Functional analysis of a truncating variant of RAD51B and two ultraconserved genomic elements in gametogenesis

Abstract

Mammalian gametogenesis, whose ultimate aim is to generate haploid gametes from diploid cells, represents one of the most complex differentiation processes in developmental biology and requires an accurate regulation of gene expression. Although a vast knowledge has been generated in this field throughout decades, several aspects regarding its regulation are still far away to be thoroughly understood In the present thesis, we have carried out functional analysis of a family with hereditary infertility and two mammalian ultraconserved elements, HS205 and HS1442, potentially involved in gametogenesis. Primary ovarian insufficiency (POI) causes female infertility by abolishing normal ovarian function. Although the genetic etiology of this disorder has been extensively investigated, most POI cases remain unexplained. RAD51B is a member of the RAD51 family of recombinases. Results of whole-exome sequencing of a family with POI identified a homozygous truncating variant in RAD51B-(c.92delT) in two affected sisters. In vitro studies revealed that this variant leads to translation reinitiation at AUG codon 64. Here, we show that this is a pathogenic hypomorphic variant in a mouse model. Rad51bc.92delT/c.92delT mice exhibited meiotic DNA repair defects due to γH2AX, RAD51 and HSF2BP/BMRE1 accumulation in the chromosome axes leading to a reduction in the number of crossovers. Interestingly, the interaction of RAD51B-c.92delT with RAD51C and with its newly identified interactors RAD51 and HELQ was abrogated or diminished. Repair of Mitomycin-C-induced chromosomal aberrations was impaired in RAD51B/Rad51b-c.92delT human and mouse somatic cells in vitro and in explanted mouse bone marrow cells. Accordingly, RAD51B-c.92delT variant reduced pluripotent reprogramming efficiency of primary mouse embryonic fibroblasts. Finally, Rad51bc.92delT/c.92delT mice displayed increased incidence of pituitary gland hyperplasia. These results provide new mechanistic insights into the role of RAD51B not only in meiosis but in the maintenance of somatic genome stability. Ultraconserved elements are genomic sequences showing extremely high levels of similarity across distant species. To date, no compelling functional explanation has been proposed which would result in such high level of conservation. However, the distribution of these elements within and across vertebrate genomes suggest that they could have many roles as transcriptional regulatory elements during the differentiation associated with development. To directly explore a functional role of these ultraconserved elements in the regulation of spermatogenesis, we have selected two ultraconserved elements, HS205 and HS1442, potentially involved in germ cell biology according to its restricted enhancer activity during mouse development to the genital tubercle. In order to analyze their role, we have further characterized its enhancer activity either in vitro different cell types, and in vivo by testis electroporation. In addition, we have deleted both hs205 and hs1442 in the mouse using CRISPR/Cas9 system and analyzed its consequences during spermatogenesis. Our results provide in vivo evidence that HS205 and HS1442 are not essential for male gametogenesis and thus, fertility in the mouse.