Novel Rad18 functions in Histone Modification and Regulation of Gene Expression

描述（由申请人提供）：我们对DNA损伤如何感知和修复以及DNA修复如何与其他动态染色质功能（例如转录，DNA复制）协调以维持基因组稳定性的理解存在重大空白。DNA修复可以防止正常细胞中环境诱导的癌症，并可以赋予癌细胞对化疗和放疗的抵抗力。因此，了解基因组维持机制对我们理解、预防和成功治疗癌症至关重要。提出探索性项目的直接目标是确定DNA修复蛋白Rad18在染色质修饰和基因表达调控中的新作用。我们的长期目标是确定rad18介导的基因表达调控如何促进基因组维持和肿瘤抑制（在正常细胞中）以及化疗/放射抵抗（在癌细胞中）。此前，Rad18仅参与调节DNA修复蛋白。基于强有力的初步数据，核心假设是Rad18直接使K119处的组蛋白H2A单泛素化是一种有助于基因组维持的转录抑制新机制。其基本原理是，确定rad18介导的基因组维持机制将为更好地了解环境致癌作用和改善癌症治疗提供新的机会。本探索性R21提案的具体目的是：(1)确定Rad18在基因表达表观遗传控制中的新功能。(2)在体内鉴定rad18响应组蛋白标记和转录事件。对于SA1，我们将进行全基因组分析，以确定在暴露于环境和治疗性基因毒性药物（分别为多环芳烃或PAH和电离辐射）后，与原代人细胞中基因表达改变相关的rad18依赖性组蛋白标记。SA2将确定Rad18在多大程度上影响骨髓（BM）祖细胞的基因表达和组蛋白修饰，骨髓祖细胞是环境多环烃和放疗遗传毒性的已知靶点。为了确定h2a泛素化对Rad18介导的基因组维持的贡献，我们将使用“功能分离”Rad18突变体特异性地缺乏h2a导向的E3连接酶活性。所提出的想法和研究是创新的，因为它们寻求提供新的范式，而不存在：Rad18的染色质修饰是新颖的，表明Rad18可能具有涉及转录抑制的新的基因组维持功能。提出的小鼠研究具有创新性，因为以前没有在BM祖细胞（或其他体细胞）体内研究过Rad18在基因组维持中的生理功能。这项工作意义重大，因为

DESCRIPTION (provided by applicant): There are major gaps in our understanding of how DNA damage is sensed and repaired and how DNA repair is coordinated with other dynamic chromatin functions (e.g. transcription, DNA replication) to maintain genome stability. DNA repair prevents environmentally-induced cancer in normal cells and can confer resistance to chemotherapy and radiotherapy in cancer cells. Therefore, knowledge of genome maintenance mechanisms is crucial for us to understand, prevent, and successfully treat cancer. The immediate objective of the proposed exploratory project is to define a new role for the DNA repair protein Rad18 in chromatin modification and regulation of gene expression. Our long-term goal is to determine how Rad18-mediated regulation of gene expression contributes to genome maintenance and tumor suppression (in normal cells) and chemo/radio-resistance (in cancer cells). Previously, Rad18 has been implicated only in regulating DNA repair proteins. Based on strong preliminary data, the central hypothesis is that direct mono-ubiquitination of Histone H2A at K119 by Rad18 is a novel mechanism for transcriptional repression that contributes to genome maintenance. The rationale is that defining mechanisms of Rad18-mediated genome maintenance will provide a better understanding of environmental carcinogenesis and new opportunities for improving cancer therapy. The Specific Aims (SAs) of this exploratory R21 proposal are: (1) Define novel Rad18 functions in epigenetic control of gene expression. (2) Identify Rad18-responsive histone marks and transcriptional events in vivo. For SA1 we will perform genome-wide analyses to identify Rad18-dependent histone marks associated with altered gene expression following exposure to environmental and therapeutic genotoxic agents (Polycyclic Aryl Hydrocarbons, or PAH, and Ionizing Radiation respectively) in primary human cells. SA2 will determine the extent to which Rad18 influences gene expression and histone modifications in bone marrow (BM) progenitor cells, known targets of genotoxicity from environmental PAH and radiotherapy. To determine the contribution of H2A-ubiquitination to Rad18-mediated genome maintenance we will use 'separation-of-function' Rad18 mutant specifically lacking H2A-directed E3 ligase activity. The proposed ideas and research are innovative because they seek to provide new paradigms where none exist: chromatin modification by Rad18 is novel and indicates that Rad18 may have new genome maintenance functions involving transcriptional repression. The proposed mouse studies are innovative because physiological Rad18 functions in genome maintenance have not previously been studied in BM progenitor cells (or other somatic cells) in vivo. The work is significant because it will define new Rad18 functions that protect normal cells against environmental exposures, and confer resistance to chemotherapy and radiotherapy in cancer cells. This work will identify new biomarkers of environmental and therapeutic genotoxicity. We seek to validate Rad18 and its effectors as druggable targets whose inhibition sensitizes cancer cells to chemotherapy and radiation.