Mouse DNA methyltransferase (MTase) deletion mutants that retain the catalytic domain display neither de novo nor maintenance methylation activity in vivo.

The mammalian genome encodes a DNA cytosine-5-methyltransferase (MTase) of about 170 kDa that is apparently responsible for both de novo and maintenance methylation at CpG sites. Both methylation activities have to be regulated accurately to ensure correct developmental and cell type-specific gene activity. Distorted DNA methylation patterns have been associated with cell aging and diseases such as cancer and fragile X syndrome. Structural and functional in vitro studies of the mouse MTase have indicated that the enzyme has both a regulatory and a catalytic region located in the N-terminal and C-terminal parts of the protein, respectively. The regulatory region includes the nuclear localization signal (NLS), the sequence for DNA targeting and the Zn-binding domain. The catalytic domain carries the ten consensus sequence motifs specific for all known pro- and eukaryotic DNA cytosine-5-methyltransferases. In an attempt to separate regulatory and catalytic functions of the enzyme in vivo, we have tested various deletion mutations by means of transient and stable cell transfection experiments. Expression of the transgenes, all of which retained the C-terminal catalytic domain, was monitored by immunofluorescence staining, Northern blot analysis and SDS gel electrophoresis. Despite high levels of transgene expression, the truncated MTase molecules exhibited neither de novo nor maintenance methylation activity. These findings might indicate that in vivo, an efficient control mechanism prevents the ectopic activity of the DNA MTase that is structurally compromised in its N-terminal regulatory region.