Somatostatin is a small peptide synthesized within multiple organs, including the pancreatic islets, intestine and central and peripheral nervous systems where it serves both hormonal and paracrine functions in the regulation of secretions; its actions are essential for metabolic homeostasis. During embryonic development, the specific hormone-producing islet cells derive from a common stem cell. Relative to the early expression of the glucagon and insulin genes on day 10 and day 12, respectively, the expression of the somatostatin gene is delayed (day 17) indicating the involvement of both positive and negative mechanisms of transcriptional control. Importantly, transcription of the rat somatostatin gene is stimulated by cAMP. A cAMP-response element of the gene, TGACGTCA, resides in the sequence at -48 to -41, a cAMP-dependent enhancer and a silencer element resides upstream at -120 to -90, and -240 to -220, respectively. We plan to investigate at a molecular level the cellular mechanisms involved in the expression of the somatostatin gene. The hypothesis to be tested is that the cell-specific and cAMP-regulated expression of the somatostatin gene is mediated by the complex coordinate interactions of several different transcriptional factors, some of which (CREB and CREB-like) bind to the cAMP response element and to a more upstream enhancer element and others, related to the insulin E2 enhancer binding proteins (Isl's) bind to more upstream both enhancer and silencer elements.
Aims of this proposal are 1) to characterize the cis-acting enhancer and silencer sequences in the 5'-flanking region of the rat somatostatin gene that specify islet cell-specific gene expression using fusion genes of the regulatory region of the somatostatin gene and the bacterial reporter gene chloramphenicol acetyl transferase (CAT). 2). To identify the DNA-binding proteins in islet cells that initiate cell-specific transcription of the somatostatin gene using mutational analyses, DNA footprinting and electrophoretic mobility shift-assays. These DNA sequences will be used as probes to select cDNAs encoding the DNA-binding proteins by direct screening of islet expression libraries. Complementary DNAs so obtained will be sequenced and the encoded proteins expressed in bacteria and in cultured islet cells will be used to examine DNA-binding and transcriptional transactivational activities of the proteins, respectively. The structure-function properties of the proteins will be analyzed by site-directed mutagenesis and expression studies. The results of these studies may provide insight into the nature of the transcription factors involved in islet cell differentiation as well as information relevant to the pathogenesis of diabetes mellitus and other disorders of hormone deficiencies.
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