De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation

DNA methyltransferases, Dnmt3a and Dnmt3b, are required for do novo methylation in embryonic stem (ES) cells and postimplantation embryos. However, the mechanism of do novo methylation is largely unknown. In this study, we have analyzed the sequence specificity of Dnmt3a and Dnmt3b during do novo methylation of murine Maloney leukemia virus provirus DNA in virus-infected ES cells. Provirus DNA from infected wild-type (J1), Dmnt1-/- (c/c), and Dnmt3a3b-/- (3a3b-/-) ES cells were analyzed using the bisulfite sequencing method. We demonstrate that Dnmt3 enzymes methylate predominantly CpG sites in vivo and confirm that Dnmt3 enzymes, but not Dnmt1, are responsible for do novo methylation. However, the sequence context and CpG density do not appear to influence de novo methylation, though strand bias is detectable. Interestingly, non-CpG methylation is detected as a component of do novo methylation. CpA methylation was detected at similar to 1.4% of all sites in J1 and similar to 1.0% in c/c, but only -0.2% in 3a3b-/-. Few methylated CpT or CpC sites were detected. Similar results from nearest neighbor analysis of global endogenous methylation levels indicated a correlation between Dnmt3a and Dnmt3b presence and CpA methylation. These results demonstrate that the Dnmt3 enzymes methylate predominantly CpG sites and at a low frequency CpA sites with no apparent sequence preferences. (C) 2002 Elsevier Science B.V. All rights reserved.