Accurate chromosome segregation during meiosis is ensured by using recombination to create crossovers (COs) between homologous chromosomes. Recombination is initiated by a DNA double-stranded break (DSB) that can be repaired either as a CO or a noncrossover (NCO), but how any given DSB is slated for CO or NCO repair has remained enigmatic despite a detailed understanding of the genetic networks involved. In the current proposal, we describe our approach for addressing how the CO/NCO decision is made and how genome-wide CO patterning mechanisms act locally at a DSB to influence repair outcome. We will use chromosome structural variants as a system for manipulating DSB repair outcome in Drosophila melanogaster. We recently showed29 that heterozygous inversions suppress COs locally outside the inversion breakpoint by altering repair outcome in favor of NCOs and that they simultaneously trigger a genome-wide increase in COs by altering repair outcome in favor of COs. We are building two research areas based on these results. First, we are exploring how heterozygous inversions shuttle DSB repair away from a CO repair outcome by carrying out a genetic analysis of recombination in these zones of suppression. This genetic analysis has two parts, a candidate gene approach to look for genes that change the distribution of COs and NCOs near inversion breakpoints, and a molecular genetic analysis of recombination events to determine which recombination pathways are used. We will take a complementary approach to these studies and use Hi-C and super- resolution imaging to ask if there are local chromosome structure and/or synaptonemal complex changes at the inversion breakpoint. Our second research area will ask how heterozygous inversions trigger a genome- wide increase in COs. To determine the mechanisms that mediate this increase, we will cytologically assay the kinetics of CO formation in heterozygous inversions, determine if normal CO patterning mechanisms are bypassed in order to facilitate the increase in COs, and determine which recombination pathways are used to form the increased COs. In sum, we will leverage our understanding of how heterozygous inversions influence the CO/NCO decision into general models that address how meiotic recombination and crossover patterning intersect to create the final recombination landscape. Furthermore, these experiments will elucidate how structural variants lead to chromosome aneuploidy and subsequent infertility in the human population.
Aneuploidy, the state of having too many or too few chromosomes, is a leading cause of developmental diseases, miscarriages, and failed pregnancies using assisted reproductive technology. The cell has many mechanisms to ensure correct chromosome segregation, including creating crossovers between chromosomes. This grant proposes to study how crossovers are made and the underlying causes of chromosome aneuploidy.
