Carnitine palmitoyltransferase-Iβ (CPT-Iβ) catalyses the transfer of long-chain fatty acids to the enzymes of β-oxidation of muscle and heart. Transcriptional control of this regulatory protein is relevant to disorders of fatty acid oxidation and the switch to glucose metabolism that occurs in cardiac pathology. The presence of a transcriptional enhancer sequence in the first untranslated exon and first intron of the CPT-Iβ gene was identified using deletional and mutational analysis, and by ligation of an oleate responsive element (fatty acid response element) to a minimal promoter. The enhancer sequences are contained in the first 40 bases downstream of the transcription start site and increase CPT-Iβ reporter gene expression independent of any 5′ cis-acting elements. Deletion of the first 40 bases of the 3′-untranslated region does not affect the up-regulation of transcription by 10μM phenylephrine. However, mutation and/or deletion of bases between +11 and +30 dramatically decreases reporter gene expression. Electrophoretic mobility-shift assays reveal two DNA (+11 to +36)—protein complexes that appear cardiac specific. The exon/intron element enhances activation of the heterologous thymidine kinase promoter in a position- and orientation-dependent manner. Therefore we have identified a novel region in the first exon/intron of the CPT-Iβ gene that acts as a non-classical transcriptional enhancer downstream of regulatory elements characterized previously in the 5′-flanking region of the minimal promoter.
Abbreviations used: BCS, bovine-calf serum; CPT-I, carnitine palmitoyltransferase I; CPT-Iα, liver isoform of CPT-I (L-CPT-I); CPT-Iβ, muscle isoform of CPT-I (M-CPT-I); EMSA, electrophoretic mobility-shift assay; DMEM, Dulbecco's modified Eagle's medium; DPE, downstream promoter element; FARE, fatty acid response element; hsv, herpes simplex virus; MED, multiple start site element downstream; MEF, myocyte enhancer factor; MZF, myeloid zinc finger; NCX, Na+/Ca2+ exchanger; Nkx, muscle homologue of the Drosophila gene tinman; NKE, Nkx-binding element; PPARα, peroxisomal-proliferator-activated receptor α; Sp1, specificity protein 1; SRE, serum response element; SRF, serum response factor; TFIID, transcription factor IID; TK, thymidine kinase.