Cloning of human acetyl-CoA carboxylase beta promoter and its regulation by muscle regulatory factors

The 280-kDa beta -isoform of acetyl-CoA carboxylase (ACC beta) is predomina ntly expressed in heart and skeletal muscle, whereas the 265-kDa alpha -iso form (ACC alpha) is the major ACC in lipogenic tissues. The ACC beta promot er showed myoblast-specific promoter activity and was strongly induced by M yoD in NIH3T3 cells. Serial deletions of the promoter revealed that MyoD ac ts on the E-boxes located at positions -498 to -403 and on the proximal reg ion including the 5'-untranslated region. Destruction of the E-boxes at pos itions -498 to -403 by site-directed mutagenesis resulted in a significant decrease of MyoD responsiveness. The "TGAAA" at -32 to -28 and the region a round the transcription start site play important roles in basal transcript ion, probably as a TATA box and an Inr element, respectively. Mutations of another E-box at -14 to -9 and a "GCCTGTCA" sequence at +17 to +24 drastica lly decreased the MyoD responsiveness. The novel cis-element GCCTGTCA was p referentially bound by MyoD homodimer in EMSA and conferred MyoD responsive ness to a luciferase reporter, which was repressed by the overexpression of E12, This finding is unique since activation via E-boxes is mediated by he terodimers of MyoD and E-proteins. We screened a human skeletal muscle cDNA library to isolate clones expressing proteins that bind to the region arou nd the GCCTGTCA (+8 to +27) sequence, and isolated Myf4 and Myf6 cDNAs. Ele ctrophoretic mobility shift assay showed that recombinant Myf4 and Myf6 bin d to this novel cis-element. Moreover, transient expression of MyfG induced significant activation on the ACC beta promoter or an artificial promoter harboring this novel vis-element, These findings suggest that muscle regula tory factors, such as MyoD, Myf4, and MyfG, contribute to the muscle-specif ic expression of ACC beta via E-boxes and the novel cis-element GCCTGTCA.