DNMT2 IS NOT REQUIRED FOR DE-NOVO AND MAINTENANCE METHYLATION OF VIRAL-DNA IN EMBRYONIC STEM-CELLS

We have shown previously that de novo methylation activities persist i n mouse embryonic stem (ES) cells homozygous for a null mutation of Dn mt1 that encodes the major DNA cytosine methyltransferase. In this stu dy, we have cloned a putative mammalian DNA methyltransferase gene, te rmed Dnmt2, that is homologous to pmt1 of fission yeast. Different fro m 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. Howeve r, baculovirus-expressed Dnmt2 protein failed to methylate DNA in vitr o. To investigate whether Dnmt2 functions as a DNA methyltransferase i n vivo, we inactivated the Dnmt2 gene by targeted deletion of the puta tive 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 mut ant ES cells as efficiently as in wild-type cells. These results indic ate that Dnmt2 is not essential for global de novo or maintenance meth ylation of DNA in ES cells.