Chromatin-mediated maintenance of genomic integrity in germ cells

染色质介导的生殖细胞基因组完整性的维持

基本信息

批准号：
10291840
负责人：
Paula M Checchi
金额：
$ 34.64万
依托单位：
MARIST COLLEGE
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：
2021-09-10 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10291840
关键词：
ATP phosphohydrolase ATPase Domain Affect Alkylating Agents Alleles Animals Antimetabolites Apoptosis Architecture Area Binding Biological Assay Biological Process CHD4 gene Caenorhabditis elegans Cancer Etiology Catalytic Domain Cell physiology Cells Cellular Stress Chemicals Chromatin Chromatin Remodeling Factor Chromatin Structure Chromosome Segregation Chromosome abnormality Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Complex Congenital Abnormality Congenital Disorders Cytology DNA DNA Damage DNA Repair DNA Repair Pathway DNA biosynthesis DNA damage checkpoint DNA lesion Deacetylase Defect Disease Down Syndrome Dyes Education Engineering Ensure Environment Epitopes Etiology Exposure to Failure Female Fertility Foundations Frequencies Genetic Genetic Engineering Genetic Materials Genetic Models Genetic Variation Genome Stability Genomics Germ Cells Germ Lines Goals Health Heritability Homologous Gene Human Immunologic Deficiency Syndromes Impairment Infertility Intellectual functioning disability Knowledge acquisition Learning Left Lesion Life Cycle Stages Maintenance Malignant Neoplasms Mediating Meiosis Metabolic stress Microscopy Mission Mitochondria Modeling Molecular Monitor Mutation Neurodevelopmental Disorder Nuclear Nucleosomes Oocytes Organism Outcome Pathologic Pathway interactions Phenotype Pregnancy loss Prevention Proteins Radiation Regulation Reporter Reproduction Research Resolution Role Science Sequence Analysis Sexual Reproduction Site Source Spontaneous abortion Stress Structural defect Testing Therapeutic Transgenic Organisms United States National Institutes of Health Work advanced maternal age age related aged autism spectrum disorder career chromatin modification chromatin remodeling chromosome missegregation developmental disease disease diagnosis early pregnancy loss egg experience experimental study genome integrity genome-wide homologous recombination idiopathic infertility in vivo insight mutant offspring ovarian failure oxidative damage prevent protein complex recruit repaired reproductive reproductive senescence reproductive success reproductive system disorder response sperm cell therapeutic target undergraduate student whole genome

项目摘要

PROJECT SUMMARY Accumulation of DNA damage is a threat to genomic integrity in all organisms. DNA damage can result from external sources (e.g. radiation or certain chemicals) and can also occur spontaneously as a result of metabolic stress or errors in DNA replication. This area of study is directly relevant to age-related disease and reproductive failure. To prevent these conditions, it is crucial that DNA repair pathways are intact. Repair of DNA lesions is critically important in germ cells, the precursors of egg and sperm. In humans, defects in the recognition or response to DNA damage in germ cells manifest as infertility and miscarriage and are also a cause of developmental disorders (e.g. Down Syndrome and Autism Spectrum Disorder). Multiple repair pathways have been identified that respond to specific DNA lesions, yet we lack a full understanding of the underlying molecular mechanisms that guide appropriate repair pathway selection depending on cellular context. This is highly significant, as repair pathways differ in their efficacy. During sexual reproduction, for example, only one type of repair pathway can accommodate exchanges in genetic material necessary for proper chromosome segregation and genetic diversity in offspring. In all scenarios, efficient DNA repair requires a specialized chromatin environment to enable access to lesions and to recruit appropriate repair machinery. Our experiments utilize the facile, genetic model Caenorhabditis elegans to determine the role of chromatin remodelers in the germ line, a pipeline of dividing cells that give rise to eggs or sperm. We discovered that successful DNA repair in germ cells requires the Nucleosome Remodeling and Deacetylase (NuRD) complex, one of several conserved protein complexes important for dynamic regulation of eukaryotic chromosomes. Our long-term goal is to learn how NuRD senses and responds to DNA damage and prevents the accumulation of harmful mutations. C. elegans is ideal for these studies due to its short (3-4 day) life-cycle, prolific reproduction, and a germ line conducive to genetically- and cytologically-tractable study of large numbers of egg or sperm. The objective of this application is to establish how the catalytic subunit of NuRD, LET-418 (CHD4 in humans), ensures the fidelity of DNA repair and limits errors from being transmitted to offspring. In Aim 1, we will use genetically-engineered strains to pinpoint how LET-418 repairs DNA lesions in germ cells, and we will use high throughput sequence analysis to quantify genome-wide mutation rates in response to damage. In Aim 2, we will define the role of LET-418 in promoting DNA repair in response to spontaneous forms of DNA damage, and we will quantify DNA lesions formed by endogenous (internal) cellular stress. Using reporter strains and high-resolution microscopy, we will determine the consequences of elevated endogenous stress in let-418 mutant germ lines. It is expected that our results will also inform how limiting stress-induced DNA damage prevents missegregation of chromosomes in females of advanced reproductive age. Once completed, our work will provide critical insight into the mechanistic causes of human reproductive disorders and will generate findings that inform therapeutic strategies for infertility.

