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

基本信息

批准号：
10596072
负责人：
Gonen Memisoglu
金额：
$ 7.38万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
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/10596072
关键词：
Acute Address Affect Alzheimer&apos s Disease Architecture Ataxia Binding Biological Biological Assay Calibration Cell Cycle Cell Cycle Progression Cell Line Cell physiology Chromatin Co-Immunoprecipitations Complex DNA DNA Binding DNA Damage DNA Double Strand Break DNA Repair Data Disease Disparate Double Strand Break Repair Down-Regulation Elements Genes Genetic Genetic Epistasis Genetic Transcription Genome 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 Modification Molecular Movement Mutate Mutation Neurodegenerative Disorders Nucleosomes Parkinson Disease Pathway interactions Phosphorylation Phosphotransferases Protein Complex Subunit Proteins RNA Polymerase II Regulation Repression Research Proposals Role Signal Pathway Signal Transduction Site Stable Isotope Labeling Substrate Specificity Tail Testing Transcript Transcriptional Activation Transcriptional Regulation Yeasts biological adaptation to stress chromatin immunoprecipitation cohesin differential expression gene repression genome integrity genome-wide genome-wide analysis histone modification homologous recombination in vivo insight live cell imaging mutant mutation screening novel phosphoproteomics promoter protein complex repaired response response biomarker transcription factor transcriptome sequencing tumorigenesis ultraviolet damage

项目摘要

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.

项目概要转录介体是一种多亚基蛋白质复合物，是 RNA 聚合酶的关键组成部分 II 介导的转录机制，调节基本上所有基因的转录。 Mediator包含一个头、中、尾模块，以及一个可逆的辅助激酶模块（CKM）与核心介体结合 CKM 结合的核心介体通常具有拮抗作用。然而，它可以在某些情况下选择性地促进特定转录本的转录。除了对转录产生相反作用外，CKM 还影响多种细胞过程，包括应激然而，CKM 如何整合尚不清楚。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及其与核心介体的相互作用通过酵母中的全基因组无偏相互作用筛选来识别。有助于调节 DNA 损伤反应的新途径，这是一种复杂的信号传导途径在参与保护基因组完整性的过程中，我发现了与中介者之间有趣的遗传相互作用。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 亚基以及 CKM 的激酶活性对于 DNA 断裂后细胞周期的重新进入至关重要。突变体不会通过同源重组损害紫外线损伤修复或DNA双链断裂修复，表明 CKM 专门影响 DNA 损伤反应。此外，我证明了 CKM。有助于 DNA 损伤后转录的整体下调，以解决串扰问题。在目标 1 中，我将研究 CKM 和 DNA 损伤信号传导之间的关系并揭示 CKM 调节的基本方面通过免疫染色，活体询问 CKM 是否直接与酵母和哺乳动物细胞中的 DNA 损伤因子相互作用我会问DNA损伤反应途径中的蛋白质是否是细胞成像和免疫沉淀。通过采用磷酸化蛋白质组质谱分析来确定 CKM 激酶活性的直接靶标。我将研究 DNA 损伤反应如何改变 CKM 功能，重点关注其亚基组成、激酶活性和底物范围，以及通过体内和体外组合进行全基因组定位最后，目标 3 将重点关注酵母中 CKM 和 DNA 损伤串扰的转录方面；具体来说，组蛋白转录物的 DNA 损伤和 CKM 依赖性下调，我会问这是否如此。下调导致整体核小体耗尽并增加染色质流动性以促进同源性使用内部校准的染色质免疫沉淀和 DNA 断裂活细胞成像进行搜索这些研究将揭示DNA之间先前未表征的联系的分子机制。损坏和 CKM，同时解决有关 CKM 调制的长期问题。