Identification of a skeletal muscle-specific regulatory domain in the rat GLUT4/muscle-fat gene.
To identify sequences responsible for the muscle-specific expression of the rat GLUT4/muscle-fat gene, we examined the transcriptional regulation of this gene in the differentiating murine C2C12 skeletal muscle cell line. Differentiated myofibers displayed a 4-5-fold increase in GLUT4 mRNA compared with undifferentiated myoblasts which paralleled the conversion from non-muscle beta-actin mRNA to muscle-specific alpha-actin mRNA expression. Transient transfection of progressive 5' and 3' deletions of the GLUT4 5'-flanking DNA identified a 281-base pair region located between -517 and -237 relative to the transcription start site which conferred myotube-specific expression. This region increased reporter activity in the context of the GLUT4 minimal promoter in an orientation-independent manner and, in addition, onto the heterologous thymidine kinase promoter. Myotube-specific expression of both GLUT4 reporter constructs and the endogenous mouse GLUT4 mRNA was also observed to be thyroid hormone-dependent. Further, cotransfection of reporter constructs containing the 281-base pair GLUT4 differentiation-specific enhancer with the thyroid hormone receptor specifically increased luciferase activity in myotubes approximately 12-fold. Thus, these data demonstrate the presence of a proximal skeletal muscle-specific activation domain that is necessary for both myotube-specific GLUT4 expression and thyroid hormone responsiveness.
Richardson
J M
JM
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Pessin
J E
JE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004277:DNA, Single-Stranded; D005786:Gene Expression Regulation; D051275:Glucose Transporter Type 4; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009124:Muscle Proteins; D009132:Muscles / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D013937:Thymidine Kinase / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology
D004277:DNA, Single-Stranded; D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D012333:RNA, Messenger; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D014284:Triiodothyronine; D013937:Thymidine Kinase