The proposed studies seek to determine the molecular events involved in the tissue-specific, regulated expression of the glycoprotein hormone alpha-subunit gene. Attention will be focused on specific DNA sequences which have been shown to be important for alpha-subunit gene expression and on the transcription factors which interact with these sequences. In particular attention will center on a pituitary-specific glycoprotein hormone basal element (PGBE) and a GnRH response element (GnRH-RE) which we have recently identified.
The specific aims i nclude: 1) Prepare transgenic animals with alpha-subunit reporter genes which contain mutations in the PGBE and the GnRH-RE.
These aims seeks to assess the physiological contributions of these two DNA elements to expression and regulation of the alpha-subunit gene. 2) Further characterize the role of the LIM-homeodomain protein, LH-2 in stimulating alpha-subunit expression. This will include immunoassays for the presence of LH-2 in endogenous, PGBE-binding proteins, disruptions of the LH-2 gene to provide a test of the role of LH-2 in activation of the alpha-subunit gene, analysis of the pituitary cell types in which LH-2 is expressed and initiation of LH-2 structure/function studies. These studies aim to provide definitive information concerning the role of LH-2 in regulating alpha-subunit gene expression as well as insights into the mechanisms of LH-2 action. 3) Search for proteins which interact with LH-2. Alternatively search for proteins which interact with other PGBE-binding proteins. 4) Further characterize DNA sequences which are required for a GnRH response of the alpha-subunit gene. This will involve fine scale mutation of this region of DNA. 5) Several different approaches will be used to search for proteins which bind to the GnRH-RE. 6) Test the role of the c-Raf protein kinase in mediating GnRH effects on alpha-subunit gene expression. Overall these studies aim to increase understanding of the structure, function and interactions of transcription factors which are important for regulating expression of the glycoprotein hormone alpha-subunit gene.
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