Chromatin-mediated maintenance of genomic integrity in germ cells

项目摘要 DNA损伤的积累是对所有生物体基因组完整性的威胁。DNA损伤可能是由 外部来源（如辐射或某些化学品），也可以作为代谢的结果自发发生 DNA复制中的压力或错误。这一研究领域与年龄相关疾病和生殖健康直接相关。 失败为了防止这些情况，DNA修复途径的完整性至关重要。DNA损伤的修复是 在生殖细胞中至关重要，是卵子和精子的前体。在人类中，识别缺陷或 对生殖细胞中DNA损伤的反应表现为不育和流产，也是导致 发育障碍（例如唐氏综合症和自闭症谱系障碍）。多种修复途径 已经确定了对特定DNA损伤的反应，但我们缺乏对潜在分子的全面了解。 这些机制根据细胞环境指导适当的修复途径选择。这是高度 重要的是，修复途径的功效不同。例如，在有性生殖过程中，只有一种类型的 修复途径可以调节染色体分离所必需的遗传物质的交换 和后代的遗传多样性。在所有情况下，有效的DNA修复都需要专门的染色质， 环境，使访问病变和招聘适当的修复机械。我们的实验利用了 一个简单的遗传模型秀丽隐杆线虫，以确定染色质重塑在生殖系中的作用， 产生卵子或精子的分裂细胞的管道。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酶（NuRD）复合物， 对真核生物染色体的动态调节很重要的复合物。我们的长期目标是 NuRD可以感知并响应DNA损伤，防止有害突变的积累。C. elegans 由于其生命周期短（3-4天），繁殖能力强，生殖系有利于 对大量卵子或精子进行的遗传学和细胞学上易于处理的研究。本申请的目的 是为了确定NuRD的催化亚基LET-418（人类的CHD 4）如何确保DNA修复的保真度 并限制错误传递给后代。在目标1中，我们将使用基因工程菌株， 我们将使用高通量序列分析来确定LET-418如何修复生殖细胞中的DNA损伤， 量化全基因组对损伤的突变率。在目标2中，我们将定义LET-418的作用， 促进DNA修复，以应对自发形式的DNA损伤，我们将量化DNA损伤 由内源性（内部）细胞应激形成。使用报告菌株和高分辨率显微镜，我们将 确定let-418突变生殖系中升高的内源性应激的后果。预期我们的 结果还将告知如何限制压力诱导的DNA损伤，防止染色体的错误分离， 育龄妇女。一旦完成，我们的工作将提供关键的洞察力， 研究人类生殖障碍的原因，并将产生为不孕症治疗策略提供信息的研究结果。

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.