Identification of a tissue-specific regulatory element within the human muscle glycogen phosphorylase gene.
Northern blot analysis showed that human muscle glycogen phosphorylase is developmentally regulated in human adult and fetal skeletal muscle. Furthermore, muscle phosphorylase mRNA expression is temporally regulated in the C2C12 mouse muscle cell line. To define regulatory elements that control expression of the human muscle glycogen phosphorylase gene, the structure of the 5' end of the gene was determined, and 1,129 base pairs of the 5'-flanking region were subcloned and sequenced. Primer extension, RNase protection, and S1 nuclease protection experiments mapped the transcription start site to 76 base pairs upstream from the starting methionine. Sequential deletions of the 5'-flanking region were tested for the ability to activate chloramphenicol acetyltransferase (CAT) expression in fused or proliferating C2C12 cells. Inclusion of the 43 base pairs between -612 and -570 led to a 9-fold increase in CAT activity in fused myotubes. No increase was observed in proliferating myoblasts. This region contains a 10-base pair sequence, CTCCAAAAGG, at -592, which is also repeated at -252. Mutation of the sequence at -592 results in a 50% decrease in CAT activity compared with the amount of CAT activity observed with the normal or a control mutation. These results indicate that a regulatory element is found within -612 to -570 of the 5'-flanking DNA of the human muscle glycogen phosphorylase gene which activates transcription only in differentiated muscle cells.
Lockyer
J M
JM
Human Genetics Program, Tulane University School of Medicine, New Orleans, Louisiana 70112.
McCracken
J B
JB
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000502:physiology; D009132:Muscles / Q000201:enzymology*; D009154:Mutation; D017570:MyoD Protein; D006005:Phosphorylases / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D009124:Muscle Proteins; D017570:MyoD Protein; D012333:RNA, Messenger; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D006005:Phosphorylases