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 是最近描述的编码假定转录因子的基因，其结构特征、表达谱和染色体定位表明它对睾丸发育和功能很重要。 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缺陷而无法生育。 拟议研究的目标是确定负责 Dmrt1 睾丸特异性表达的转录机制以及 Dmrt1 调节睾丸分化的方式。目标 I 提议鉴定 Dmrt1 细胞特异性表达和激素调节所需的调节元件和结合蛋白。 将通过瞬时转染分析和随后的蛋白质结合研究来鉴定和表征元件，以鉴定相关的调节蛋白。将生成携带由不同长度的 Dmrt1 启动子驱动的 LacZ 报告基因的转基因小鼠，并确定 β 半乳糖苷酶在时间、空间和激素调节方面的表达谱。 目标 I 还建议评估 Dmrt1 启动子的 FSH 调节并确定负责的调节元件和蛋白质。 这将涉及在添加激素存在和不存在的情况下通过瞬时转染分析来表征启动子突变体，并评估相关的 DNA-蛋白质相互作用。 目标 II 将使用体外结合测定和瞬时转染分析来检查 Dmrt1 的 DNA 结合和转录特性。 这将采用基于 PCR 的检测来鉴定 Dmrt1 结合的随机 DNA 序列，并采用共转染实验来评估 Dmrt1 转录活性。最后，下游靶基因将通过使用已确定的 Dmrt1 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比较 Dmrt1 表达和非表达细胞系之间的表达谱来鉴定。 Dmrt1 作为睾丸功能重要调节因子的作用表明，鉴定其上游和下游的因子将有助于深入了解调节睾丸基因表达的遗传过程，并增强我们对睾丸分化和生育力所需分子事件的理解。