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Characterization of the Regulatory Regions of the Human Aromatase (P450arom) Gene Involved in Placenta-Specific Expression

Departments of Biochemistry (A.K., J.L.A., C.K., C.R.M.) and Obstetrics-Gynecology (C.R.M.), The Cecil H. and Ida Green Center for Reproductive Biology Sciences University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75235

Aromatase P450 (P450arom), a product of the CYP19 gene, catalyzes the conversion of C₁₉-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 isolated from midterm human placenta spontaneously fuse in culture to form multinucleated syncytiotrophoblast. These morphological changes are associated with a marked induction of P450arom gene expression. The majority 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 genomic sequences required for placenta-specific P450arom expression, fusion genes containing various amounts of DNA flanking the 5'-end of placenta-specific exon I.1 linked to the human GH (hGH) gene, as reporter, were introduced into primary cultures of human trophoblast cells and other cell types. Since the trophoblast cells manifest high levels of aromatase P450 expression, we believe that this provides a physiologically relevant system for characterizing the regulatory regions of this gene. Expression of the fusion genes increased as a function of time in culture in concert with syncytiotrophoblast differentiation and induction 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-fold greater than those of fusion genes containing 2400 bp of exon I.1 5'-flanking DNA, suggesting the presence of an upstream silencer element(s). Expression of these fusion genes was undetectable in cell lines that do not express aromatase or that express aromatase utilizing a nonplacental P450arom promoter. By contrast, P450arom I.1-hGH fusion genes containing 246, 201, or 125 bp of exon I.1 5'-flanking sequence were expressed both in trophoblast cells and in other cell lines. These findings demonstrate that 501 bp of exon I.1 5'-flanking DNA contain response elements required for trophoblast-specific expression of P450arom. These results also suggest the presence of regulatory elements between -501 bp and -246 bp of exon I.1 5'-flanking sequence that bind inhibitory transcription factors expressed in nontrophoblast cells. Deletion and site-directed mutagenesis experiments further suggest that cis-acting elements, including a GC box and two hexameric sequences present within 246 bp of sequence flanking the 5'-end of exon I.1, contribute to the high levels of P450arom promoter activity in primary cultures of placental cells. By competitive and supershift electrophoretic mobility shift assays, it was observed that the ubiquitously expressed transcription factor Sp1 comprises one of the proteins binding to the GC box in the 5'-flanking sequence of P450arom exon I.1.

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