Synthesis of LH, FSH, TSH, and CG requires expression of the primate alpha subunit gene at discrete and different times in cytotrophoblasts of the placenta, cells of the hypophyseal placode, and in gonadotropes and thyrotropes of the pituitary. This complex pattern probably involves both activated and restricted expression dictated by combinatorial arrays of regulatory elements and trans-acting factors. Deciphering the critical elements responsible for correct temporal and spatial expression of the human alpha subunit gene represents the major goal of this proposal. Accordingly, we propose the following Specific Aims: 1. Determine the Identity and Interactive Nature of the Elements and Factors that Target Expression of the alpha SubunIt Gene to Placenta. Placenta-specific expression requires the binding of CREB and several additional proteins to a composite enhancer (PSE). While the functional significance of CREB is well established, the Identity and functional role of the other factors remains unclear. We propose that CREB interacts with some of these proteins to define a unique placenta- specific transcriptional complex. Experimental approaches include transfection, cDNA cloning, and functional analysis of chimeric DNA- binding proteins. 2. Define Regulatory Elements Required for GnRH- and Gonadotrope-Specific Expression of the Human alpha Subunit Gene. Our previous transgenic studies indicate that the 1500 bp human alpha subunit promoter targets expression of reporter genes specifically to gonadotropes but not to thyrotropes. This promoter also renders the transgene responsive to GnRH and sex steroids. Gonadotrope-specific expression probably requires interaction between multiple elements located in the promoter-proximal region. Furthermore, as the receptor for GnRH couples to Galpha/q, we postulate that the PI-PLC pathway plays a role in alpha subunit gene expression and gonadotrope proliferation. Approaches include transfection and a refined transgenic strategy that includes targeted over-expression of components of the Galpha/q pathway. 3. Define Requirements for Complete Temporal and Spatial Expression of the Human alpha Subunit Gene. We postulate that thyrotrope-specific expression of the human alpha subunit gene requires a specific interaction between regulatory elements located in the promoter-proximal region and a distal region that resides between -5000 and -1500 bp relative to the start of transcription. We also suspect that interactions between distal and proximal segments of the promoter affect temporal patterns of expression. Testing this hypothesis requires shielding transgenes from integration site effects as well as defining a transcriptional locus that targets expression to both gonadotropes and thyrotropes. This will involve both transfection and transgenic strategies.
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