Despite the important effects of androgens on the male phenotype and the breakthrough in cloning the androgen receptor (AR), the precise mechanisms by which the effects of androgens and their receptors on gene expression are mediated remain unresolved The objective of this proposal is to provide understandings regarding these mechanisms using the well- characterized androgen response of mouse beta-glucuronidase (GUS) as a model. Several lines of evidence, including results from DNase I hypersensitivity, gel binding, DNA sequence, and transgene analyses, suggest that the strong response of GUS to androgen observed in kidney of certain strains of mice is controlled by elements within intron 9 of the GUS structural gene (Gus-s), and requires the induction of chromatin changes, an intact androgen response element (ARE) and the cooperative binding of an androgen-inducible, kidney-specific factor (KSAIF) to sequences within a 58 bp region of complex dyad symmetry. The proposed work will test this hypothesis and examine the mechanism by which the androgen regulatory elements exert control.
Three specific aims will be pursued. First, one or more kidney tubule cell culture systems will be established as appropriate models for examining the androgen response of Gus-s. Deletion analysis will he used to analyze a construct containing the entire Gus-s gene plus 3.8 kb of 5' flanking sequence for its ability to confer responsiveness on a luciferase reporter gene. Targeted regions will be the focus of site-directed mutagenesis experiments. Particular attention will be given to a region within intron 9 which includes the ARE and the region of complex dyad symmetry.
The second aim i s to examine whether formation of an androgen-dependent hypersensitive (HS) site within intron 9 is a prerequisite for the androgen response of GUS and whether its formation requires expression of the AR and the presence of the ARE located in intron 9. AR dependence will be examined in mice genetically deficient for AR while ARE-dependence will be examined by in vitro mutagenesis of the ARE in luciferase reporter constructs containing intron 9 sequences.
The third aim addresses whether KSAIF expression underlies the tissue-specific response of GUS to androgen. Upon final purification of KSAIF, a partial sequence of the polypeptide(s) will be used to generate cognate cDNAs to be used for several purposes including analyses of KSAIF gene expression. Several approaches are proposed to examine whether KSAIF is required for the androgen response of Gus-s, including cotransfection of KSAIF and AR expression vectors into cultured cell lines to determine whether endogenous GUS becomes androgen-responsive. Results of this work should provide new and important information regarding the role of the AR and other transcriptional activators, and of the ARE and other DNA sequences that are involved in the specificity of androgen- regulated gene expression.
Showing the most recent 10 out of 11 publications
