Dynamics of demethylation and activation of the alpha-actin gene in myoblasts.

Transient transfection into L8 myoblasts has been used to study the rat alpha-actin gene promoter. Demodification of specific sites occurs in two stages, with a hemimethylated intermediate formed within a few hours after entry of the alpha-actin gene construct into the cell. The removal of the methyl moiety from the complementary strand takes place after a delay of at least 48 hr, and both events are actively carried out in the absence of DNA replication. By assaying gene activity during the course of the transfection, it was possible to demonstrate that demethylation of both strands at the critical CpG loci is essential to activate transcription. Genetic analysis revealed the existence of cis-acting elements required for demethylation. The recognition of these sites early in the differentiation process probably leads to the demodification events required to make the gene accessible to its transcription factors.