STEROIDOGENIC FACTOR-I, AN ORPHAN NUCLEAR RECEPTOR, REGULATES THE EXPRESSION OF THE RAT AROMATASE GENE IN GONADAL TISSUES

In a concerted analysis of the genes encoding three mouse steroid hydr oxylases, we identified and characterized a transcriptional regulatory protein, designated steroidogenic factor 1 (SF-1), that contributes t o the coordinate expression in adrenocortical cells. SF-1, an orphan m ember of the nuclear receptor family, binds to PyCAAGGPyCPu motifs ups tream of the steroid hydroxylases to regulate their expression. In the present study, we extend these findings by examining the role of SF-1 in regulation of the rat P450 aromatase gene in gonadal tissues. The 5'-flanking region of the rat aromatase gene was isolated by a polymer ase chain reaction-based approach, using primers corresponding to the 5'- and 3'-ends of a published aromotase sequence. DNA sequence analys is revealed three differences between our sequence and the previously published sequence, including a 44-base pair (bp) insertion. Moreover, the transcription initiation site, as determined by primer extension analysis, differed from that previously proposed. The new transcriptio n initiation site is located 23 bp 3' of a putative TATA box. When a r evised rat sequence was compared to that of the human aromatase PII pr omoter by BEST-FIT analysis, a region of about 300 bp was identified t hat was 80% conserved between the two promoters. A potential SF-1 site , CCAAGGTCA, was identified st position -82 within this region. An oli gonucleotide probe containing this putative SF-1 site was used in gel mobility shift assays. Consistent with previous studies, a specific co mplex was observed with nuclear extracts from gonadal steroidogenic ti ssues but was absent with nuclear extracts from nonsteroidogenic tissu es. The role of SF-1 in this steroidogenic cell-specific complex was n ext addressed more directly. Bacterial extracts containing an SF-1-glu tathione S-transferase fusion protein interacted specifically with the putative SF1 site, and polyclonal antisera against SF-1-glutathione S -transferase specifically abolished the complex formed with nuclear ex tracts from rat ovaries or R2C rat Leydig tumor cells. Finally, the ar omatase SF-1 element increased expression of an SV40 promoter/lucifera se construct in transient transfection experiments in a steroidogenic cell-selective manner. Collectively, these studies implicate SF-1 in t he regulation of steroid hydroxylase gene expression in nonadrenal tis sues, significantly extending previous studies in adrenocortical cells .