TRANSCRIPTION FACTOR INTERACTIONS AT THE GH PROMOTER

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Growth hormone (GH) is a powerful regulator of linear growth and metabolic homeostasis that acts at almost all organ systems. Exogenous GH therapy is costly but effective at counteracting the profound metabolic effects associated with idiopathic GH-deficiency and, seemingly, the normal age-related decline in GH gene transcription. GH synthesis is restricted to the somatotroph cells of the anterior pituitary gland where the transcription of the GH gene is tightly regulated in distinct physiologic, age-related and circadian patterns by systemic and hypothalamic hormones, many of which operate via protein kinase systems. Our long-term goal is to identify the molecular mechanisms of hormone- regulated, somatotroph-specific GH gene expression as a first step in the design of cost-effective therapies targeting GH transcription. The transcription factor C/EBPalpha cooperates with the pituitary-specific transcription factor Pit-1 and the coactivator CBP to control protein kinase A and C-regulated GH gene transcription. Cooperative activation likely arises from mutual or complementary, kinase-regulated actions of C/EBPalpha, Pit-1 and CBP at specific basal factor and/or structural targets. C/EBPalpha, Pit-1 and CBP each interact with, and colocalize to a nuclear substructure with, the basal transcription factor TBP that participates in activation by those transcription factors and cofactor. The molecular basis for cooperative action will be assessed by examining C/EBPalpha, CBP, Pit-1, and TBP activities required for kinase-regulated GH promoter activation by, nuclear localization of, and physical interactions between, those factors. We have already developed a series of innovative in vivo cellular assays which will allow us to determine the: 1. structures in C/EBPalpha and CBP affecting kinase-regulated cooperative activation of the GH promoter, 2. structures in TBP required for kinase-regulated C/EBPalpha, CBP and Pit-1 GH promoter activation, 3. nuclear positioning of C/EBPalpha, CBP, Pit-1, TBP and TFIIB and the spatiotemporal relationships of interactions between those factors under the same cellular conditions used in the functional assays. 4. the role of C/EBPalpha in the embryonic onset of GH gene transcription in C/EBPalpha knockout mice which die shortly after birth.