In order to analyze the transcriptional regulation of the muscle-specific 
subunit of the human phosphoglycerate mutase (PGAM-M) gene, chimeric genes 
composed of the upstream region of the PGAM-M gene and the bacterial 
chloramphenicol acetyltransferase (CAT) gene were constructed and transfected 
into C2C12 skeletal myocytes, primary cultured cardiac muscle cells, and 
C3H10T1/2 fibroblasts. The expression of chimeric reporter genes was restricted 
in skeletal and cardiac muscle cells. In C2C12 myotubes and primary cultured 
cardiac muscle cells, the segment between nucleotides -165 and +41 relative to 
the transcription initiation site was sufficient to confer maximal CAT activity. 
This region contains two E boxes and one MEF-2 motif. Deletion and substitution 
mutation analysis showed that a single MEF-2 motif but not the E boxes had a 
substantial effect on skeletal and cardiac muscle-specific enhancer activity and 
that the cardiac muscle-specific negative regulatory region was located between 
nucleotides -505 and -165. When the PGAM-M gene constructs were cotransfected 
with MyoD into C3H10T1/2, the profile of CAT activity was similar to that 
observed in C2C12 myotubes. Gel mobility shift analysis revealed that when the 
nuclear extracts from skeletal and cardiac muscle cells were used, the PGAM-M 
MEF-2 site generated the specific band that was inhibited by unlabeled PGAM-M 
MEF-2 and muscle creatine kinase MEF-2 oligomers but not by a mutant PGAM-M 
MEF-2 oligomer. These observations define the PGAM-M enhancer as the only 
cardiac- and skeletal-muscle-specific enhancer characterized thus far that is 
mainly activated through MEF-2.