项目摘要 DNA损伤的积累是对所有生物中基因组完整性的威胁。 DNA损伤可能是由外部来源（例如辐射或某些化学物质），也可以自发地发生 DNA复制中的应力或误差。该研究领域与年龄有关的疾病和生殖直接相关失败。为了防止这些条件，至关重要的是DNA修复途径完好无损。 DNA病变的修复是在生殖细胞，卵和精子的前体中至关重要。在人类中，识别或对生殖细胞中DNA损伤的反应表现为不育和流产，也是原因发育障碍（例如唐氏综合症和自闭症谱系障碍）。多个维修途径已经确定对特定的DNA病变有反应，但我们对基本分子缺乏完全的理解指导适当修复途径选择的机制取决于细胞环境。这是高度的重要的是，由于修复途径的功效有所不同。例如，在有性复制期间，只有一种维修途径可以容纳适当染色体隔离所需的遗传材料的交换和后代的遗传多样性。在所有情况下，有效的DNA修复需要专门的染色质环境可以进入病变并招募适当的维修机械。我们的实验利用轻松的遗传模型Caenorhabditis秀丽隐杆线虫确定染色质重塑剂在生殖系中的作用，A 分裂细胞的管道，产生卵或精子。我们发现生殖细胞中成功的DNA修复需要核小体重塑和脱乙酰基酶（NURD）复合物，这是几种保守蛋白之一复合物对于真核染色体的动态调节很重要。我们的长期目标是了解如何 Nurd感应并应对DNA损伤，并防止有害突变的积累。秀丽隐杆线由于其短（3-4天）生命周期，多产的繁殖以及有利于生物的生命周期，因此是这些研究的理想选择。大量卵或精子的基因和细胞学上可削减的研究。此应用的目的是建立Nurd的催化亚基，Let-418（人类中的CHD4），确保DNA修复的保真度并限制了将错误传输到后代的错误。在AIM 1中，我们将使用基因工程的应变来查明Let-418如何修复生殖细胞中的DNA病变，我们将使用高通量序列分析到响应损害的响应量化全基因组突变率。在AIM 2中，我们将定义Let-418的作用促进DNA修复以响应自发形式的DNA损伤，我们将量化DNA病变由内源性（内部）细胞应激形成。使用报告基因菌株和高分辨率显微镜，我们将确定let-418突变生殖系中内源性应激升高的后果。预计我们的结果还将告知限制应力诱导的DNA损伤如何阻止染色体的错误分析高级生殖年龄的女性。完成后，我们的工作将提供对机械的重要见解人类生殖疾病的原因，将产生与不育治疗策略有关的发现。