Identification of a novel retinoid-responsive element in the promoter region of the medium chain acyl-coenzyme A dehydrogenase gene.

To study the mechanisms involved in regulation of nuclear genes encoding mitochondrial enzymes in oxidative energy pathways, the promoter region of the medium-chain acyl-CoA dehydrogenase (MCAD) gene was analyzed. A series of hexamer sequences known to bind and confer responsiveness to a subset of members of the nuclear receptor superfamily of transcription factors was identified. Cotransfection of an MCAD promoter-chloramphenicol acetyltransferase (CAT) reporter plasmid with retinoic acid receptor (RAR)alpha, beta, or retinoid X receptor alpha (RXR alpha) resulted in 10-15-fold transcriptional activation in response to retinoic acid. The retinoic acid-induced activation was 3-4-fold higher with RXR alpha than with either RAR alpha or RAR beta. Deletional analysis confirmed that a region between -341 and -308 base pairs upstream of the MCAD gene cap site conferred the RA-responsive transcriptional activation to homologous and heterologous promoters. Gel mobility shift assays demonstrated that the MCAD RARE interacted directly with overexpressed receptors. Mutational analysis of the RARE delineated three hexamer binding sequences with unique orientation and spacing compared to other reported retinoid responsive elements. These results indicate that the MCAD gene promoter region contains a novel regulatory element that interacts with members of the retinoid receptor family, with preferential activation by RXR alpha. This element likely plays a role in the transcriptional regulation of this gene and perhaps others involved in oxidative energy metabolism.