Combinatorial Signals & Gene Expression in Trophoblasts

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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.