Hormonal and cell-specific regulation of Dmrt1

DMRT1是最近发现的一个编码转录因子的基因，其结构特征、表达谱和染色体定位表明它对睾丸的发育和功能具有重要意义。此外，缺乏DMRT1的雄性小鼠由于支持细胞分化和生殖细胞存活方面的缺陷而不育。本研究的目的是确定DMRT1基因在睾丸中特异表达的转录机制，以及DMRT1调控睾丸分化的途径。目的I，建议确定DMRT1的细胞特异性表达和激素调节所必需的调控元件和结合蛋白。这些元件将通过瞬时转染分析进行鉴定，然后进行蛋白质结合研究，以鉴定相关的调节蛋白。将产生携带由不同长度的DMRT1启动子驱动的LacZ报告基因的转基因小鼠，并从时间、空间和激素调节方面确定β-半乳糖苷酶的表达谱。目的评价DMRT1启动子对卵泡刺激素的调控作用，确定其调控元件和蛋白。这将包括在存在和不存在附加激素的情况下通过瞬时转染分析来表征启动子突变的特征，以及评估相关的DNA-蛋白质相互作用。目的研究DMRT1的DNA结合和转录特性，采用体外结合实验和瞬时转染分析。这将使用基于聚合酶链式反应的测试来确定DMRT1与之结合的随机DNA序列，并使用共转染实验来评估DMRT1的转录活性。最后，下游靶基因将通过使用DMRT1的高亲和力结合位点搜索候选基因的序列数据库和微阵列分析来识别，以比较DMRT1表达和非表达细胞系的表达谱。DMRT1‘S作为睾丸功能的重要调节因子，其上游和下游作用因子的鉴定将有助于我们深入了解调控睾丸基因表达的遗传过程，并加深对睾丸分化和生育所必需的分子事件的理解。

Dmrt1 is a recently described gene encoding a putative transcription factor whose structural features, expression profile, and chromosomal localization suggest it is important for testis development and function. Furthermore, male mice lacking Dmrt1 are infertile due to defects in Sertoli cell differentiation and germ cell survival. The goals of the proposed research are to identify both the transcriptional mechanisms responsible for testis-specific expression of Dmrt1 and the means by which Dmrt1 regulates testis differentiation. Aim I, proposes to identify the regulatory elements and binding proteins necessary for cell-specific expression and hormonal regulation of Dmrt1. Elements will be identified and characterized by transient transfection analysis followed by protein binding studies to identify relevant regulatory proteins. Transgenic mice carrying a LacZ reporter driven by various lengths of the Dmrt1 promoter will be generated and the expression profile for betagalactosidase determined with respect to temporal, spatial, and hormonal regulation. Aim I also proposes to evaluate FSH regulation of the Dmrt1 promoter and identify the responsible regulatory elements and proteins. This will involve characterization of promoter mutants by transient transfection analysis in the presence and absence of added hormone and evaluation of relevant DNA-protein interactions. Aim II, will examine the DNA binding and transcriptional properties of Dmrt1 using in vitro binding assays and transient transfection analysis. This will employ a PCR-based assay to identify randomized DNA sequences to which Dmrt1 binds and co-transfection experiments to evaluated Dmrt1 transcriptional activity. Finally, downstream target genes will be identified by both searching sequence databases for candidate genes using an identified high-affinity binding site for Dmrt1 and microarray analysis to compare the expression profile between Dmrt1 expressing and non-expressing cell lines. Dmrt1's role as an important regulator of testis function suggests that identification of factors acting upstream and downstream of it will provide insight into the genetic processes that regulate gene expression in the testis and enhance our understanding of the molecular events necessary for testis differentiation and fertility.