DE-NOVO DNA CYTOSINE METHYLTRANSFERASE ACTIVITIES IN MOUSE EMBRYONIC STEM-CELLS

It has been a controversial issue as to how many DNA cytosine methyltr ansferase mammalian cells have and whether de novo methylation and mai ntenance methylation activities are encoded by a single gene or two di fferent genes, To address these questions, we have generated a null mu tation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-s omite stage, Surprisingly, the null mutant embryonic stem cells are vi able and contain low but stable levels of methyl cytosine and methyltr ansferase activity, suggesting the existence of a second DNA methyltra nsferase in mammalian cells, Further studies indicate that de novo met hylation activity is not impaired by the mutation as integrated provir us DNA in MoMuLV-infected homozygous embryonic stem cells become methy lated at a similar rate as in wild-type cells. Differentiation of muta nt cells results in further reduction of methyl cytosine levels, consi stent with the de novo methylation activity being down regulated in di fferentiated cells, These results provide the first evidence that an i ndependently encoded DNA methyltransferase is present in mammalian cel ls which is capable of de novo methylating cellular and viral DNA in v ivo.