The role of conserved chromatin-associated LEX-1 in meiotic DNA repair and germline maintenance in Caenorhabditis elegans

Abstract

In recent years, modulation of the germline chromatin environment has emerged as a central regulator of the formation, distribution, and successful repair of programmed DNA double-strand breaks (DSBs) in meiosis; however, in-depth study of the factors responsible for this complex regulation remains limited. Here we characterize the molecular function of LEX-1, a conserved chromatin-associated protein highly expressed in the germline of the metazoan Caenorhabditis elegans, in germline DNA repair and germ cell maintenance. LEX-1 (C. elegans) is a chromatin-associated protein conserved from budding yeast (Yta7) to humans (ATAD2) containing a AAA+ ATPase domain and a bromodomain that we identified in a RNAi screen for germline-enriched DNA repair factors. LEX-1 homologs bind histones and act in chromatin-templated processes, but their functions in meiosis remain largely unknown despite high expression in germline tissue in worms, mice, and humans. In Chapter 2, we utilize cytological and genetic approaches to demonstrate that LEX-1 promotes timely DSB repair and the off-centered biased DSB distribution observed along meiotic chromosomes in C. elegans. LEX-1 further promotes timely crossover (CO) formation and off-center CO position between homologous chromosomes, a key event that initiates asymmetric chromosome remodeling at late prophase I and ensures accurate segregation of homologs at meiosis I. Delayed DSB repair in lex-1 mutants results in p53-dependent DNA damage checkpoint activation in late pachytene. Through biochemical, molecular, and genomic approaches, we further demonstrate that LEX-1 is a novel regulator of the germline chromatin environment. Germline-specific RNA sequencing reveals that transcription is highly dysregulated in the absence of LEX-1 function. lex-1 mutants also exhibit increased chromosome axis length and alterations in multiple histone post-transcriptional modifications associated with transcriptional regulation. ATAC sequencing of germ cells revealed that chromatin compaction and accessibility are disrupted in the absence of LEX-1, with functional consequences for regulation of gene expression. We identified physical interactors of LEX-1 via biochemical and in silico methods and propose that LEX-1 may interact with members of transcriptional repression complexes in the germline. These findings support a critical role for LEX-1 in the regulation and repair of meiotic DSBs and germline transcriptional states through chromatin-based mechanisms. In Chapter 3, we investigate the role of LEX-1 in the mitotic germline using cytological methods. We find that LEX-1 promotes mitotic cycling and regulates the timing of entry into meiosis. In the absence of LEX-1, the mitotic DNA damage checkpoint is activated and mitochondrial membrane potential is significantly decreased, suggesting mitochondrial dysfunction and possible increase in oxidative stress. These studies reveal additional roles for LEX-1 in the maintenance and protection of the mitotic germline, a critical starting point in the formation of healthy germ cells. In Appendix I, we describe preliminary studies into the function of LEX-1 in the male germline. Our initial findings suggest that LEX-1 is required for male fertility and may exhibit sexually dimorphic effects in C. elegans. This dissertation identifies a critical function of LEX-1 as a regulator of germline chromatin states and meiotic DSB repair in the female germline as well as a promotor of normal mitotic germline processes. More broadly, this work underscores the need for further investigation into chromatin regulators as central actors in the basic biology of meiotic DNA repair and as important contributors to reproductive health.