COOPERATIVE BINDING OF ANDROGEN RECEPTORS TO 2 DNA-SEQUENCES IS REQUIRED FOR ANDROGEN INDUCTION OF THE PROBASIN GENE

The functional and structural interactions of two androgen receptor-bi nding sites in the 5'-flanking DNA of the rat probasin gene were deter mined, Deletion mapping and DNase I footprinting analysis had previous ly identified two androgen receptor-binding sites (ARBS) necessary for androgen induction of the probasin gene: ARBS-1, which resembled a gl ucocorticoid-responsive element, and ARBS-2, which had a unique sequen ce, In this study, maximal androgen induction in transient transfectio n studies only occurred when both sites were present, Neither binding site functioned independently, and deletion of the DNA sequence betwee n the sites resulted in a 60% loss of androgen inducibility, Moreover, point mutations in either ARBS-1 or ARBS-2 led to >90% loss in activi ty. Scatchard analysis indicated that ARBS I and ARBS-2 bound a synthe tic androgen receptor, AR2, with K-d values of 20.0 and 6.7 nm, respec tively, Consistent with the higher affinity, ARBS-2 bound AR2 at half the threshold concentration (200 ng) of that required in reciprocal DN ase I footprinting experiments with ARBS-1, By comparison, protection occurred at a much lower threshold concentration of AR2 (60 ng) and to the same extent over each site when both sites were present, suggesti ng a cooperative interaction between the two sites, The cooperative ef fect was further substantiated when a point mutation in ARBS-1 blocked AR2 binding not only to ARBS-1, but also to ARBS-2, Similarly, a poin t mutation in ARBS-2 also prevented receptor binding to both sites, An drogen-specific: regulation of probasin gene transcription therefore r equired an androgen-responsive region (positions -286 and +28) contain ing two androgen receptor-binding sites, where the binding of the andr ogen receptor to both sites occurred in a cooperative, mutually depend ent manner,