A MUSCLE-SPECIFIC ENHANCER WITHIN INTRON-1 OF THE HUMAN DYSTROPHIN GENE IS FUNCTIONALLY DEPENDENT ON SINGLE MEF-1 E BOX AND MEF-2/AT-RICH SEQUENCE MOTIFS/

In previous studies we have described a 5.0 kb HindIII fragment downst ream of muscle exon 1 that exhibits properties consistent with a muscl e-specific transcriptional enhancer. The goal of this study has been t o identify the sequence elements responsible for muscle-specific enhan cer activity. Functional studies indicated that this enhancer is activ e in pre- and pest-differentiated H962(2-1) myoblasts but functions po orly in L6 and C2C12 myotubes. The core enhancer region was delimited to a 195 bp Spel-Accl fragment and sequence analysis identified three MEF-1/E box and two MEF-2/AT-rich motifs as potential muscle-specific regulatory domains. EMSA competition and DNase footprinting indicated that sequences within a 30 bp region containing single adjoining MEF-1 /E box and MEF-2/AT-rich motifs are target binding sites for transacti ng factors expressed in H9C2(2-1) myotubes but not in L6 or C2C12 myot ubes. Site-specific mutations within these motifs resulted in a signif icant reduction in enhancer activity in H9C2(2-1) myotubes. These resu lts suggest that the mechanisms governing DMD gene expression in muscl e are similar to those identified in other muscle-specific genes. Howe ver, the myogenic profile of enhancer activity and transacting factor binding suggests a more specialized role for this enhancer that is con sistent with its potential involvement in dystrophin gene regulation i n cardiac muscle.