Elucidating the mechanisms of non-canonical CDK8 function in DNA damage signaling

项目摘要 转录介体是一种多亚基蛋白复合物，是RNA聚合酶的重要组成部分 II-介导的转录机制，调节基本上所有基因的转录。核心 Mediator包含一个头部、中部和尾部模块，以及一个辅助激酶模块（CKM）， 与核心调解人联系。CKM结合的核心介体通常具有拮抗作用， 然而，在某些情况下，它可以选择性地促进特定转录物的转录。在 除了对转录有相反的影响外，CKM还影响多种细胞过程，包括应激 反应、基因组组织和肿瘤发生。然而，尚不清楚CKM如何将 这些不同途径的信号，以及CKM的这些功能是否依赖于其激酶活性 及其与核心中介的交互。通过在酵母中进行全基因组无偏相互作用筛选， 新的途径，有助于调节DNA损伤反应，一个复杂的信号通路 在参与保护基因组完整性的过程中，我发现了一个有趣的与中介者的遗传相互作用。 通过对酵母中Mediator-CKM亚基的集中突变筛选，我发现所有四个CKM亚基都是 以及CKM的激酶活性对于DNA断裂后重新进入细胞周期是必不可少的。值得注意的是，CKM 突变体不通过同源重组损害UV损伤修复或DNA双链断裂修复， 表明CKM特异性地影响DNA损伤反应。此外，我还证明了CKM 有助于DNA损伤后转录的整体下调。为了解决串扰问题 CKM和DNA损伤信号之间的联系，并揭示CKM调节的基本方面，在目标1中，我将 询问CKM是否通过免疫染色直接与酵母和哺乳动物细胞中的DNA损伤因子相互作用， 细胞成像和免疫沉淀。我会问DNA损伤反应途径中的蛋白质是否 CKM的激酶活性的直接目标，通过采用磷酸蛋白质组学质谱分析。在目标2中， 我将研究DNA损伤反应如何改变CKM功能，重点是其亚基组成，激酶 活性和底物范围，以及通过使用体内和体外组合的全基因组定位 接近。最后，在目标3将集中在酵母中CKM和DNA损伤串扰的转录方面; 具体而言，DNA损伤和CKM依赖性下调组蛋白转录本。我会问这是否 下调导致整体核小体耗竭和染色质迁移率增加以促进同源性 使用内部校准的染色质免疫沉淀和DNA断裂的活细胞成像进行搜索 运动这些研究将揭示DNA之间以前未被表征的连接的分子机制。 损害和CKM，同时解决长期存在的问题CKM调制。

PROJECT SUMMARY The Mediator of transcription is a multi-subunit protein complex that is a critical component of RNA polymerase II-mediated transcriptional machinery which regulates the transcription of essentially all genes. The core Mediator contains a head, middle and tail module, and an accessory kinase module (CKM) which can reversibly associate with the core Mediator. CKM-bound core Mediator has generally an antagonizing effect on transcription; however, it can selectively to promote the transcription of specific transcripts in certain contexts. In addition to its opposing effects on transcription, CKM influences a variety of cellular processes including stress responses, genome organization and tumorigenesis. However, it is not well-understood how CKM incorporates signals from these divergent pathways, and whether these functions of CKM are dependent on its kinase activity and its interactions with core Mediator. Through a genome wide unbiased interaction screen in yeast to identify novel pathways that contribute to the regulation of DNA damage response, an intricate signaling pathway involved in protecting the integrity of the genome, I discovered an intriguing genetic interaction with the Mediator. Through a focused mutational screen of Mediator-CKM subunits in yeast, I found that all of the four CKM subunits as well as CKM’s kinase activity are essential for cell cycle re-entry following a DNA break. Notably, the CKM mutants did not impair UV damage repair or DNA double-strand break repair through homologous recombination, indicating that CKM specifically impinges on DNA damage response. Furthermore, I demonstrated that CKM contributes to the global downregulation of transcription following DNA damage. To address the crosstalk between CKM and DNA damage signaling and uncover fundamental aspects of CKM regulation, in Aim 1, I will ask if CKM directly interacts with DNA damage factors in yeast and mammalian cells by immunostaining, live cell imaging and immunoprecipitation. I will ask whether the proteins in DNA damage response pathway are direct targets of CKM’s kinase activity by employing phosphoproteomic mass spectrometry analyses. In Aim 2, I will examine how the DNA damage response alters CKM function, focusing on its subunit composition, kinase activity and substrate scope, and genome-wide localization by using a combination of in vivo and in vitro approaches. Lastly, in Aim 3 will focus on the transcriptional aspect of CKM and DNA damage crosstalk in yeast; specifically, the DNA damage- and CKM-dependent downregulation of histone transcripts. I will ask whether this downregulation leads to global nucleosome depletion and increased chromatin mobility to promote homology searching by using internally-calibrated chromatin immunoprecipitation and live cell imaging of DNA break movement. These studies will reveal the molecular mechanisms of previously uncharacterized link between DNA damage and CKM while addressing long-standing questions on CKM modulation.