Friday, March 14, 2025
3/14/2025
PROJECT SUMMARY Diploid organisms, including humans, create haploid gametes for sexual reproduction through the specialized cell division program of meiosis. For meiosis to occur successfully, Crossover (CO) repair of DNA double- strand breaks (DSBs) is essential, as it creates temporary connections between homologous chromosome pairs that promote orderly homolog segregation at the first division. Failure to form COs results in aneuploidy, the leading cause of miscarriage and birth defects in humans. Yet, CO formation relies on the creation of DSBs, which pose a significant danger to genomic integrity. Meiotic cells must therefore balance the risks imposed by DSBs with the requirement for COs to promote orderly chromosome segregation. Further, CO formation is highly limited and constrained: In many organisms, the majority of DSBs are repaired as non-COs (which restore genomic integrity but do not connect homologous chromosomes), whereas CO recombination occurs at only a single DSB site per chromosome (or per chromosome arm). This restriction of CO formation necessitates that each site selected to become a CO must reliably mature as a CO. The paradox of requiring, yet limiting, CO recombination leads to several major outstanding questions: How are a subset of DSBs selected to become COs? What are the mechanisms that operate to ensure and limit CO formation? How is robustness conferred to the process of CO maturation? The proposed research will investigate the mechanisms that promote and ensure reliable formation of meiotic COs, exploiting genetic and cytological features of the C. elegans experimental system that make it especially amenable to addressing these issues. The strategy will focus on COSA-2 (CO-Site Associated), a newly-discovered intrinsically-disordered protein that I identified as a critical component of the CO machinery. COSA-2 exhibits striking localization to CO- designated recombination sites and is required for the retention/accumulation of additional CO-promoting factors at these sites. My preliminary data have led to the hypothesis that COSA-2 promotes CO formation by acting as a hub protein to concentrate CO-promoting factors, stabilize CO-site architecture, and promote formation of a protected spatial compartment to ensure CO-specific repair of DSBs. I will test this hypothesis by: (1) using innovative cytological methodologies and super-resolution microscopy to probe interrelationships between COSA-2 and known CO factors, CO-site architecture, and meiosis-specific chromosome structures; (2) identifying COSA-2 protein partners and features of the COSA-2 protein that contribute to its CO-promoting activity, and (3) using temporally-controlled protein degradation and ectopic recruitment assays to test the mechanistic role of COSA-2 in CO maturation at natural CO sites and to evaluate whether COSA-2 can drive formation of CO-site-like compartments and/or stimulate CO-specific repair. Experiments in this proposal will illuminate fundamental mechanisms that promote reliable formation of the COs needed to direct orderly chromosome segregation and ensure the faithful inheritance of genomes.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).