Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene.

作者：Paul S. Rennie, Nicholas Bruchovsky, Kevin J. Leco, Patricia C. Sheppard, Shelley A. McQueen, Helen Cheng, Robert Snoek, Andre L. Hamel, Margaret E. Bock, Barbara S. MacDonald, Barbara E. Nickel, Chawnshang Chang, Shutsung Liao, Peter A. Cattini, Robert J. Matusik · 发表于：Molecular Endocrinology · 年份：1993 · DOI：10.1210/mend.7.1.8446105 · 被引用次数：321 · 研究领域：DNA Repair Mechanisms、DNA and Nucleic Acid Chemistry、Prostate Cancer Treatment and Research

The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined. The DNA- and steroid-binding domains of the rat androgen receptor [glutathione-S-transferase (GST)-AR1] and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography. Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies. DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2). Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3). PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins. Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation. A single base mutation in either ARE resulted in a more than 95% loss of andro...