FUNCTIONAL-ROLE OF A CONFORMATIONALLY FLEXIBLE HOMOPURINE HOMOPYRIMIDINE DOMAIN OF THE ANDROGEN RECEPTOR GENE PROMOTER INTERACTING WITH SP1AND A PYRIMIDINE SINGLE-STRAND DNA-BINDING PROTEIN/

The androgen receptor (AR) gene promoter does not contain the TATA or CAAT box, but it contains a long (similar to 90-bp) homopurine/homopyr imidine (pur/pyr) stretch immediately upstream of the Sp1-binding GC b ox site. This pur/pyr stretch is conserved at the same proximal positi on in the rat, mouse, and human AR gene promoters. Mutation of this re gion results in a 3-fold decline in promoter activity, indicating an i mportant regulatory function. Examination of the conformational state of the AR pur/pyr region with the single-strand-specific S1 nuclease s howed that it is capable of forming a non-B DNA structure involving un paired single strands. Fine mapping of the S1-sensitive site revealed an unsymmetric cleavage pattern indicative of an intramolecular triple helical H-form DNA conformation. Electrophoretic mobility shift analy ses showed that the pur/pyr region of the AR promoter can bind a novel pyrimidine single-strand-specific protein (ssPyrBF) and also a double -strand DNA-binding protein. Both oligonucleotide cross-competition an d antibody supershift experiments established that the double-strand b inding protein is equivalent to Sp1. Deoxyribonuclease I (DNase I) foo tprinting analysis showed multiple Sp1-binding to the pur/pyr site and a weaker Sp1 interaction to this region compared with the adjacently located GC box, where Sp1 functions to recruit the TFIID complex. Thes e results suggest that the pur/pyr domain of the AR gene can serve to attract additional Sp1 molecules when it exists in the double-stranded B-DNA conformation. However, binding of ssPyrBF and the resultant sta bilization of the non-B DNA structure is expected to prevent its inter action with Sp1. We speculate that in the TATA-less AR gene promoter, multiple weak Spl sites at the pur/pyr region adjacent to the GC box c an provide a readily available source of this transcription factor to the functional GC box, thereby facilitating the assembly of the initia tion complex.