CHARACTERIZATION OF THE REGULATORY REGIONS OF THE HUMAN AROMATASE (P450AROM) GENE INVOLVED IN PLACENTA-SPECIFIC EXPRESSION

Aromatase P450 (P450arom), a product of the CYP19 gene, catalyzes the conversion of C-19-steroids to estrogens. Human P450arom is expressed in placental syncytiotrophoblast, ovarian granulosa cells, and adipose stromal cells by use of tissue-specific promoters that are located 5' of unique untranslated first exons. Mononuclear cytotrophoblasts isol ated from midterm human placenta spontaneously fuse in culture to form multinucleated syncytiotrophoblast. These morphological changes are a ssociated with a marked induction of P450arom gene expression. The maj ority of P450arom transcripts in placental syncytiotrophoblast contain sequences encoded by exon I.1, which lies more than 35 kb upstream of the translation initiation site in exon II. To functionally map genom ic sequences required for placenta-specific P450arom expression, fusio n genes containing various amounts of DNA flanking the 5'-end of place nta-specific exon I.1 linked to the human GH (hGH) gene, as reporter, were introduced into primary cultures of human trophoblast cells and o ther cell types. Since the trophoblast cells manifest high levels of a romatase P450 expression, we believe that this provides a physiologica lly relevant system for characterizing the regulatory regions of this gene. Expression of the fusion genes increased as a function of time i n culture in concert with syncytiotrophoblast differentiation and indu ction of aromatase activity and of P450arom gene expression. P450arom- hGH fusion genes containing 923 and 501 bp of exon I.1 5'-flanking DNA were expressed at comparable levels; these levels were more than 3-fo ld greater than those of fusion genes containing 2400 bp of exon I.1 5 '-flanking DNA, suggesting the presence of an upstream silencer elemen t(s). Expression of these fusion genes was undetectable in cell lines that do not express aromatase or that express aromatase utilizing a no nplacental P450arom promoter. By contrast, P450arom I.1-hGH fusion gen es containing 246, 201, or 125 bp of exon I.1 5'-flanking sequence wer e expressed both in trophoblast cells and in other cell lines. These f indings demonstrate that 501 bp of exon I.1 5'-flanking DNA contain re sponse elements required for trophoblast-specific expression of P450ar om. These results also suggest the presence of regulatory elements bet ween -501 bp and -246 bp of exon I.1 5'-flanking sequence that bind in hibitory transcription factors expressed in nontrophoblast cells. Dele tion and site-directed mutagenesis experiments further suggest that ci s-acting elements, including a GC box and two hexameric sequences pres ent within 246 bp of sequence flanking the 5'-end of exon I.1, contrib ute to the high levels of P450arom promoter activity in primary cultur es of placental cells. By competitive and supershift electrophoretic m obility shift assays, it was observed that the ubiquitously expressed transcription factor Spl comprises one of the proteins binding to the GC box in the 5'-flanking sequence of P450arom exon I.1.