All glycoprotein hormones are heterodimers composed of an identical a subunit and a distinct beta subunit. Synthesis of chorionic gonadotropin (CG) occurs only in placenta of primates and horses, whereas synthesis of the other glycoprotein hormones is restricted to the pituitary in all other mammals. Consequently, in primates and horses, the alpha subunit gene must be expressed in two locations-- pituitary and placenta. Placental-specific expression of the human a subunit gene requires at least two DNA sequence elements. One confers responsiveness to cAMP (CRE), whereas the other (URE) acts in conjunction with the CRE to specify expression in the placenta. It is unclear, however, whether additional elements are required for placental-specific expression and whether any of these elements mediate the action of sex steroids or hypothalamic-releasing hormones. It is also unknown whether different combinations of cis-acting elements are required for pituitary-specific expression of the a subunit gene. To address these questions, we propose three specific aims. First, we will determine whether additional cis-acting elements are required for placental-specific expression and then characterize the molecular mechanisms underlying the interaction between the CRE and URE and any newly detected cis-acting element. The former will involve analysis of a series CAT vectors linked to alpha subunit promoter-regulatory regions containing 10 bp transversions between positions -170 and +44. Interactions between the factors that bind to cis-acting regulatory sequences will bc evaluated by using a gel-shift assay to construct saturation isotherms. Analysis by the methods of Scatchard and Hill should indicate whether these interactions involve homotropic or heterotropic cooperativity. In the second aim, transgenic mice expressing chimeric human and bovine alpha subunit genes will be used to detect cis-acting sequences required for pituitary-specific expression and to determine whether these or other cis-acting elements are required for regulation by sex-steroids or hypothalamic-releasing hormones. The goal of the third aim is to establish a pituitary-specific in vitro transcription assay and identify, characterize, and clone the trans-acting factor required for pituitary-specific expression of the alpha subunit gene.
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