MULTIPLE STEROIDOGENIC FACTOR-1 BINDING-ELEMENTS IN THE HUMAN STEROIDOGENIC ACUTE REGULATORY PROTEIN GENE 5'-FLANKING REGION ARE REQUIRED FOR MAXIMAL PROMOTER ACTIVITY AND CYCLIC-AMP RESPONSIVENESS

A proximal element from the human StAR gene promoter, containing the s equence (-105)TATCCTTGAC(-95), was shown to confer responsiveness to 8 -Br-cAMP in the presence of steroidogenic factor 1 (SF-1) when placed behind a minimal thymidine kinase promoter or an SV40 promoter and tra nsfected into BeWo cells which normally lack StAR and SF-1. This eleme nt was also transactivated by SF-1 in a yeast one-hybrid system. The - 105 to -95 sequence was protected by SF-1 in footprint analysis and a double-stranded oligonucleotide containing the element bound SF-1 spec ifically in electrophoretic mobility shift assays. Another SF-1-bindin g sequence 35 bp upstream of the transcription start site (-(42)CAGCCT TC(-35)) was identified in the DNase 1 footprint analysis and, when mu tated, markedly reduced SF-1-dependent and 8-Br-cAMP-stimulated StAR p romoter activity in BeWo cells. The two proximal SF-1 response element s were shown to be critical for StAR promoter function in human granul osalutein cells, which express SF-1 and respond to cAMP with increased transcription of the StAR gene. Mutation of either element substantia lly reduced basal and forskolin-stimulated promoter activity, although mutation of the -105 to -95 element had more pronounced effects. Muta tion of a third, more distal, SF-l-binding site at -926 to -918 also r educed basal but not forskolin-stimulated promoter activity in the gra nulosa-lutein cells. These findings demonstrate that multiple SF-1 res ponse elements are required for maximal StAR promoter activity and reg ulation by cAMP.