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 multi nucleated myotubes and transcript levels are increased a further 10-fo ld in mature skeletal muscle, In previous studies we have demonstrated that the core muscle promoter of the human DMD gene contains sequence s that regulate the induction of DMD gene expression with myoblast dif ferentiation. However, direct injection studies have indicated that th e activity of the core muscle promoter in mature skeletal muscle is 30 -fold lower than in immature myotubes, This discrepancy between endoge nous transcript levels and core promoter activity suggested that addit ional transcriptional elements are involved in the regulation of DMD g ene expression in muscle. In this report we present evidence for the e xistence of a muscle-specific enhancer within intron 1 of the human DM D gene. Functional analysis of HindIII fragments from within a 36 kb r egion surrounding muscle exon 1 of the human DMD gene resulted in the identification of a 5.0 kb fragment within muscle intron 1 that consis tently provided high levels of reporter gene expression in both immatu re and mature skeletal muscle. Sequences within this 5 kb fragment wer e shown to be functionally independent of position and orientation and to be inactive in fibroblasts, properties that are consistent with th e definition of a muscle-specific enhancer. Although this enhancer pro vided a 30-fold increase in transcription from a SV40 viral promoter i n mature skeletal muscle, only a 3-fold increase was observed from the DMD core muscle promoter. Intron 1 enhancer activity alone is therefo re insufficient to account for the discrepancy between endogenous tran script levels and core muscle promoter activity in immature and mature skeletal muscle and points to the existence of additional enhancer el ements in other regions of the DMD gene. This report provides the firs t evidence for the involvement of a transcriptional enhancer in DMD ge ne regulation in muscle and impacts on our understanding of the functi onal consequences of mutations at the 5'-end of gene. In this regard, deletions in this region in X-linked dilated cardiomyopathy patients p rovides indirect evidence for a role for this enhancer in regulating D MD gene expression in cardiac muscle.