2 DIFFERENT, OVERLAPPING PATHWAYS OF TRANSCRIPTION INITIATION ARE ACTIVE ON THE TATA-LESS HUMAN ANDROGEN RECEPTOR PROMOTER - THE ROLE OF SP1

In this study, the minimal promoter requirements of the TATA-less huma n androgen receptor (hAR) gene promoter are described. The hAR promote r is characterized by a short GC-box (-59/-32) and a long homopurine s tretch (-117/-60). Two major transcription initiation sites, AR transc ription initiation site I (AR-TIS I, (+1/2/3)) and AR transcription in itiation site II (AR-TIS II, (+12/13)) are located in a 13-base pair r egion (Faber, P. W., van Rooij, H. C. J., van der Korput, J. A. G. M., Baarends, W. M., Brinkmann, A. O., Grootegoed, J. A., and Trapman, J. (1991) J. Biol. Chem. 266, 10743-10749). Transient transfection of CO S cells with hAR promoter deletion and mutant constructs, followed by RNA isolation and S1 nuclease protection analysis showed that the proc ess of transcription initiation through AR-TIS I and AR-TIS II is regu lated by different promoter sequences. The GC-box directed initiation from AR-TIS II but did not affect AR-TIS I utilization, which is depen dent upon sequences between positions -5 and +57. Band shift analysis identified the transcription factor Sp1 as the protein interacting wit h the GC-box. A single Sp1 binding sequence was found to be present in the GC-box. Footprint analysis confirmed the interaction of Sp1 with this sequence. The differential initiation through AR-TIS I and AR-TIS II was substantiated by the introduction of point mutations in the Sp 1 binding sequence: only mutations that specifically abolished Sp1 bin ding interfered with AR-TIS II utilization, but all mutations left AR- TIS I initiation intact.