels that may be regulated by a recently identified mechanism of Pol II nuclear import. In pilot experiments I will assess potentially altered subcellular localizations of the Pol II import adaptor Iwr1 in different environmental conditions and attempt to establish a tuneable Pol II nuclear import system to assess the interdependence of nuclear Pol II levels and transcription in a defined system.Combined, the two projects hold the promise to add the concept of global regulation to our understanding of cellular transcription.