Nuclear receptors mediate differential transcriptional regulation of a gene involved in mitochondrial energy production

Expression of the nuclear gene encoding medium-chain acyl-CoA dehydrogenase (MCAD), which encodes the initial step in mitochondrial fatty acid oxidation, parallels βoxidation rates among tissues and during development. We have shown recently in transgenic mice that a nuclear receptor response element (NRRE-1) within the MCAD promoter is required for high level expression in tissues with high energy demands such as heart and slow-twitch skeletal muscle (SKM). To identify the nuclear receptors that interact with NRRE-1 in vivo, electrophoretic mobility shift (EMSA) and antibody *supershift* mobility (SMSA) assays were performed with nuclear protein extracts prepared from adult rat tissues with known high and low MCAD expression. EMSA performed with a labeled NRRE-1 oligonucleotide probe demonstrated that two specific NRRE-1 tprotein complexes (low mobility=complex I; high mobility=complex II) formed with all tissue extracts tested. The intensity of complex II was greatest in tissues with high β-oxidative capacity and MCAD expression (heart and slow-twitch SKM) whereas the amount of complex II was greatest in tissues with low MCAD expression (brain, lung, fast-twitch SKM). SMSA performed with a panel of antibodies to known nuclear receptors revealed that the orphan receptor COUP-TF, a known transcriptional represser, is present in complex I. The protein(s) in complex II (the putative activator complex) were not identified by SMSA. Transient transfections of wild-type and NRRE-1 mutant MCAD promoter constructs confirmed that NRRE-1 conferred transcriptional activation in primary cardiocytes but not in NIH 3T3 cells. These data indicate that the binding of differential proportions of represser and activator NRTFs to NRRE-1 provides a mechanism whereby the expression of this gene can be tightly modulated in response to different tissue requirements for fatty acid β-oxidation.