The dynamics of myogenin site-specific demethylation is strongly correlated with its expression and with muscle differentiation

The molecular mechanisms underlying the activation of tissue-specific genes have not yet been fully clarified. We analyzed the methylation status of s pecific CCGG sites in the 5'-flanking region and exon 1 of myogenin gene, a very important myogenic differentiation factor. We demonstrated a loss of methylation, at the onset of C2C12 muscle cell Line differentiation, limite d to the CCGG site of myogenin 5'-flanking region, which was strongly corre lated with the transcriptional activation of this gene and with myogenic di fferentiation. The same CCGG site was also found to be hypomethylated, in v ive, in embryonic mouse muscle (a myogenin-expressing tissue), as opposed t o nonmuscle (nonexpressing) tissues that had a fully methylated site. In a C2C12-derived clone with enhanced myogenic ability, demethylation occurred within 2 h of induction of differentiation, :suggesting the involvement of some active demethylation mechanism(s) that occur in the absence of DNA rep lication. Exposure to drugs that inhibit DNA methylation by acting on the S -adenosylmethionine metabolism produced a further reduction, to a few minut es, in the duration of the demethylation dynamics, These effects suggest th at the final site-specific DNA methylation pattern of tissue-specific:genes is defined through a continuous, relatively fast interplay between active DNA demethylation and re-methylation mechanisms.