Transient transfection into L8 myoblasts has been used to study the rat α-actin gene promoter. Demodification of specific sites occurs in two stages, with a hemimethylated intermediate formed within a few hours after entry of the α-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.
Bibliographical noteFunding Information:
We would like to thank D. Yaffe and U. Nude1 for making available the a-actin gene and the L8 myoblasts used in this study. Some of the deletion mutations were prepared by ltai Hovers. We are grateful to Irene Bloom and Sarah Selig for help in preparing this manuscript, This research was supported by grants from the National Institutes of Health and the Israel Cancer Research Foundation.