The parent grant focuses on elucidating the function of the testis-specific double bromodomain-containing gene Brdt in the male germ line. We now plan to expand the scope of the original grant to include studies on two related members of the mouse BET family, Brd2 and Brd4, which similarly to the testis-specific BET gene Brdt, are expressed in a stage- and lineage-specific manner during germ cell differentiation. Brd2 was in fact initially discovered (by us) during a screen of testicular cDNA libraries and is quite abundant in both the male and female germ lines. During spermatogenesis its expression is restricted to meiotic prophase cells, whereas Brd4 expression is restricted to spermatogonia. Unlike testis-specific Brdt, both Brd2 and Brd4 are expressed in a variety of tissues, and targeted mutagenesis has revealed that both are essential genes: Brd2-deficient embryos die at mid-gestation while embryos lacking Brd4 die even earlier, around implantation. The striking stage-specific expression of the BET family genes in the male germ line, the critical role played by Brdt as shown in our studies to date, and the observation that Brd2 and Brd4 perform important functions during embryonic development lead us to hypothesize that Brd2 and Brd4 will also be important for spermatogenesis, but at stages distinct from the function of Brdt. The embryonic lethality of null mutations in Brd4 and Brd2 complicates efforts to understand their in vivo functions and completely restricts understanding their function in adult tissues, including the testis. Therefore, we will generate strains of mice carrying floxed alleles of Brd2 and Brd4 and ablate their function uniquely in the male germ line by mating the floxed allele-bearing strains with mice expressing Cre recombinase in typeA spermatogonia. Understanding the function of Brdt, Brd2, and Brd4 during spermatogenesis will provide a powerful developmental model system for elucidating the role of the BET genes during normal differentiation and in chromatin remodeling. Once generated, these strains will provide valuable resources not only for our own studies focused on spermatogenesis but will also be a novel resource for other investigators studying the BET family genes and investigators in the fields of chromatin remodeling and epigenetics more generally.
Brdt is a member of a sub-family of bromodomain-containing proteins which have recently been shown to have essential functions in diverse basic cellular functions from DNA replication to transcription to chromatin remodeling. Our targeted mutational analysis has shown that deletion of the first of the two bromodomains in Brdt in the mouse model leads to male sterility, but the animals are otherwise viable and the females are fertile. Our studies will provide important insight into the potential mis-function of human BRDT in cases of unexplained (or idiopathic) infertility in men and may provide a new and novel target for male contraception.
Showing the most recent 10 out of 11 publications
