Co-regulation of tissue-specific alternative human carnitine palmitoyltransferase I beta gene promoters by fatty acid enzyme substrate

Carnitine palmitoyltransferase I (CPT-I) catalyzes the rate-determining ste p in mitochondrial fatty acid beta-oxidation. CPT-I has two structural gene s (alpha and beta) that are differentially expressed among tissues. Our CPT -I beta isolates from a human cardiac cDNA library contained two different extreme 5'-sequences derived from short alternative first untranslated exon s that utilize a common splice acceptor site in exon 2. Primer extension id entified single dominant start sites for each transcript, and ribonuclease protection assays showed the presence of one 5'-exon in liver, muscle, and heart mRNAs, indicating that the cognate promoter U (upstream/ubiquitous) i s active in each of these tissues. By contrast, mRNAs containing the altern ative 5'-exon were present only in muscle and heart, indicating a muscle-sp ecific promoter M (muscle). CPT-I beta mRNA levels increased markedly in ti ssues of fasted rats, when circulating free fatty acid concentrations are e levated. Using CPT-I beta promoter/reporter transient transfection of murin e C2C12 myotubes and HepG2 hepatocyte, fatty acids were found to increase p romoter activity in a peroxisome proliferator-activated receptor alpha (PPA R alpha)-dependent fashion. A promoter fatty acid response element (FARE) w as mapped, mutation of which ablated fatty acid-mediated production of both transcripts. PPAR alpha/retinoid X receptor a formed specific complexes wi th oligonucleotides containing the FARE, and anti-PPAR alpha antibody shift ed nuclear protein-DNA complexes, confirming the role of this factor in reg ulating the expression of this critical metabolic enzyme gene. The constitu tive repressor chicken ovalbumin upstream promoter transcription factor com petitively binds at the FARE and modulates fatty acid induction of the prom oters.