Function of the Bromodomain Protein Brdt in Spermatogenesis

描述（由申请人提供）：溴结构域是一个进化上保守的基序，结合组蛋白和其他蛋白质中的乙酰化赖氨酸。哺乳动物含溴结构域蛋白的BET亚类是独特的，它的成员包含两个溴结构域（BD1和BD2）和一个额外的末端（ET）结构域。在小鼠（和人类）中有四个BET家族成员——Brd2、Brd3、Brd4和Brdt，它们在雄性生殖系中以惊人的动态模式表达。我们在小鼠Brdt基因中产生了一个突变，命名为Brdt BD1，该突变产生了一个缺失两个溴结构域中的第一个的截断蛋白。纯合子Brdt BD1后代是可以存活的，但雄性是不育的，产生较少的精子，这些精子在形态上是异常的。目的1将测试以下假设：i) Brdt作为转录复合体的一部分，调节一组基因的表达，这些基因的表达对精子发生至关重要；ii) Brdt的BD1是这种调节所必需的。我们将通过微阵列分析确定在缺乏BD1的情况下表达改变的基因，并同时检查H1t启动子的染色质修饰状态，作为brdt复合物结合区域的模型。目的2将测试另一种假设，但不是相互排斥的假设，即含brdt复合物的功能是标记精子细胞基因组的区域，以便随后被参与精子发生过程中染色质结构独特变化的复合物识别。将使用带有抗brdt抗体的ChIP，然后使用Solexa/Illumina 1g技术进行全基因组测序。目的3将验证以下假设：首先，Brdt的两个溴结构域在精子发生过程中调节转录和/或染色质重构中具有不同的功能；其次，除了精子发生外，Brdt可能在精子发生的几个阶段和过程中起作用；第三，Brdt bd1突变等位基因是一个半形等位基因。我们将产生一个突变等位基因，产生含有BD1但缺乏BD2的Brdt蛋白（Aim 3a）和一个完全缺乏功能蛋白的突变Brdt等位基因（Aim 3b）。我们预测，由此产生的表型将部分重叠，但将不同于bd1缺陷突变体。了解Brdt在精子发生过程中的功能，将为阐明BET基因在正常分化过程中的作用提供一个强大的发育模型系统。重要的是，精子发生也是一个生理相关系统，其中组蛋白乙酰化与染色质重塑明显相关。公共卫生相关性：Brdt是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的各种基本细胞功能中具有重要功能。我们的目标突变分析表明，在小鼠模型中，Brdt中两个溴结构域中的第一个被删除会导致雄性不育，但动物在其他方面是可存活的，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）不育症中的潜在功能障碍提供重要见解，并可能为男性避孕提供新的靶点。

DESCRIPTION (provided by applicant): The bromodomain is an evolutionarily conserved motif that binds acetylated lysines in histones and other proteins. The BET sub-class of mammalian bromodomain-containing proteins is unique in that its members contain two bromodomains (BD1 and BD2) and an extra terminal (ET) domain. There are four BET family members in mouse (and human) - Brd2, Brd3, Brd4, and Brdt - and they are expressed in a striking and dynamic pattern in the male germ line. We have generated a mutation in the mouse Brdt gene, designated Brdt BD1, that yields a truncated protein lacking the first of the two bromodomains. Homozygous Brdt BD1 progeny are viable but the males are sterile, producing fewer sperm that are morphologically abnormal. Aim 1 will test the hypotheses that i) Brdt functions as part of a transcription complex that regulates a set of genes whose expression is essential for spermatogenesis, and ii) that the BD1 of Brdt is required for this regulation. We will identify genes whose expression is changed in the absence of BD1 by microarray analysis and concomitantly, examine the chromatin modification status of the H1t promoter as a model for Brdt-complex binding regions. Aim 2 will test the alternative, but not mutually exclusive, hypothesis that Brdt-containing complexes function to mark regions of the spermatid genome for subsequent recognition by complexes that are involved in the unique changes in chromatin structure during spermiogenesis. ChIP with anti-Brdt antibodies followed by genome-wide sequencing using the Solexa/Illumina 1 G technology will be used. Aim 3 will test the hypotheses that first, the two bromodomains of Brdt have distinct functions in modulating transcription and/or chromatin re-modeling during spermiogenesis; second, that Brdt may function in several stages and processes of spermatogenesis, in addition to spermiogenesis; and third, that the Brdt BD1-mutant allele is a hypomorphic allele. We will generate a mutant allele producing Brdt protein containing BD1 but lacking BD2 (Aim 3a) and a mutant Brdt allele completely lacking functional protein (Aim 3b). We predict that the resulting phenotypes will overlap in part but will be distinct from the BD1-deficient mutant. Understanding the function of Brdt during spermatogenesis will provide a powerful developmental model system for elucidating the role of the BET genes during normal differentiation. Importantly, spermatogenesis is also a physiologically relevant system in which histone acetylation is clearly linked to chromatin remodeling. 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.