Development and expression of the male phenotype are dependent on androgen binding to its receptor and subsequent activation of gene expression. Androgen receptor (AR) binds to hormone response elements (HREs) of specific genes and acts as a transcription factor. In the case of androgen-regulated genes, these HREs may be simple or complex in their DNA sequence and mode of activation. The main objective of the work described here is to understand the mechanism of androgen-dependent regulation of the prostatic 20 kDa protein gene through AR or nuclear protein interactions with the intron 1 complex androgen response element (ARE) of this gene. The characterization of androgen receptor and nuclear protein binding sites in an intronic complex ARE is fundamental to our understanding of the mechanisms of androgen-regulated gene expression. This objective will be accompanied by completing the following specific aims: I Define the DNA sequences of the complex ARE of the 20kDa protein gene that interacts with (A) the androgen receptor (AR), and (B) prostate-specific nuclear binding proteins; II Use site- directed mutagenesis to establish the role of specific sequences in AR or nuclear protein binding and androgen-induced transcriptional activity of the complex ARE. DNA footprinting and mobility shift assays with AR- DNA binding domain and full-length AR will be used to define the AR binding sites and to study the effects of prostate nuclear extract factors on AR binding to the ARE. Nuclear protein binding sites will be defines by DNase I footprinting in ventral prostate nuclei using ligation-mediated PCR. Site-directed mutagenesis will be used to mutate the AR binding sites and nuclear protein binding sites and the effects of these mutations on protein binding and transcriptional activity will be determined by mobility shift and cotransfection assays, respectively. Information from these experiments will be important in determining the mechanism of gene activation through an AR-specific complex ARE, and will result in a more complete understanding of androgen-regulated prostate function.