Hormones provide acute and long-term regulation of complex metabolic processes such as gluconeogenesis and glycolysis. The long term changes are generally the result of alterations of the amount of critical enzymes. The rate of synthesis of an enzyme is generally related to the amount of cytoplasmic mRNA, and the steady level of mRNA is determined by its rate of synthesis and degradation. The objectives of my research program are to understand how several hormones, each with a unique mechanism of action, combine to regulate gene expression and mRNA metabolism. We are also interested in learning how a single hormone can have opposing effects on genes in the same cell. Transcriptional regulation is achieved by the interaction of trans-acting protein factors which are targeted to the transcription complex by their respective cis-acting DNA elements. Hormones modulate this interaction and thereby influence transcription in a positive or negative direction. Our studies emphasize the regulation of the P-enolpyruvate carboxykinaae (PEPCK) gene which directs the synthesis of PEPCK, the rate-controlling enzyme in gluconeogenesis. The PEPCK gene is regulated positively by glucocorticoids and cAMP, and negatively by insulin. The regulation of gene transcription by insulin is a major emphasis of the laboratory so we also study gene 33 (which encodes a protein of unknown function) because it is positively regulated by insulin.
Three Specific Aims, each based on observations made during the current project period, are proposed.
Aim 1 : """"""""How does cAMP regulate PEPCK gene transcription?"""""""" concerns the purification and cloning of the trans-acting factor involved in this action of cAMP, and studies of the mechanism of action of the protein.
Aim 2 : """"""""How do glucocorticoids regulate mRNAPEPCK metabolism?"""""""" concerns the structure of the DNA element (and possible accessory protein factors) involved in the action of glucocorticoids on the PEPCK gene, and with the mechanism of mRNAPEPCK stabilization by glucocorticoids.
Aim 3 : """"""""How does insulin affect gene transcription?"""""""" concerns the identification of the insulin-responsive DNA elements in the PEPCK and gene 33 promoters; the identification, isolation and mechanism of action of the protein factors that bind to these elements will be explored.
These Aims will employ many techniques of contemporary molecular biology including: DNA-mediated transfection, mobility shift and footprinting assays to detect DNA-protein interactions, and in vitro transcription. The studies proposed address questions that have not been answered in any other system, so should provide new information.
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