Function of the Bromodomain Protein Brdt in Spermatogenesis

描述（由申请人提供）：溴结构域结合组蛋白和其他蛋白质中的乙酰化赖氨酸，存在于许多染色质相关蛋白、转录因子和几乎所有已知的组蛋白乙酰转移酶中。BET亚家族的独特之处在于其成员包含两个溴结构域（BD1和BD2）和一个额外的末端结构域（ET）。我们在小鼠BET家族基因Brdt中产生了一个突变，命名为Brdt¿BD1，该突变产生了一个缺乏BD1的n端截断的Brdt蛋白。纯合子Brdt¿BD1的雄性后代是不育的，具有明显异常的细长精子。我们已经发现了圆形和细长精子的异常，特别是在细长精子中存在多色中心和异常染色质。此外，我们已经产生了完全丧失BRDT功能的突变体（指定为BRDT -/-），这与我们最初的假设一致，表现出与BRDT¿BD1突变体不同的表型：BRDT -/-雄性小鼠也不育，但精子发生在减数分裂后期的前期精母细胞中被阻止，精子不形成。我们将通过研究BRDT在精子发生过程中在几个不同但不相互排斥的水平上调节基因表达的基本功能来扩展这些新的研究，利用我们已经产生的不同小鼠模型，这些模型在生殖细胞分化的不同阶段表现出截然不同的表型。目的1将验证BRDT形成功能性转录抑制复合物的假设，特别是我们已经确定与BRDT相互作用并已知具有抑制功能的3种蛋白质- prmt5， HDAC1和TRIM28。我们将确定负责形成这些复合物的BRDT的功能域，鉴定体内复合物的其他成分，并鉴定由于BRDT功能部分或完全丧失而表达上调的特定基因集，其错误表达可能导致我们在两个突变模型中观察到的生精缺陷。Aim 2将继续挖掘、验证和研究我们在微阵列和ChIP-Seq数据中鉴定的候选基因的表达，并通过我们初步ChIP-Seq分析确定的候选激活转录因子，通过RNA-Seq在两个突变模型中建立“全转录组”，从而跟踪越来越多的证据，证明BRDT对精子发生过程中的转录激活也很重要。并通过免疫沉淀和质谱法鉴定体内相互作用蛋白。目的3将探讨BRDT在精子细胞和精子细胞中建立高阶染色质/染色体组织中的功能，特别是研究表达缺乏BD1的截断BRDT蛋白（BRDT¿BD1）和完全缺乏BRDT蛋白（BRDT -/-）的精子细胞和精子细胞中染色质凝聚、色中心形成、染色体动力学和核结构的变化。这些研究将为BRDT在精子发生过程中的功能以及BET蛋白在分化过程中的功能提供重要的新见解。

DESCRIPTION (provided by applicant): The bromodomain binds acetylated lysines in histones and other proteins and is found in many chromatin- associated proteins, transcription factors, and in nearly all known histone acetyltransferases. The BET sub- family is unique in that its members contain two bromodomains (BD1 and BD2) and an extra terminal (ET) domain. We have generated a mutation in the mouse BET family gene Brdt, designated Brdt¿BD1, which yields an N-terminal truncated BRDT protein lacking BD1. Homozygous Brdt¿BD1 male progeny are sterile, with strikingly aberrant elongating spermatids. We have identified abnormalities in round and elongating spermatids, specifically the presence of multiple chromocenters and abnormal chromatin in elongating spermatids. In addition, we have generated complete loss of BRDT function mutants (designated Brdt-/-), which, in concordance with our original hypothesis, exhibit a phenotype distinct from that of the Brdt¿BD1 mutants: Brdt-/- male mice are also sterile but spermatogenesis is arrested in late meiotic prophase spermatocytes and spermatids do not form. We will extend these novel studies by examining BRDT's essential function in regulating gene expression during spermatogenesis at several distinct but not mutually exclusive levels, taking advantage of the distinct mouse models we have generated which exhibit dramatically distinct phenotypes at distinct stages of germ cell differentiation. Aim 1 will test the hypothesis that BRDT forms functional transcriptional repression complexes, specifically with 3 proteins we have identified as interacting with BRDT and known to have repressor function-PRMT5, HDAC1, and TRIM28. We will determine the functional domains of BRDT responsible for forming these complexes, identify other components of the complexes in vivo and identify specific gene sets whose expression would be up-regulated due to partial or complete loss of BRDT function and whose mis-expression could contribute to the spermatogenic defects observed in our two mutant models. Aim 2 will follow up on increasing evidence that BRDT is also important for transcriptional activation during spermatogenesis by continuing to mine, validate and study the expression of candidate genes identified in our microarray and ChIP-Seq data and pursue candidate activating transcription factors identified by our preliminary ChIP-Seq analysis, establishing the "full transcriptomes" in the two mutant models by RNA-Seq, and identifying in vivo interacting proteins by immunoprecipitation followed by mass spectrometry. Aim 3 will explore the function of BRDT in establishing higher order chromatin/chromosome organization in spermatocytes and spermatids, specifically examining changes in chromatin condensation, chromocenter formation, chromosome dynamics and nuclear architecture in spermatocytes and spermatids expressing a truncated BRDT protein lacking BD1 (Brdt¿BD1) and in spermatocytes that lack BRDT protein altogether (Brdt-/-). These studies will provide critical new insight into the functions of BRDT during spermatogenesis and the function of the BET proteins in general during differentiation.