Chromatin-mediated maintenance of genomic integrity in germ cells

项目总结

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.