Function of the Bromodomain Protein Brdt in Spermatogenesis

描述（由申请人提供）：父母资助的重点是阐明睾丸特异性双溴结构域基因Brdt在男性生殖系中的功能。我们现在计划扩大原始拨款的范围，以包括对小鼠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异性BET基因Brdt，在生殖细胞分化期间以阶段和谱系特异性方式表达。Brd2事实上最初是在睾丸cDNA文库的筛选过程中被发现的，并且在男性和女性生殖系中都非常丰富。在精子发生过程中，其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。与睾丸特异性Brdt不同，Brd2和Brd4都在各种组织中表达，靶向诱变显示两者都是必需基因：Brd2缺陷的胚胎在妊娠中期死亡，而缺乏Brd4的胚胎在着床前后死亡更早。BET家族基因在雄性生殖系中的显著阶段特异性表达，Brdt所起的关键作用，如我们迄今为止的研究所示，以及Brd2和Brd4在胚胎发育过程中发挥重要作用的观察结果，使我们假设Brd2和Brd4对精子发生也很重要，但与Brdt的功能不同。Brd4和Brd2中无效突变的胚胎致死性使理解其体内功能的努力变得复杂，并且完全限制了对它们在成人组织（包括睾丸）中的功能的理解。因此，我们将产生小鼠品系携带的floxed等位基因的Brd2和Brd4和消融其功能独特的雄性生殖系交配floxed等位基因轴承株与小鼠表达Cre重组酶在A型精原细胞。了解Brdt，Brd2和Brd4在精子发生过程中的功能将提供一个强大的发育模型系统，阐明BET基因在正常分化和染色质重塑过程中的作用。一旦产生，这些菌株将提供宝贵的资源，不仅为我们自己的研究集中在精子发生，但也将是一个新的资源，为其他研究人员研究BET家族基因和研究人员在染色质重塑和表观遗传学领域更普遍。 公共卫生相关性：Brdt是含溴结构域蛋白质的亚家族的成员，其最近已被证明在从DNA复制到转录到染色质重塑的多种基本细胞功能中具有必需的功能。我们的靶向突变分析表明，在小鼠模型中，Brdt中两个溴结构域中的第一个的缺失导致雄性不育，但动物在其他方面是可行的，雌性是可育的。我们的研究将为不明原因（或特发性）男性不育症患者中人类BRDT的潜在误功能提供重要的见解，并可能为男性避孕提供新的和新颖的靶点。

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.