Abstract 830 Inborn Errors of Metabolism Platform, Monday, 5/3

VLCAD is a mitochondrial flavoenzyme encoded on the nuclear genome which catalyzes the first reaction in the β-oxidation spiral of the very long chain fatty acid. We subcloned 4 Kb upstream 5′-flanking region of the human VLCAD gene. Sequence analysis of this promoter region showed that it lacks TATA or CAAT boxes, but is extremely GC-rich. Chimeric plasmids containing various 5′-flanking genomic fragments fused upstream of the luciferase reporter gene of PGL2-basic vector were transiently transfected into human hepatoma cells, neonatal rat cardiomyocytes, mouse 3T3 fibroblast cells and C2C12 myotube cells. The minimal promoter is 0.1 Kb of the 5′-flanking region. Transfection of constructs containing 4 Kb, 2.3 Kb, 1.0 Kb, 0.3 Kb, 0.2 Kb, 0.16 Kb, and 0.12 Kb of the 5′-flanking region activated VLCAD gene transcription activity from 10 to 30 fold as compared with transfection with the 0.1 Kb construct in HepG2 cells, and from 11 to 20 fold in transfection of neonatal cardiomyocytes. Compared with a promoterless PGL2 basic plasmid, these constructs activated luciferase activates from 276 to 482 fold in HepG2 cells and 45 to 95 fold in neonatal rat cardiomyocytes. These results indicate that the 20 bp between 0.12 and 0.1 Kb contains important transcription factor binding sites. By computer search, there are two transcription factor consensus binding sites: AP-2, IL-6 motifs in this region. Cotransfection study with 1-3 µg of AP-2α expression vector in HepG2 cells showed that AP-2α activated VLCAD gene transcription from 21 to 60 fold. VLCAD 120-100 TK luciferase construct activated VLCAD transcription 9.1 fold, and VLCAD 120-100 TK luciferase construct with the AP-2 binding site mutation activated VLCAD transcription only 2.1 fold, indicating that the mutational construct is much less active when compared with the wild type construct. We conclude that activation of VLCAD transcription is most likely mediated by the 20 bp sequence, between 0.12 kb and 0.1 kb, especially the AP-2 binding site. Thus, the AP-2 or AP-2 like transcription factor are critical for VLCAD gene regulation of expression.