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

Abstract

DNA methyltransferases, Dnmt3a and Dnmt3b, are required for de novo methylation in embryonic stem (ES) cells and postimplantation embryos. However, the mechanism of de novo methylation is largely unknown. In this study, we have analyzed the sequence specificity of Dnmt3a and Dnmt3b during de novo methylation of murine Maloney leukemia virus provirus DNA in virus-infected ES cells. Provirus DNA from infected wild-type (J1), Dnmt1−/− (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 de 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 de novo methylation. CpA methylation was detected at ∼1.4% of all sites in J1 and ∼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.

Introduction

DNA methylation is essential for embryonic development in mammals and has been implicated in numerous human pathological conditions, yet its regulation is poorly understood (Robertson and Wolffe, 2000). Early studies revealed the presence of both a maintenance and de novo methylase activity in mammals (Jahner et al., 1982, Wigler et al., 1981). Four mammalian DNA cytosine methyltransferases, Dnmt1, Dnmt2, Dnmt3a, and Dnmt3b, have been isolated (Bestor et al., 1988, Okano et al., 1998a, Yoder and Bestor, 1998). Dnmt1 preferentially methylates hemi-methylated substrates in vitro and is associated with replication foci (Leonhardt et al., 1992). Inactivation of Dnmt1 leads to a genome-wide loss of methylation, indicating Dnmt1 functions as a maintenance methylase (Li et al., 1992, Lei et al., 1996). Unlike Dnmt1, Dnmt3a and Dnmt3b lack a preference for hemi-methylated substrates in vitro, and are required for de novo methylation in ES cells and early postimplantation embryos, suggesting Dnmt3a and Dnmt3b function as de novo methylases (Okano et al., 1999). No direct evidence exists that Dnmt2, the only mammalian Dnmt lacking a large N-terminal domain, is a functional cytosine methyltransferase.

There is evidence for methylation of cytosine at non-symmetric dinucleotides in both plants and mammals. In plants a direct link between RNA-directed DNA methylation and methylation of non-CpG sites has been demonstrated (Pelissier et al., 1999). Evidence supports non-CpG methylation in plants as a functional mechanism for initiation of transcriptional gene silencing, probably due to the activity of homologues of the Arabidopsis thaliana CHROMOMETHYLASE3 gene (Jones et al., 2001, Lindroth et al., 2001). Clark et al. (1995) described the presence of CpNpG methylation in mammals. Recently, data has been presented that non-CpG methylation also occurs in mouse ES cells and might be the result of Dnmt3a activity (Ramsahoye et al., 2000). Further evidence that Dnmt3a possesses an intrinsic non-CpG methylase activity comes from the introduction of mouse Dnmt3a into Drosophila where while CpG methylation predominated, CpA and CpT methylation were both also detected at levels above endogenous methylation (Ramsahoye et al., 2000, Lyko et al., 1999).

To determine whether Dnmt3 enzymes have any sequence preferences in vivo and whether non-CpG methylation occurs during de novo methylation of CpG sites by Dnmt3a and Dnmt3b, we examined de novo methylation of MMLV provirus DNA in wild-type J1 mouse ES cells, Dnmt1 null (c/c) ES cells, and Dnmt3 null (3a3b−/−) ES cells 8 days post-MMLV infection by sodium bisulfite sequencing. Our results showed that Dnmt3 enzymes are predominantly CpG methylases in vivo. In addition, Dnmt3 enzymes are also responsible for most non-CpG (especially CpA) methylation.