Errors in homologous chromosome segregation during meiosis are the leading cause of birth defects, spontaneous abortions, and contribute to infertility. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes via crossovers, generated by homologous recombination (HR)-mediated repair of double-strand DNA breaks (DSBs). Chromatin regulates the accessibility to DSBs and, in turn, DSB repair. Chromatin remodeling complexes are required to repair DSBs in meiotic and mitotic dividing cells, but how they control recruitment and activity of the HR repair machinery at DSBs is unclear. The PBAF and BAF chromatin remodelers are connected to histone modifications occurring at meiotic DSBs, and function in recruiting HR repair factors to DSBs. Our central hypothesis is that PBAF and BAF link histone marks surrounding DSBs with the recruitment and activation of the meiotic HR machinery.
Our Specific Aims will test this hypothesis by addressing the following questions: (i) How do PBAF and BAF regulate HR-mediated repair of DSB and, in turn, the number and position of HR intermediates and crossovers? (ii) Do specific histone marks control the recruitment of PBAF/BAF and the HR machinery to HR hotspot sites on meiotic chromosomes? (iii) Do changes in chromatin architecture around DSBs (i.e. condensation) influence the efficiency of DSB repair? The first Aim will investigate the roles for the PBAF- specific subunit Baf200, and the BAF-specific subunit Baf250A, in meiotic DSB repair, crossover formation, and the association and disjunction of homologous chromosomes. We will employ established tools, such as imaging and genetically modified mice, to discern the functions of PBAF and BAF as meiotic regulators.
In Aim 2, we will elucidate the relationship between PBAF/BAF and HR repair by generating high-resolution genome- wide binding profiles for PBAF/BAF and HR repair factors (Dmc1/Rad51) in mouse spermatocytes. To assess whether PBAF/BAF are present and required for HR hotspot formation, we will generate and compare genome-wide binding profiles of Dmc1/Rad51 in spermatocytes that lack Brg1, Baf200 (PBAF) or Baf250A (BAF) to wild-type spermatocytes. To investigate whether PBAF/BAF are sufficient to influence local DSB repair and crossover formation, we developed a lacO-lacI approach to target lacI fusion proteins (e.g. lacI- Brg1) to ectopic lacO arrays.
In Aim 3, we will determine which specific chromatin modifications around DSBs are required to recruit PBAF/BAF and the HR machinery. These experiments will test our model that the chromatin landscape around DSBs control PBAF/BAF recruitment, and illuminate how PBAF/BAF interact with the HR pathway to influence DSB repair efficiency. Experiments in Aim 1 and 2 will also inform the molecular aspects of how changes in chromatin structure influence meiotic recombination for the first time. The outcomes are expected to be significant because they will unravel the epigenetic mechanisms underlying proper DNA repair and homologous chromosome segregation during meiosis, which safeguards the next generation.
Errors in the process of distributing chromosomes to the gametes are the major cause of spontaneous abortions and birth defects, and are an important contributor to infertility. This project uses the mouse meiosis model and combines genome-wide, genetic, and cellular approaches to unravel chromatin based mechanisms controlling the function of the DNA recombination machinery, and ultimately the association of meiotic chromosomes, which is of paramount importance in preventing the chromosome segregation errors that lead to birth defects.
