The central goal of this proposal is to achieve further insight into the molecular events responsible for generation of specific cell types and homeostatic regulation of trophic factors in the anterior pituitary gland. The cloning and functional analysis of several cell-specific transcription factors, and the identification of a large family of tissue-specific transcription factors containing a novel motif, the POU- domain, permits analysis of strategies of transcriptional activation and regulation of neuroendocrine genes and the mechanisms of establishing distinct cellular phenotypes within an organ. The functions of the pituitary-specific factor, Pit-1, in pituitary development will be investigated by detailed ontogenic analysis of genetic dwarf mice with defects in the Pit-1 gene, and by targeted ecotopic expression in transgenic animals. Additional Pit-1, DNA contacts, the nature of the Pit-1 response element, and a full characterization of the mutative trans- activation domains will be performed in mammalian cells and in yeast. Protein-protein interactions and the effects of hormone-induced covalent modifications of Pit-1 will be evaluated with respect to DNA binding and transcriptional activation. The combinational code responsible for cell-specific patterns of activation and restriction of growth hormone and prolactin gene expression will be studied by characterizing and cloning additional factors exerting functions on expression of the promoter and enhancer elements of these genes. Two such factors that have been identified and cloned will be characterized in detail. The cis-active elements responsible for activation of the Pit-1 gene in thyrophs, lactotrophs, and somatotrophs will be established both by transgenic animal and cell transfection studies, and the nature of the transacting factors will be investigated. We have recently defined a novel calcium-mediated pathway of transcriptional regulation dependent upon the actions of a calcium, calmodulin-dependent protein kinase. We plan to identify the responsive cis-active elements in the rat prolactin gene, and other transcription units, and to clone and characterize the responsible transacting factor(s), utilizing constitutive variants of the calcium, calmodulin-dependent protein kinase alpha created by mutagenesis. The molecular basis of DNA site- specific positive and negative regulation of gene expression will be further defined by evaluating the role of protein-protein interaction domains and various high-affinity binding sites for several classes of transcription factors, including ligand-dependent transcription factors that prevent them from acting as response elements. The structural domains of the T3 receptor and Pit-1 that dictate element-dependent negative transcriptional responses will be defined to the level of single amino acid residues. Both retinoic acid and thyroid hormone exert important functions in growth hormone and prolactin gene expression. The retinoic acid receptor appears to require a series of factors distinct in different cell types, to form high-affinity heterodimers required to bind to retinoic acid response elements. These factors will be purified, characterized, and encoding cDNAs in pituitary cells will be cloned using several strategies in order to permit study of their putative functional roles in morphogenesis and transcriptional regulation. These studies should provide insights into the complex interactions of regulatory factors that control development and homeostatic characteristics of specific cell types within an endocrine organ.
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