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

阐明 DNA 损伤信号传导中非经典 CDK8 功能的机制

• 批准号: 10390046
• 负责人: Gonen Memisoglu
• 金额: $ 6.98万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10390046
• 关键词: Acute; Address; Affect; Alzheimer' s Disease; Architecture; Ataxia; Binding; Biological; Biological Assay; Cell Cycle; Cell Cycle Progression; Cell physiology; Cells; Chromatin; Complex; DNA; DNA Binding; DNA Damage; DNA Double Strand Break; Data; Disease; Double Strand Break Repair; Down-Regulation; Elements; Genes; Genetic; Genetic Epistasis; Genetic Transcription; Genome; Genomics; Global Change; Goals; Head; Histones; Human; Immunofluorescence Immunologic; Immunoprecipitation; Impairment; In Vitro; Lead; Link; Malignant Neoplasms; Mammalian Cell; Mammals; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Modification; Molecular; Movement; Mutate; Mutation; Neurodegenerative Disorders; Nucleosomes; Parkinson Disease; Pathway interactions; Phosphotransferases; Protein Complex Subunit; Proteins; RNA Polymerase II; Regulation; Repression; Research Proposals; Role; Signal Pathway; Signal Transduction; Site; Stable Isotope Labeling; Stress; Substrate Specificity; Tail; Testing; Transcript; Transcriptional Activation; Yeasts; base; biological adaptation to stress; chromatin immunoprecipitation; cohesin; differential expression; genome integrity; genome-wide; histone modification; homologous recombination; in vivo; insight; live cell imaging; mutant; mutation screening; novel; phosphoproteomics; promoter; protein complex; repaired; response; transcription factor; transcriptome sequencing; tumorigenesis; ultraviolet damage

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.

项目总结 转录介体是一种多亚单位蛋白质复合体，是RNA聚合酶的重要组成部分 II介导的转录机制，基本上调节所有基因的转录。其核心是 介体包括头、中、尾三个模块，以及一个可以可逆地 与核心调解人关联。CKM结合的核心介体一般具有对抗作用 转录；然而，它可以选择性地促进特定转录本在某些上下文中的转录。在……里面 除了对转录的相反作用外，CKM还影响包括应激在内的各种细胞过程 反应、基因组组织和肿瘤发生。然而，对于长江基建是如何整合的，目前还不是很清楚。 来自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 以及它与Core Mediator的交互。通过全基因组无偏互作筛选在酵母中进行鉴定 参与调控DNA损伤反应的新途径--一种复杂的信号途径 在参与保护基因组完整性的过程中，我发现了与介体之间有趣的基因相互作用。 通过对酵母中Mediator-CKM亚基的集中突变筛选，我发现这四个CKM亚基都是 以及CKM的激酶活性对于DNA断裂后细胞周期的重新进入是必不可少的。值得注意的是，CKM 突变体没有通过同源重组损害紫外线损伤修复或DNA双链断裂修复， 提示CKM特异性地影响DNA损伤反应。此外，我还展示了CKM 有助于DNA损伤后转录的全球下调。要解决串扰问题 在CKM和DNA损伤信号之间，并揭示CKM调控的基本方面，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不是 用磷蛋白组质谱分析直接靶点CKM的激酶活性。在目标2中， 我将研究DNA损伤反应是如何改变CKM功能的，重点是它的亚单位组成，即激酶 活性和底物范围，以及利用体内和体外相结合的全基因组定位 接近了。最后，在目标3中，将集中在酵母中CKM的转录方面和DNA损伤串扰； 具体地说，依赖于DNA损伤和CKM的组蛋白转录下调。我会问这是不是 下调导致全球核小体耗尽和染色质流动性增加以促进同源性 用内标染色质免疫沉淀和DNA断裂的活细胞成像进行搜索 有动静。这些研究将揭示以前未被描述的DNA之间联系的分子机制 损害和CKM，同时解决关于CKM调制的长期问题。