Hormonal and cell-specific regulation of Dmrt1

Dmrt1 的激素和细胞特异性调节

• 批准号: 6624218
• 负责人: LESLIE L. HECKERT
• 金额: $ 30.38万
• 依托单位: UNIVERSITY OF KANSAS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6624218
• 关键词: DNA binding protein; antibody; binding sites; cell differentiation; cell growth regulation; cell line; developmental genetics; fertility; follicle stimulating hormone; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; genetic transcription; genetically modified animals; hormone regulation /control mechanism; laboratory mouse; laboratory rat; microarray technology; polymerase chain reaction; protein structure function; reporter genes; reproductive development; stainings; testis; transcription factor; transfection /expression vector

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.

DMRT1是一个最近描述的基因，编码了一个推定的转录因子，其结构特征，表达曲线和染色体定位表明它对睾丸发育和功能很重要。 此外，由于Sertoli细胞分化和生殖细胞存活中的缺陷，缺乏DMRT1的雄性小鼠是不育的。 拟议的研究的目标是确定导致DMRT1特异性表达的转录机制以及DMRT1调节睾丸分化的手段。目的I提议确定DMRT1细胞特异性表达和激素调节所必需的调节元件和结合蛋白。 将通过短暂转染分析进行鉴定和特征，然后进行蛋白结合研究以鉴定相关的调节蛋白。将生成由DMRT1启动子的各个长度驱动的LACZ报告基因的转基因小鼠，并根据时间，空间和激素调节确定的贝加乳糖苷酶的表达谱。 目的我还建议评估DMRT1启动子的FSH调节，并确定负责任的调节元素和蛋白质。 这将涉及在存在和不存在添加激素的情况下通过短暂转染分析和相关DNA-蛋白质相互作用的评估来表征启动子突变体。 AIM II将使用体外结合测定和瞬时转染分析检查DMRT1的DNA结合和转录特性。 这将采用基于PCR的测定方法来识别DMRT1结合并共转染实验与评估DMRT1转录活性的随机DNA序列。最后，将使用鉴定出的高亲和力结合位点进行DMRT1和微阵列分析来比较DMRT1表达和非表达细胞系之间的表达曲线，从而通过搜索序列数据库为候选基因识别下游靶基因。 DMRT1作为睾丸功能的重要调节剂的作用表明，鉴定在其上游和下游作用的因素可以洞悉调节睾丸中基因表达的遗传过程，并增强我们对睾丸分化和生育能力所需的分子事件的理解。