Characterization of the human very-long-chain acyl-CoA dehydrogenase gene promoter region: a role for activator protein 2.
Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of a family of nuclear-encoded enzymes that catalyze the initial step in mitochondrial fatty acid beta-oxidation (FAO). Previous studies have indicated that two other members of the AD gene family (medium-chain AD and long-chain AD) are controlled at the transcriptional level by nuclear hormone receptors. In this study, we have cloned and characterized the human VLCAD gene promoter region to identify cis-acting elements involved in its transcriptional control. VLCAD gene promoter-luciferase reporter (VLCAD-Luc) constructs were found to be transcriptionally active in a variety of mammalian cell lines and in primary rat cardiomyocytes when driven by varying lengths of the VLCAD promoter region. Removal of a 20-bp DNA segment of the proximal VLCAD gene promoter markedly reduced the transcriptional activity of VLCAD-Luc constructs. Gel mobility shift assays identified a DNA-binding activity in nuclear extracts prepared from human hepatoma G2 cells that interacted with the 20-bp regulatory region. Competition studies revealed that this DNA-binding activity could be abolished by a molar excess of unlabeled specific oligonucleotide as well as a DNA fragment containing an activator protein 2 (AP-2)-binding site but not by an unrelated nonspecific DNA fragment. These results provide an initial characterization of the human VLCAD gene promoter, identify AP-2 as a candidate activator of VLCAD gene transcription, and suggest that VLCAD gene transcription may be regulated by pathways distinct from that of other AD genes.
Zhou
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Department of Pediatrics, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, Missouri 63110, USA.
Kelly
D P
DP
Strauss
A W
AW
Sims
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H
Zhang
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Z
D044942:Acyl-CoA Dehydrogenase, Long-Chain / Q000235:genetics*; D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008928:Mitochondria / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C083115:activator protein-2 binding element; D008156:Luciferases; D044942:Acyl-CoA Dehydrogenase, Long-Chain