DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation

Abstract

DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.

This work was supported by NIH fellowship GM 19767/01to M.L., a fellowship from the Rett Syndrome Research Foundation to D.S., and DK44746, HL57620, and CA54337 to M.G.

We thank E. Li for the ES cell lines and helpful discussions, Eric Bouhassira for the RL5 and RL6 MEL cell lines, and the members of the Groudine laboratory for suggestions; Wendy Paulsene and Urszula Maliszewski for technical assistance; and Tomoyuki Sawado, Mike Teitell, and Fred van Leeuwen for comments on the manuscript.