Medium-chain acyl-CoA dehydrogenase (MCAD), a nuclear encoded mitochondrial enzyme, catalyzes the initial step in the fatty acid beta- oxidation cycle. The importance of this enzyme in cellular energy metabolism is underscored by the severe and often fatal consequences of inherited human MCAD deficiency. Expression of the MCAD gene is highly regulated in parallel with fatty acid oxidation rates among tissues, during development and in response to fasting and physiologic stimuli. This competitive renewal is designed to test the hypothesis that human MCAD gene promoter sequences identified in vitro, including several novel nuclear hormone receptor response elements and Sp1 binding sites, confer transcriptional regulation of MCAD gene expression in vivo. This hypothesis will be tested by comparing the transcriptional activity of human MCAD gene promoter fragments containing point mutations targeted to known regulatory elements with that of the wild-type promoter in transgenic mice. Elements involved in tissue- and developmental-stage specific expression of the MCAD gene will be delineated. Following localization, the tissue and developmental regulatory elements will be evaluated upstream of a minimal promoter in transgenic mice. The transgenic mice will also be used to identify and localize cis-acting elements that confer regulation of MCAD gene expression in response to fasting, perturbations in mitochondrial fatty acid beta-oxidation, and chronic dietary changes. Transgenic mice containing the fasting and metabolic response elements upstream of heterologous promoters will be produced and characterized as an initial step in the production of tissue- restricted, expression systems responsive to dietary and physiologic stimuli known to increase cellular demands. Lastly, cDNAs encoding the transcription factors that bind the MCAD gene fasting responsive elements will be cloned and characterized. This work should lead to an improved understanding of the pathogenesis of inborn errors in fatty acid oxidation and the development of expression strategies that should be useful in the study and treatment of a variety of inborn and acquired diseases due to abnormalities in cellular energy metabolism.
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