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 downstream of muscle exon 1 that exhibits properties consistent with a muscle-specific transcriptional enhancer. The goal of this study has been to identify the sequence elements responsible for muscle-specific enhancer activity. Functional studies indicated that this enhancer is active in pre- and pest-differentiated H962(2-1) myoblasts but functions poorly 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 transacting factors expressed in H9C2(2-1) myotubes but not in L6 or C2C12 myotubes. Site-specific mutations within these motifs resulted in a significant reduction in enhancer activity in H9C2(2-1) myotubes. These results suggest that the mechanisms governing DMD gene expression in muscle are similar to those identified in other muscle-specific genes. However, the myogenic profile of enhancer activity and transacting factor binding suggests a more specialized role for this enhancer that is consistent with its potential involvement in dystrophin gene regulation in cardiac muscle.