De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation
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.
Section snippets
Perl script algorithms
The template MMLV sequence was obtained from GenBank, accession number AF033811. Perl script I, ‘find_c’, was employed to generate a Microsoft Excel spreadsheet with all cytosines, their genomic position in column 1, and a 0 or 1 in each of columns 2–5 corresponding to their 3′ base-A, C, G, or T. Next, Perl script II, ‘score_mec’, was used to show where cytosines from sequences previously aligned to a sodium bisulfite template were retained, (the bisulfite template was obtained by using the
5-Methylcytosine analysis of MMLV using sodium bisulfite sequencing
We previously showed that Dnmt3a and Dnmt3b were essential for de novo methylation of CpG dinucleotides in ES cells and postimplantation embryos (Okano et al., 1999). However, it has not been determined whether these enzymes specifically methylate CpG sites or they can also methylate non-CpG sites during de novo methylation in vivo. We are also curious as to whether Dnmt3 enzymes have any sequence preferences in vivo. To address these questions, we analyzed de novo methylation of MMLV provirus
Discussion
DNA methylation patterns are established by de novo methylation during embryonic development. However, how sequence specific de novo methylation is regulated remains largely unknown. In this study, we try to define the intrinsic properties of the Dnmt3 enzymes in vivo by analyzing de novo methylation of MMLV proviral DNA in wild-type, Dnmt1−/− and Dnmt3a3b−/− mutant ES cell lines using the bisulfite sequencing method. We demonstrated that Dnmt3 enzymes, but not Dnmt1, are essential for de novo
Acknowledgments
This work was supported by NIH grants (CA82389 and GM52106) to E.L. M.O. is a special fellow of the Leukemia and Lymphoma Society, and J.E.D. is supported by an institutional NRSA postdoctoral fellowship award to the Cardiovascular Research Center at Massachusetts General Hospital.
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