SF-1 INDEPENDENT TRANSCRIPTIONAL REGULATION OF P450SCC

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DESCRIPTION (provided by applicant) The cholesterol side-chain cleavage enzyme, cytochrome P450scc, initiates the biosynthesis of all steroid hormones and is the rate-limiting and hormonally regulated step. Many studies have shown that P450scc gene transcription in the adrenal and gonad are essentially identical, and depend on the orphan nuclear receptor SF-1, but the transcription of the P450scc gene in the placenta employs cis-acting elements different from those used in the adrenal and gonad, and is independent of SF-1. P450scc is expressed in human placental JEG-3 cells in a cAMP-responsive fashion that is very similar to the regulation of P450scc in primary cultures of cytotrophoblast cells, but SF-1 is not detectable by RT-PCR in JEG-3 cells. Previous work in Dr. Miller?s laboratory has shown that placental-specific factors are involved in the transcriptional regulation of P450scc in the placenta, but these factors were not identified. My recent work in Dr. Miller?s laboratory has lead to the cloning of novel LBP transcription factors: LBP-1b and LBP-9, that regulate P450scc transcription through these placental-specific elements. LBP-1b, which is similar to several other members of this newly-described family of LBP transcription factors, appears to induce the placental transcription of P450scc. By contrast LBP-9 is wholly novel and appears to repress placental transcription of P450scc; a mouse homologue of LBP-9 was reported in February 2001. The discovery of these LBP proteins that interact with the-155/-131 region of the P450scc promoter permits me to propose the following Specific Aims: Aim 1: Characterize LBP-1b and LBP-9. a) Determine whether LBP-9 possesses a repression domain or an activation domain. b) Analyze whether LBP-1b-EGFP and LBP-9-EGFP compete for binding to the-155/-131 element of P450scc. c) Determine whether LBP-1b and LBP-9 affect each other?s transcriptional activity using our recently constructed stable cell lines. Aim 2: Delineate the tissue-specific and ontogenic expression patterns of LBP-related proteins. Aim 3: Identify co-factors that may facilitate the transactivation of P450scc gene by LBP-1b or by LBP-9. Completion of these aims will provide new information about the nature of SF-1-independent placental transcription of the human P450scc gene.