REGULATED DEMETHYLATION OF THE MYOD DISTAL ENHANCER DURING SKELETAL MYOGENESIS

myoD is one of a family of four related basic helix-loop-helix transcr iption factors involved in the specification and differentiation of sk eletal muscle. We previously identified a 258-bp distal enhancer that is sufficient for embryonic activation of myoD and is highly conserved between humans and mice, In this paper, we show using a modified bisu lfite deamination/PCR amplification method that the distal myoD enhanc er is completely unmethylated at all the CpG sites tested in myogenic cells and a subpopulation of somite cells. Conversely, the distal enha ncer 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 d istal enhancer in somites of mouse embryos precedes myoD transcription , suggesting that demethylation of the distal enhancer is an active, r egulated process that is essential for myoD activation. We also show b y analysis of transgenic mice carrying a human distal enhancer/reporte r construct in which the three enhancer CpG sites have been mutated th at methylation of the distal enhancer is not required to prevent preco cious or ectopic embryonic myoD expression. We propose that a subset o f somite cells demethylate the distal enhancer in response to specific developmental signals, thus making the enhancer accessible and able t o respond to subsequent signals to activate the myoD gene. (C) 1996 Ac ademic Press, Inc.