Combinatorial Signals & Gene Expression in Trophoblasts

描述(申请人提供)：绒毛膜促性腺激素(CG)是关键 灵长类动物发育中的胚胎分泌的促黄体激素。内分泌 有助于最大CG亚单位合成的机制是重要的 成功怀孕的决定因素。初步证据表明， Cgα亚单位基因以胎盘特异的方式协同诱导 通过由几个顺式元件(两个)组成的复杂转录“单位” CRES和JRE)是整合多个细胞内信号所必需的。 Cre结合复合体由几种激活剂(CREB、AP-1、CBP)和 辅阻遏子分子(HDAC2)。JRE是一个同源异型盒结合位点， 与无远端3(D1x3)交互。这项提案详细说明了一系列复杂的 实验检验了转录激活物和 抑制子联合作用于细胞特异性调节 α亚基基因。此外，滋养层细胞的分化依赖于基因 DLX3诱导的程序。总体目标是全面了解 CG合成的调控机制及调控因素分析 滋养层细胞分化。目标是：目标1：继续分析 Cre依赖的α亚单位基因调控机制 滋养层细胞。Cre依赖的α亚单位基因调控及其作用 EGF和cAMP将在原代人类滋养层细胞中进行检测。生化 突变研究调查了Cre中的协同调节因子的作用。 结合复合体，并定义α染色质重塑的程度 亚基启动子。目的2：探讨DLX3在细胞周期调控中的作用 α亚基基因。研究将分析DLX3在灵长类动物中的表达模式 胎盘和滋养层细胞体外分化过程中 免疫细胞化学。生化和诱变研究将检查 Cre和JRE结合复合体在α亚基内的相互作用 推动者。影响α亚基基因的DLX3相互作用伙伴 调控和对滋养层细胞分化的作用将被确定。目标3： 研究DLX3在胎盘发育和分化中的作用。DLX3 基因敲除小鼠将用于鉴定DLX3在小鼠体内诱导的基因程序 使用最先进的基因图谱分析滋养层细胞。来补充 这些研究，对调解所需因素的调查 DLX3基因启动子的转录激活将导致 识别滋养层细胞分化的“上游”效应因子将是 指挥。

DESCRIPTION (provided by applicant): Chorionic gonadotropin (CG) is a key luteotropic hormone secreted by the developing conceptus in primates. Endocrine mechanisms contributing to maximal CG subunit synthesis are important determinants of a successful pregnancy. Preliminary evidence suggests that the CG alpha subunit gene is synergistically induced in a placental-specific manner via a complex transcriptional "unit" comprised of several cis elements (two CREs and the JRE) necessary for integration of multiple intracellular signals. The CRE binding complex consists of several activator (CREB, AP-1, CBP) and corepressor molecules (HDAC2). The JRE is a homeobox-binding site that interacts with Distal-less 3 (D1x3). This proposal details a complex series of experiments examining the hypothesis that transcriptional activators and repressors contribute combinatorially to the cell-specific regulation of the alpha subunit gene. Further, trophoblast differentiation depends upon gene programs induced by Dlx3. The overall goal is a comprehensive understanding of mechanisms governing CG synthesis and analysis of factors regulating trophoblast differentiation. The aims are: Aim 1: Continued analysis of the mechanisms that govern CRE-dependent alpha subunit gene regulation in trophoblasts. CRE-dependent alpha subunit gene regulation and the effects of the EGF and cAMP will be examined in primary human trophoblasts. Biochemical and mutagenesis studies investigate the role of coregulators within the CRE binding complex and defines the extent of chromatin remodeling within the alpha subunit promoter. Aim 2: Explore the role of Dlx3 in the regulation of the alpha subunit gene. Studies will analyze the Dlx3 expression pattern in primate placenta and during trophoblast differentiation in vitro by immunocytochemistry. Biochemical and mutagenesis studies will examine interactions between CRE and JRE binding complexes within the alpha subunit promoter. Interacting partners of Dlx3 that contribute to alpha subunit gene regulation and to trophoblast differentiation will be identified. Aim 3: Examine the role of Dlx3 in placental development and differentiation. The Dlx3 knockout mouse will be used to identify gene programs induced by Dlx3 in murine trophoblasts using state-of-the-art gene profiling analysis. To complement these studies, an investigation of the factors required to mediate transcriptional activation of the Dlx3 gene promoter that will lead to identification of "upstream" effectors of trophoblast differentiation will be conducted.