Cloning of human acetyl-CoA carboxylase beta promoter and its regulation by muscle regulatory factors.
The 280-kDa beta-isoform of acetyl-CoA carboxylase (ACCbeta) is predominantly expressed in heart and skeletal muscle, whereas the 265-kDa alpha-isoform (ACCalpha) is the major ACC in lipogenic tissues. The ACCbeta promoter showed myoblast-specific promoter activity and was strongly induced by MyoD in NIH3T3 cells. Serial deletions of the promoter revealed that MyoD acts on the E-boxes located at positions -498 to -403 and on the proximal region including the 5'-untranslated region. Destruction of the E-boxes at positions -498 to -403 by site-directed mutagenesis resulted in a significant decrease of MyoD responsiveness. The "TGAAA" at -32 to -28 and the region around the transcription start site play important roles in basal transcription, 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 drastically decreased the MyoD responsiveness. The novel cis-element GCCTGTCA was preferentially bound by MyoD homodimer in EMSA and conferred MyoD responsiveness to a luciferase reporter, which was repressed by the overexpression of E12. This finding is unique since activation via E-boxes is mediated by heterodimers of MyoD and E-proteins. We screened a human skeletal muscle cDNA library to isolate clones expressing proteins that bind to the region around the GCCTGTCA (+8 to +27) sequence, and isolated Myf4 and Myf6 cDNAs. Electrophoretic mobility shift assay showed that recombinant Myf4 and Myf6 bind to this novel cis-element. Moreover, transient expression of Myf6 induced significant activation on the ACCbeta promoter or an artificial promoter harboring this novel cis-element. These findings suggest that muscle regulatory factors, such as MyoD, Myf4, and Myf6, contribute to the muscle-specific expression of ACCbeta via E-boxes and the novel cis-element GCCTGTCA.
Lee
J J
JJ
Department of Biochemistry and Molecular Biology, the Institute of Genetic Science, Yonsei University College of Medicine, 134 Shinchon-dong Seodaemun-gu, Seoul, 120-752, Korea.
Moon
Y A
YA
Ha
J H
JH
Yoon
D J
DJ
Ahn
Y H
YH
Kim
K S
KS
D000103:Acetyl-CoA Carboxylase / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D017570:MyoD Protein / Q000378:metabolism*; D018006:Myogenic Regulatory Factors / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding
D017570:MyoD Protein; D018006:Myogenic Regulatory Factors; D000103:Acetyl-CoA Carboxylase