Function of the Bromodomain Protein Brdt in Spermatogenesis

描述(申请人提供)：父母资助的重点是阐明睾丸特异的双溴结构域基因BRDT在雄性生殖系中的功能。我们现在计划扩大最初拨款的范围，包括对小鼠BET家族的两个相关成员Brd2和Brd4的研究，这两个成员类似于睾丸特有的BET基因BRDT，在生殖细胞分化过程中以阶段和谱系特有的方式表达。事实上，Brd2最初是(由我们)在筛选睾丸cDNA文库时发现的，在雄性和雌性生殖系中都相当丰富。在精子发生过程中，Brd4的表达仅限于减数分裂前期细胞，而Brd4的表达仅限于精原细胞。与睾丸特异的BRDT不同，Brd2和Brd4都在多种组织中表达，定向突变表明这两个基因都是必需的基因：Brd2缺陷的胚胎在妊娠中期死亡，而缺乏Brd4的胚胎甚至在植入前后死亡更早。BET家族基因在雄性生殖系中显著的阶段特异性表达，BRDT在我们的研究中所发挥的关键作用，以及Brd2和Brd4在胚胎发育中发挥重要作用的观察，使我们假设Brd2和Brd4对精子发生也将是重要的，但在不同于BRDT的功能的阶段。Brd4和Brd2零突变的胚胎致命性使了解它们在体内的功能的努力复杂化，并完全限制了对它们在成人组织中的功能的了解，包括睾丸。因此，我们将产生携带Brd2和Brd4等位基因的小鼠品系，并通过将携带Brd2和Brd4等位基因的品系与在A型精原细胞中表达Cre重组酶的小鼠交配，在雄性生殖系中独一无二地消除它们的功能。了解BRDT、Brd2和Brd4在精子发生过程中的功能将为阐明BET基因在正常分化和染色质重塑中的作用提供一个强大的发育模型系统。一旦产生，这些菌株不仅将为我们自己专注于精子发生的研究提供宝贵的资源，而且还将为研究BET家族基因的其他研究人员以及染色质重塑和更广泛的表观遗传学领域的研究人员提供一个新的资源。 与公共卫生相关：BRDT是一个含溴结构域蛋白亚家族的成员，最近被证明在从DNA复制到转录到染色质重塑等多种基本细胞功能中具有重要功能。我们的定向突变分析表明，在小鼠模型中，BRDT中两个溴结构域中的第一个缺失会导致雄性不育，但动物在其他方面是可行的，雌性是可生育的。我们的研究将为人类BRDT在男性不明原因(或特发性)不育病例中潜在的功能障碍提供重要的见解，并可能为男性避孕提供一个新的目标。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.