SF-1 INDEPENDENT TRANSCRIPTIONAL REGULATION OF P450SCC

描述(由申请人提供) 胆固醇侧链裂解酶，细胞色素P450scc，启动 所有类固醇激素的生物合成，是限速和激素 规定的步骤。许多研究表明，P450scc基因在人体内转录。 肾上腺和性腺本质上是相同的，并依赖于孤儿的核 受体SF-1，但胎盘中P450scc基因转录 使用不同于肾上腺和性腺的顺式作用元件， 并且独立于SF-1。P450scc在人胎盘JEG-3细胞中表达 以与P450scc的监管非常相似的CAMP响应方式 在细胞滋养层细胞的原代培养中，但SF-1不被检测到 在JEG-3细胞中进行RT-PCR。米勒？S博士实验室之前的研究表明 胎盘特异性因子参与转录调控 P450scc在胎盘中的表达，但这些因子尚未被鉴定。我最近的作品 在米勒·S博士的实验室里，已经克隆出了新的LBP转录 调节P450scc转录的因子：LBP-1b和LBP-9 胎盘特有的元素。LBP-1b，与其他几个成员类似 在这个新描述的LBP转录因子家族中，似乎可以诱导 胎盘中P450scc的转录。相比之下，LBP-9是完全新颖的 似乎抑制胎盘P450scc的转录； LBP-9是2001年2月报告的。这些LBP蛋白的发现 与P450scc启动子的-155/-131区域相互作用允许我提出 以下是具体目标： 目的1：鉴定LBP-1b和LBP-9。 A)确定LBP-9是否具有抑制结构域或激活 域。 B)分析LBP-1b-EGFP和LBP-9-EGFP是否竞争结合 P450scc的-155/-131元素。 C)确定LBP-1b和LBP-9是否相互影响？S转录 使用我们最近构建的稳定细胞系进行活性检测。 目的2：描述组织特异性和个体发育的表达模式 LBP相关蛋白。 目标3：确定可能促进P450scc反式激活的辅助因素 基因由LBP-1b或LBP-9表达。完成这些目标将提供新的 关于SF-1非依赖性胎盘转录的性质的信息 人P450scc基因。

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.