Genotoxic stress response and mutagenesis in normal tissues of mice deficient in homology directed repair

缺乏同源定向修复的小鼠正常组织中的基因毒性应激反应和诱变

基本信息

批准号：
10211496
负责人：
James Jackson
金额：
$ 34.2万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-09 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10211496
关键词：
Address Adult Age Animal Model Apoptosis BRCA1 gene Biological Models Cell Culture Techniques Cell Cycle Arrest Cells Code DNA DNA Damage DNA Double Strand Break DNA Repair DNA Repair Pathway Data Dependence Development Disease Double Strand Break Repair Embryo Embryonic Development Exposure to Failure Gene Expression Genetic Transcription Genetically Engineered Mouse Genome Genomics Genotoxic Stress Health Human Individual Knock-out LIG4 gene Ligase Link Longevity Maintenance Malignant Neoplasms Mammals Mammary gland Measures Mediating Mediator of activation protein Messenger RNA Mitomycin C Modeling Mus Mutagenesis Mutation Mutation Analysis Nonhomologous DNA End Joining Normal tissue morphology Organ Organism Pathology Pathway interactions Phenotype Research Role Somatic Mutation Stress TP53 gene Tissues Vertebrates biological adaptation to stress brca gene crosslink deep sequencing environmental mutagens exome gene repair genome integrity human tissue in vivo irradiation mouse model preservation prevent programs repair function repaired response senescence transcription factor transcriptomics tumor

项目摘要

Project Summary/Abstract The long-term objective of this project is to characterize the pathways and mechanisms that preserve viability and genome integrity in adult mammals exposed to environmental genotoxic stresses. DNA damage can be repaired by the high fidelity pathway homology directed repair (HDR) or by error prone non-homologous end joining (NHEJ). The consequences of HDR loss in adult vertebrates is not well understood because germline knockouts are embryonic lethal, thus, most of our understanding has come from cell culture models or inferred from tumors caused by HDR deficiency. To address this critical need, we have generated genetically engineered mice that can undergo inducible deletion of up to 99% of the essential HDR gene Brca1 in every tissue, post development. We will use this model to address critical questions on 1) the role of HDR in cell/tissue/organismal viability after different types of DNA damage; 2) the dependence of different tissues on HDR to maintain genome integrity after different types of DNA damage; 3) mutation signatures in tissues of HDR deficient mice caused by different stresses, allowing inference of repair pathway utilized in absence of HDR; 4) how the transcription factor p53 cooperates with HDR in each of these phenotypes; 5) the cooperative role of HDR and NHEJ in DNA repair. We hypothesize that in mice with deletion of the essential HDR gene Brca1, tissues known to develop HDR deficient tumors will have a higher mutation rate and favor p53 mediated programs of arrest over apoptosis compared to non-tumor prone tissues. Specific Aim 1. Determine how HDR preserves viability by mediating tissue specific cellular and transcriptional responses. In wild type or Brca1 deleted mice, we will examine how Brca1/HDR preserves near-term viability after different types of DNA damage by 3 measures: 1) the DNA damage response and cell fate (apoptosis, cell cycle arrest); 2) transcriptomic changes; 3) pathology of different tissues. Specific Aim 2. Determine tissue and DNA damage-specific role of Brca1/HDR in maintaining genome integrity. In this aim, we will examine long-term consequences of HDR deficiency on genome maintenance. Somatic mutations are known to accumulate in normal human tissues with age and are linked to disease. We will examine fidelity of repair and longevity of mice with or without functional HDR that survive different types of DNA damage. Deep sequencing and mutation signature analysis will reveal what tissues rely on HDR and what type of DNA perturbation is most mutagenic in different HDR deficient tissues. Specific Aim 3. Determine viability and stress response in mice deficient in NHEJ or both NHEJ and HDR. Because embryonic deletion of either Brca1 or the NHEJ gene Lig4 (coding for Ligase 4) is lethal, their individual and cooperative roles in adult tissues are not known. In this aim, we will examine survival and stress response after deletion of both HDR and NHEJ pathways in adult mice, leaving only SSA and alt-NHEJ.

项目概要/摘要该项目的长期目标是描述保持生存能力的途径和机制和暴露于环境遗传毒性应激的成年哺乳动物的基因组完整性。 DNA 损伤可能是通过高保真路径同源定向修复 (HDR) 或容易出错的非同源末端进行修复加入（NHEJ）。成年脊椎动物 HDR 丧失的后果尚不清楚，因为种系敲除是胚胎致死的，因此，我们的大部分理解来自细胞培养模型或推断由 HDR 缺陷引起的肿瘤。为了满足这一关键需求，我们培育出了可以进行诱导性缺失的基因工程小鼠发育后每个组织中高达 99% 的必需 HDR 基因 Brca1。我们将使用这个模型来解决以下关键问题：1) 不同类型 DNA 处理后 HDR 在细胞/组织/有机体活力中的作用损害; 2）不同类型的DNA后，不同组织对HDR维持基因组完整性的依赖损害; 3) 不同压力引起的 HDR 缺陷小鼠组织中的突变特征，可以进行推断在没有 HDR 的情况下使用的修复途径； 4）转录因子p53如何与HDR在各个方面协同作用这些表型； 5）HDR和NHEJ在DNA修复中的协同作用。我们假设在小鼠中删除必需的 HDR 基因 Brca1，已知会形成 HDR 缺陷肿瘤的组织将具有更高的与非肿瘤易发组织相比，突变率更高，并且有利于 p53 介导的细胞凋亡程序。具体目标 1. 确定 HDR 如何通过介导组织特异性细胞和转录反应。在野生型或 Brca1 缺失小鼠中，我们将检查 Brca1/HDR 如何保留通过 3 种措施评估不同类型 DNA 损伤后的近期生存能力：1) DNA 损伤反应和细胞命运（细胞凋亡、细胞周期停滞）； 2）转录组变化； 3）不同组织的病理。具体目标 2. 确定 Brca1/HDR 在维持基因组中的组织和 DNA 损伤特异性作用正直。为此，我们将研究 HDR 缺陷对基因组维护的长期影响。众所周知，体细胞突变会随着年龄的增长在正常人体组织中积累，并与疾病相关。我们将检查具有或不具有功能性 HDR 的小鼠的修复保真度和寿命，这些小鼠在不同类型的 DNA损伤。深度测序和突变特征分析将揭示哪些组织依赖 HDR 以及哪些组织依赖 HDR 在不同的 HDR 缺陷组织中，DNA 扰动类型的诱变性最强。具体目标 3. 确定缺乏 NHEJ 或同时缺乏 NHEJ 和 HDR 的小鼠的生存能力和应激反应。由于 Brca1 或 NHEJ 基因 Lig4（编码连接酶 4）的胚胎缺失是致命的，因此它们的个体成人组织中的合作作用尚不清楚。为此，我们将研究生存和压力反应在成年小鼠中删除 HDR 和 NHEJ 通路后，仅留下 SSA 和 alt-NHEJ。