The major site of estrogen production in humans include the placenta, ovaries, adipose tissue, and fetal liver. The synthesis of estrogen is catalyzed by the aromatase enzyme complex, which contains a unique form of cytochrome P450 (P450arom). The gene encoding human P450 is>75kb in size; the region encoding the P450 protein is contained within 9 exons (II-X). We have found that the 5'-untranslated region encoding the mRNAs in placenta, gonads and adipose tissue/fetal liver have distinct 5' termini that are encoded by different exons which are spliced onto a common site that lies just upstream of the translation initiation site in exon II, as a consequence of the use of tissue specific promoters that control aromatase expression in human placenta (promoter I.1), ovary/testis (promoter II), adipose tissue/fetal liver (promoter I.4). We propose that tissue-specific and multifactorial regulation of aromatase expression in placenta, gonads and adipose/fetal liver is regulated by unique combinatorial interactions of transcription factors bound to cis-acting elements within these major promoters. An objective of the proposed research is to define the cis-acting elements within each of these promoter regions that mediate P450 expression in the appropriate tissue and cell specific, as well as developmentally regulated fashion. To accomplish this objective, we will produce transgenic mice carrying fusion genes comprising various amounts of DNA flanking placenta-specific exon I.1, ovary/testis specific exon II, and adipose/fetal liver-specific exon I.4 subcloned upstream of the human growth hormone (hGH) gene, as reporter. Another objective of the proposed research is to use transfected human trophoblasts in primary culture to define the cis acting elements that are required for regulation of promoter I.1 activity. In placenta P450 is expressed only in the syncytiotrophoblast layer. Once these gene regulatory elements are defined, they will be used to isolate cDNAs encoding transcription factors that mediate syncytiotrophoblast specific P450 gene expression. There is evidence to suggest that transcription factors of the POU domain, basic helix-loop-helix and steroid receptor superfamilies serve a role in placental differentiation. PCR amplification, as well as subtraction hybridization will be used to isolate cDNAs encoding novel members of these transcription factor families that are differentially regulated during trophoblast differentiation. Effects of overexpression of these factors in placental cells on expression of the endogenous P450 gene and on expression of P450:hGH fusion genes also will be studied.
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