SIMULTANEOUS MYOGENIN EXPRESSION AND OVERALL DNA HYPOMETHYLATION PROMOTE IN-VITRO MYOBLAST DIFFERENTIATION

Two clones of the L5 myoblast line (M6 and the fusion-defective M12) w ere examined for the expression of myogenin, one of the regulatory gen es involved in the regulation of differentiation to myofibers after tr eatment with 3-deazaadenosine, a metabolic inhibitor of methyl transfe r reactions, Cultures treated with 3-deazaadenosine showed, using Nort hern blot hybridization, a conspicuous increase in myogenin expression , which in clone M6 correlated to the extent of cell differentiation u nder fusing conditions but was evident also in growth medium, although the drug was unable to start the myogenic program, We also tested the extent of total DNA methylation to verify whether the activation of t he regulatory cascade could be correlated to the decrease of the overa ll number of 5-methylcytosines present in the genome. The results show that the loss of 5-methylcytosine from newly synthesized DNA, but not from preexisting DNA, is evident in fusing conditions and enhanced by 3-deazaadenosine. It appears that there is a positive correlation bet ween the passive demethylation of newly synthesized DNA, the activatio n of the myogenin gene by demethylation, and the differentiation of my oblasts. However, in fusing conditions, the defective clone M12, altho ugh it is able to express myogenin and its DNA is hypomethylated, fuse s only in the presence of 3-deazaadenosine, suggesting some alternativ e way of induction.