Novel Rad18 functions in Histone Modification and Regulation of Gene Expression

描述(由申请人提供)：我们对DNA损伤是如何感知和修复的，以及DNA修复如何与其他动态染色质功能(如转录、DNA复制)相协调以维持基因组稳定性的理解存在重大差距。DNA修复可以防止正常细胞中环境诱导的癌症，并可以使癌细胞对化疗和放射治疗产生抵抗力。因此，基因组维持机制的知识对于我们理解、预防和成功治疗癌症至关重要。拟议的探索性项目的近期目标是确定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介导的基因表达调节如何有助于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只与调节DNA修复蛋白有关。基于强大的初步数据，中心假设是RAD18直接单一泛素化K119位的组蛋白H2A是一种新的转录抑制机制，有助于基因组的维持。其基本原理是，明确Rad18介导的基因组维持机制将提供对环境致癌的更好理解，并为改进癌症治疗提供新的机会。这个探索性的R21提案的具体目的是：(1)定义新的Rad18在表观遗传控制基因表达中的功能。(2)确定体内对Rad18反应的组蛋白标记和转录事件。对于SA1，我们将进行全基因组分析，以确定与原代人类细胞暴露于环境和治疗性遗传毒性物质(分别为多环芳烃或PAH和电离辐射)后基因表达变化相关的Rad18依赖的组蛋白标记。SA2将确定Rad18影响骨髓(BM)祖细胞基因表达和组蛋白修饰的程度，这是环境多环芳烃和放射治疗的已知遗传毒性目标。为了确定H_2A泛素化对RAD18介导的基因组维持的贡献，我们将使用缺乏H_2A指导的E3连接酶活性的‘功能分离’突变体。提出的想法和研究是创新的，因为他们试图提供不存在的新范式：Rad18对染色质的修饰是新颖的，并表明Rad18可能具有涉及转录抑制的新的基因组维持功能。拟议的小鼠研究是创新的，因为以前还没有在体内的BM祖细胞(或其他体细胞)中研究过Rad18在基因组维持中的生理功能。这项工作意义重大，因为它 将定义新的Rad18功能，保护正常细胞免受环境暴露，并赋予癌细胞对化疗和放射治疗的抵抗力。这项工作将确定环境和治疗性遗传毒性的新生物标记物。我们试图验证Rad18及其效应物是否为可用药靶点，其抑制作用可使癌细胞对化疗和放疗敏感。

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.