De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation.
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 Moloney 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 approximately 1.4% of all sites in J1 and approximately 1.0% in c/c, but only approximately 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.
Dodge
Jonathan E
JE
Department of Medicine, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Charlestown, MA 02129, USA.
Ramsahoye
Bernard H
BH
Wo
Z Galen
ZG
Okano
Masaki
M
Li
En
E
D044503:5-Methylcytosine; D000818:Animals; D018899:CpG Islands / Q000235:genetics; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009154:Mutation; D011533:Proviruses / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D000097584:DNA Methyltransferase 3B
D044503:5-Methylcytosine; D003596:Cytosine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D004279:DNA, Viral; D000097584:DNA Methyltransferase 3B; C000715535:Dnmt3a protein, mouse; C000619381:Dnmt1 protein, mouse