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.