Regulated Demethylation of themyoDDistal Enhancer during Skeletal Myogenesis

myoDis one of a family of four related basic helix-loop-helix transcription factors involved in the specification and differentiation of skeletal muscle. We previously identified a 258-bp distal enhancer that is sufficient for embryonic activation ofmyoDand is highly conserved between humans and mice. In this paper, we show using a modified bisulfite deamination/PCR amplification method that the distalmyoDenhancer is completely unmethylated at all the CpG sites tested in myogenic cells and a subpopulation of somite cells. Conversely, the distal enhancer in nonmuscle cells and tissues is methylated to an average level of >50% and we find no chromosomes in these tissues with a completely unmethylated enhancer. We present evidence that demethylation of the distal enhancer in somites of mouse embryos precedesmyoDtranscription, suggesting that demethylation of the distal enhancer is an active, regulated process that is essential formyoDactivation. We also show by analysis of transgenic mice carrying a human distal enhancer/reporter construct in which the three enhancer CpG sites have been mutated that methylation of the distal enhancer is not required to prevent precocious or ectopic embryonicmyoDexpression. We propose that a subset of somite cells demethylate the distal enhancer in response to specific developmental signals, thus making the enhancer accessible and able to respond to subsequent signals to activate themyoDgene.