Identification of a transcriptional enhancer within muscle intron 1 of the human dystrophin gene

The 14 kb muscle isoform of the Duchenne muscular dystrophy (DMD) gene is expressed primarily in skeletal and cardiac muscle, Transcription of the muscle isoform is induced as myoblasts differentiate into multinucleated myotubes and transcript levels are increased a further 10-fold in mature skeletal muscle, In previous studies we have demonstrated that the core muscle promoter of the human DMD gene contains sequences that regulate the induction of DMD gene expression with myoblast differentiation. However, direct injection studies have indicated that the activity of the core muscle promoter in mature skeletal muscle is 30-fold lower than in immature myotubes, This discrepancy between endogenous transcript levels and core promoter activity suggested that additional transcriptional elements are involved in the regulation of DMD gene expression in muscle. In this report we present evidence for the existence of a muscle-specific enhancer within intron 1 of the human DMD gene. Functional analysis of HindIII fragments from within a 36 kb region surrounding muscle exon 1 of the human DMD gene resulted in the identification of a 5.0 kb fragment within muscle intron 1 that consistently provided high levels of reporter gene expression in both immature and mature skeletal muscle. Sequences within this 5 kb fragment were shown to be functionally independent of position and orientation and to be inactive in fibroblasts, properties that are consistent with the definition of a muscle-specific enhancer. Although this enhancer provided a 30-fold increase in transcription from a SV40 viral promoter in mature skeletal muscle, only a 3-fold increase was observed from the DMD core muscle promoter. Intron 1 enhancer activity alone is therefore insufficient to account for the discrepancy between endogenous transcript levels and core muscle promoter activity in immature and mature skeletal muscle and points to the existence of additional enhancer elements in other regions of the DMD gene. This report provides the first evidence for the involvement of a transcriptional enhancer in DMD gene regulation in muscle and impacts on our understanding of the functional consequences of mutations at the 5'-end of gene. In this regard, deletions in this region in X-linked dilated cardiomyopathy patients provides indirect evidence for a role for this enhancer in regulating DMD gene expression in cardiac muscle.