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

• 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
• 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

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.

项目摘要/摘要 该项目的长期目标是表征保留生存能力的途径和机制 和暴露于环境基因毒性应激的成年哺乳动物中的基因组完整性。 DNA损伤可能是 由高保真途径同源性修复（HDR）修复或易于非同源端的错误 加入（NHEJ）。成人脊椎动物中HDR损失的后果尚不清楚，因为种系 敲除胚胎致死，因此，我们的大多数理解都来自细胞培养模型或推断 来自HDR缺乏引起的肿瘤。 为了满足这一关键需求，我们产生了可以进行诱导删除的基因工程小鼠 在每个组织中，多达99％的基本HDR基因BRCA1。我们将使用此模型 在1）解决不同类型的DNA后HDR在细胞/组织/有机生存力中的作用的关键问题 损害; 2）不同组织对HDR的依赖性在不同类型的DNA之后维持基因组完整性 损害; 3）由不同应力引起的HDR缺乏小鼠组织中的突变特征，从而推断 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合作 这些表型； 5）HDR和NHEJ在DNA修复中的合作作用。我们假设在小鼠中 删除基本HDR基因BRCA1，已知会产生HDR缺乏肿瘤的组织将具有较高的 与非肿瘤的易于凋亡相比，突变率和有利于p53介导的停滞计划，而不是凋亡。 具体目标1。确定HDR如何通过介导组织特异性细胞和 转录响应。在野生型或BRCA1删除的小鼠中，我们将研究BRCA1/HDR蜜饯 通过3次测量，不同类型的DNA损伤后的近期生存能力：1）DNA损伤响应和细胞 命运（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。在此目标中，我们将研究HDR缺乏对基因组维持的长期后果。 已知体细胞突变会随着年龄的增长而聚集在正常的人体组织中，并且与疾病有关。我们 将检查有或没有功能性HDR的小鼠维修和寿命的保真度 DNA损伤。深度测序和突变签名分析将揭示哪些组织依赖于HDR和什么 在不同的HDR缺乏组织中，DNA扰动的类型是最诱人的。 具体目标3。确定缺乏NHEJ或NHEJ和HDR的小鼠的生存力和应力反应。 因为BRCA1或NHEJ基因lig4的胚胎缺失（连接酶4）是致命的，所以他们的个体 在成人组织中的合作作用尚不清楚。在此目标中，我们将检查生存和压力反应 在成年小鼠中删除HDR和NHEJ途径后，仅留下SSA和Alt-NHEJ。