The aromatase enzyme complex is comprised of two polypeptides; NADPH- cytochrome P-450 reductase, a ubiquitous flavoprotein, and a unique form of cytochrome P-450, P-450/AROM, which is present exclusively in estrogen- producing cells. Aromatase is expressed in a number of tissue sites throughout the body; the highest levels of aromatase expression are present in the syncytiotrophoblast of the placenta of pregnant women, ovarian granulosa cells of premenopausal women, and adipose tissue of postmenopausal women. Increased estrogen production by adipose tissues of obese and aged women is believed to serve a role in the promotion of carcinomas of the endometrium and breast. Cyclic AMP appears to serve a major role in the induction of aromatase expression in human granulosa, adipose stromal and placental cells; however, there are distinct tissue- specific differences in the actions of several other regulatory factors, including phorbol esters, and a number of growth factors and cytokines. To define the molecular mechanisms whereby stimulatory and inhibitory factors regulate tissue-specific expression of the P-450AROM gene in estrogen-producing cells, as well as to characterize the trans-acting factors and cis-acting elements that are required for tissue-specific and hormonal regulation of aromatase expression, we have isolated and characterized the entire human P-450AROM structural gene, as well as flanking genomic DNA. The P-450AROM gene is greater than 72 kb in size; the region encoding the P-450AROM protein is contained within 9 exons (II- X) and spans less than or equal to 35kb of DNA. The gene appears to have two major unique promoters that direct expression in placenta and fetal liver, on the one hand, and in ovarian and adipose cells, on the other. The major placental/fetal liver transcription initiation site in exon I.1 lies - 35 kb upstream of the ovarian/adipose tissue transcription initiation site in exon II. Thus, the major regulatory region for placental aromatase expression appears to be localized upstream of exon I.1, greater than 35 kb upstream of exon II, whereas, the regulatory elements for aromatase expression in ovarian and adipose tissues appear to be localized just upstream of exon II. It is the objective of this research to use transgenic technology to define genomic elements involved in ovarian/adipose cell-specific aromatase expression, and to use transfected human placental and fetal liver cells together with transgenic technology to characterize genomic elements and tissue-specific and general transcription factors involved in placenta- and fetal liver- specific P-450AROM gene expression and its multifactorial regulation. In addition, it is proposed to purify the transcription factors involved in placenta/fetal liver-specific P-450AROM expression and to isolate and characterize cDNA and genomic clones for these transcription factors so that we can investigate the molecular mechanisms involved in their regulation. It is anticipated that this research will provide insight into the mechanisms involved in the tissue-specific expression of P- 450AROM gene, in particular and those involved in the tissue-specific regulation of eukaryotic gene expression, in general.
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