Androgen regulation of male sexual development and reproductive function is mediated by androgen receptor (AR) interactions with response elements that function as transcriptional enhancers in a network of target genes. Simple androgen response elements (ARE) belong to the class of 15 bp partial palindromic hormone response elements that lack specificity in that they mediate responses to AR and G (glucocorticoid receptor). Complex AREs are larger sequences capable of mediating AR-specific transactivation. We have identified a strong simple ARE within intron 1 of the C3 subunit gene of rat prostatein and a complex ARE in the same region, of the 20 kDa protein gene. The goal of this research is to define the mechanisms by which these elements mediate AR transactivation and delineate the protein-DNA and protein-protein interactions that determine AR-specific regulation.
Specific aims of this aspect of the proposal are to: I Characterize the interactions of full-length AR and GR with the complex androgen response element (ARE) of the 20 kDa protein gene. We will identify receptor-binding sites on the complex ARE and protein factors that influence receptor binding. II Elucidate the mechanism of AR-specific transactivation mediated by the 20 kDa complex ARE. AR DNA-binding and N-terminal domain interactions with the ARE and nuclear proteins will be investigated. III Establish the role of intron 1 AREs in androgen regulation of 20 kDa and C3 gene transcription. In vivo functions of the intron 1 AREs with their homologous gene promoters will be determined by footprinting and transcription assays in ventral prostate and epithelial cell lines derived from prostate. Gene constructs will be prepared for analysis of transcriptional regulation in stably transfected cells and in transgenic mice. IV Identify protein modulators of AR transcriptional regulation that interact with intron 1 ARE and 5' flanking 20 kDa and C3 gene promoter regions. We will search for androgen-regulated immediate-early gene products among these DNA-binding proteins. A closely-related Aim V is to further establish structure-function relationships of AR domains in vivo by analysis of human AR gene mutations causing androgen insensitivity. A genital skin fibroblast assay for androgen responsiveness will also be established. It is expected that this research will identify specific mechanisms of androgen action and lead to a better understanding of the development of male reproductive function.
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