The role of conserved chromatin-associated LEX-1 in germline DNA repair - ABSTRACT Germline aneuploidy is the leading cause of infertility and miscarriages, occurring in more than 40% of human oocytes. The underlying causes of errors in meiotic chromosome segregation are not fully understood, but a subset arise from dysregulation of programmed double-strand break (DSB) repair into crossovers (COs), the position of which enables proper chromosome remodeling and homolog tethering that are essential to accurate segregation. Mounting evidence suggests that the meiotic chromatin environment and its modulation are tightly linked to DSB formation, DSB repair (DSBR), and CO distribution, effectively governing the success of homolog segregation. However, few studies have examined how these key aspects of meiosis interact with and regulate one another. We have identified numerous germline-enriched protein factors involved in meiotic DNA repair and genomic stability, including the highly conserved chromatin-associated LEX-1 (C. elegans). Despite multiple roles for the budding yeast (Yta7), mouse (ATAD2), and human (ATAD2) homologs of LEX-1 in chromatin- templated processes including regulation of chromatin accessibility and boundaries, little is known about their roles in meiosis. There is a pressing need to elucidate mechanisms of chromatin-associated factors in the germline, as recent studies increasingly implicate such factors as critical players in successful meiotic chromosome segregation. Preliminary data indicate that lex-1 null mutants are defective in DSBR and are sensitive to gamma irradiation; however, it is presently unknown how LEX-1 functions in these key meiotic processes. This proposal will define the role of LEX-1 and its chromatin-related activity in germline DSBR and CO formation. We will utilize immunofluorescence to determine whether lex-1 mutants exhibit alterations in DSB and CO frequency and distribution, DNA damage response, and DNA repair. We will also perform laser microirradiation studies to understand how LEX-1 localization is altered in response to DNA damage induction. Through immunoprecipitation and mass spectrometry analysis, we have identified LEX-1 interactors in the presence and absence of DNA damage and will utilize RNAi and computational modeling to determine which interactions are critical for LEX-1 function. We will also define the chromatin remodeling activity of LEX-1 in the germline by assaying chromatin accessibility at specific substages in meiosis in various lex-1 mutants through a novel nuclear sorting method. We will then determine the requirement of the conserved domains of LEX-1 in germline function and meiotic progression. The proposed aims will elucidate the previously undescribed importance of a conserved chromatin-associated factor in fundamental meiotic mechanisms affecting accurate homolog segregation and will develop a novel procedure for isolating germline nuclei from specific substages of prophase I. Our findings may identify genetic risk factors that contribute to infertility, congenital chromosomal conditions, and genomic instability, improving our understanding of reproductive biology and maintenance of genomic integrity.
