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. 1998 Jun 1;26(11):2536–2540. doi: 10.1093/nar/26.11.2536

Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells.

M Okano 1, S Xie 1, E Li 1
PMCID: PMC147598  PMID: 9592134

Abstract

We have shown previously that de novo methylation activities persist in mouse embryonic stem (ES) cells homozygous for a null mutation of Dnmt1 that encodes the major DNA cytosine methyltransferase. In this study, we have cloned a putative mammalian DNA methyltransferase gene, termed Dnmt2 , that is homologous to pmt1 of fission yeast. Different from pmt1 in which the catalytic Pro-Pro-Cys (PPC) motif is 'mutated' to Pro-Ser-Cys, Dnmt2 contains all the conserved methyltransferase motifs, thus likely encoding a functional cytosine methyltransferase. However, baculovirus-expressed Dnmt2 protein failed to methylate DNA in vitro . To investigate whether Dnmt2 functions as a DNA methyltransferase in vivo , we inactivated the Dnmt2 gene by targeted deletion of the putative catalytic PPC motif in ES cells. We showed that endogenous virus was fully methylated in Dnmt2 -deficient mutant ES cells. Furthermore, newly integrated retrovirus DNA was methylated de novo in infected mutant ES cells as efficiently as in wild-type cells. These results indicate that Dnmt2 is not essential for global de novo or maintenance methylation of DNA in ES cells.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Baylin S. B., Herman J. G., Graff J. R., Vertino P. M., Issa J. P. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res. 1998;72:141–196. [PubMed] [Google Scholar]
  3. Bestor T. H. Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. EMBO J. 1992 Jul;11(7):2611–2617. doi: 10.1002/j.1460-2075.1992.tb05326.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Jones P. A., Gonzalgo M. L. Altered DNA methylation and genome instability: a new pathway to cancer? Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2103–2105. doi: 10.1073/pnas.94.6.2103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Klimasauskas S., Roberts R. J. M.HhaI binds tightly to substrates containing mismatches at the target base. Nucleic Acids Res. 1995 Apr 25;23(8):1388–1395. doi: 10.1093/nar/23.8.1388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kumar S., Cheng X., Klimasauskas S., Mi S., Posfai J., Roberts R. J., Wilson G. G. The DNA (cytosine-5) methyltransferases. Nucleic Acids Res. 1994 Jan 11;22(1):1–10. doi: 10.1093/nar/22.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laird P. W., Jaenisch R. The role of DNA methylation in cancer genetic and epigenetics. Annu Rev Genet. 1996;30:441–464. doi: 10.1146/annurev.genet.30.1.441. [DOI] [PubMed] [Google Scholar]
  9. Lei H., Oh S. P., Okano M., Jüttermann R., Goss K. A., Jaenisch R., Li E. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development. 1996 Oct;122(10):3195–3205. doi: 10.1242/dev.122.10.3195. [DOI] [PubMed] [Google Scholar]
  10. Li E., Beard C., Jaenisch R. Role for DNA methylation in genomic imprinting. Nature. 1993 Nov 25;366(6453):362–365. doi: 10.1038/366362a0. [DOI] [PubMed] [Google Scholar]
  11. Li E., Bestor T. H., Jaenisch R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell. 1992 Jun 12;69(6):915–926. doi: 10.1016/0092-8674(92)90611-f. [DOI] [PubMed] [Google Scholar]
  12. Malagnac F., Wendel B., Goyon C., Faugeron G., Zickler D., Rossignol J. L., Noyer-Weidner M., Vollmayr P., Trautner T. A., Walter J. A gene essential for de novo methylation and development in Ascobolus reveals a novel type of eukaryotic DNA methyltransferase structure. Cell. 1997 Oct 17;91(2):281–290. doi: 10.1016/s0092-8674(00)80410-9. [DOI] [PubMed] [Google Scholar]
  13. Monk M. Epigenetic programming of differential gene expression in development and evolution. Dev Genet. 1995;17(3):188–197. doi: 10.1002/dvg.1020170303. [DOI] [PubMed] [Google Scholar]
  14. Mortensen R. M., Conner D. A., Chao S., Geisterfer-Lowrance A. A., Seidman J. G. Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol. 1992 May;12(5):2391–2395. doi: 10.1128/mcb.12.5.2391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mountford P., Zevnik B., Düwel A., Nichols J., Li M., Dani C., Robertson M., Chambers I., Smith A. Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4303–4307. doi: 10.1073/pnas.91.10.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Neumann B., Barlow D. P. Multiple roles for DNA methylation in gametic imprinting. Curr Opin Genet Dev. 1996 Apr;6(2):159–163. doi: 10.1016/s0959-437x(96)80045-1. [DOI] [PubMed] [Google Scholar]
  17. Pinarbasi E., Elliott J., Hornby D. P. Activation of a yeast pseudo DNA methyltransferase by deletion of a single amino acid. J Mol Biol. 1996 Apr 12;257(4):804–813. doi: 10.1006/jmbi.1996.0203. [DOI] [PubMed] [Google Scholar]
  18. Renbaum P., Razin A. Interaction of M.SssI and M.HhaI with single-base mismatched oligodeoxynucleotide duplexes. Gene. 1995 May 19;157(1-2):177–179. doi: 10.1016/0378-1119(95)00111-i. [DOI] [PubMed] [Google Scholar]
  19. Wilkinson C. R., Bartlett R., Nurse P., Bird A. P. The fission yeast gene pmt1+ encodes a DNA methyltransferase homologue. Nucleic Acids Res. 1995 Jan 25;23(2):203–210. doi: 10.1093/nar/23.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Yang A. S., Shen J. C., Zingg J. M., Mi S., Jones P. A. HhaI and HpaII DNA methyltransferases bind DNA mismatches, methylate uracil and block DNA repair. Nucleic Acids Res. 1995 Apr 25;23(8):1380–1387. doi: 10.1093/nar/23.8.1380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yoder J. A., Bestor T. H. A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast. Hum Mol Genet. 1998 Feb;7(2):279–284. doi: 10.1093/hmg/7.2.279. [DOI] [PubMed] [Google Scholar]

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