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Volume 272, Number 28, Issue of July 11, 1997 pp. 17623-17631
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Androgen-dependent Protein Interactions within an Intron 1 Regulatory Region of the 20-kDa Protein Gene

(Received for publication, December 26, 1996, and in revised form, March 18, 1997)

From the Laboratories for Reproductive Biology, Department of Pediatrics, University of North Carolina School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

The 20-kDa protein gene is androgen regulated in rat ventral prostate. Intron 1 contains a 130-base pair complex response element (D2) that binds androgen (AR) and glucocorticoid receptor (GR) but transactivates only with AR in transient cotransfection assays in CV1 cells using the reporter vector D2-tkCAT. To better understand the function of this androgen-responsive unit, nuclear protein interactions with D2 were analyzed by DNase I footprinting in ventral prostate nuclei of intact or castrated rats and in vitro with ventral prostate nuclear protein extracts from intact, castrated, and testosterone-treated castrated rats. Multiple androgen-dependent protected regions and hypersensitive sites were identified in the D2 region with both methods. Mobility shift assays with ³²P-labeled oligonucleotides spanning D2 revealed specific interactions with ventral prostate nuclear proteins. Four of the D2-protein complexes decreased in intensity within 24 h of castration. UV cross-linking of the androgen-dependent DNA binding proteins identified protein complexes of approximately 140 and 55 kDa. The results demonstrate androgen-dependent nuclear protein-DNA interactions within the complex androgen response element D2.

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