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

在单个基因中，转录调控的许多方面在机械细节上被理解。然而，可能在全球水平上影响基因调控的机制尚未被探索。在这个研究计划中，我提出了两个项目来研究酵母在全球水平上的转录调控机制。第一个项目旨在阐明渗透胁迫反应中转录调控的机制基础。在渗透胁迫诱导后，转录组经历了快速重编程，显著地将RNA聚合酶（Pol） II占用从管家基因转移到渗透胁迫应答基因，其机制基础尚不清楚。渗透胁迫反应是由高渗透压甘油（HOG）途径及其有丝分裂原激活的蛋白激酶Hog1介导的，该蛋白激酶直接与Pol II结合。我将测试一个或多个渗透胁迫聚合酶复合物的假设，包括Pol II， Hog1和可能的其他因素形成渗透胁迫诱导的转录重编程的机制基础。我将建立一个人工Hog1激活系统，1)定义Hog1的充分性和需求，2)表征其成分，3)在不干扰高渗透压或其他信号通路的情况下量化渗透胁迫聚合酶复合物的分数。然后，进一步的功能研究将在提出的假设中找到一个机制解释，或者支持经典基因特异性转录调控介导转录应激反应的替代方案。第二个项目将探索基于核Pol II水平的转录率全球控制的假设，这可能受到最近确定的Pol II核进口机制的调节。在试点实验中，我将评估不同环境条件下可能改变的Pol II输入适配器Iwr1的亚细胞定位，并尝试建立一个可调节的Pol II核输入系统，以评估核Pol II水平和转录的相互依赖性。结合起来，这两个项目有望将全球调控的概念添加到我们对细胞转录的理解中。

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.