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 hav e to be regulated accurately to ensure correct developmental and cell type-specific gene activity. Distorted DNA methylation patterns have b een associated with cell aging and diseases such as cancer and fragile X syndrome. Structural and functional in vitro studies of the mouse M Tase have indicated that the enzyme has both a regulatory and a cataly tic region located in the N-terminal and C-terminal parts of the prote in, respectively, The regulatory region includes the nuclear localizat ion signal (NLS), the sequence for DNA targeting and the Zn-binding do main. The catalytic domain carries the ten consensus sequence motifs s pecific for all known pro- and eukaryotic DNA cytosine-5-methyltransfe rases. In an attempt to separate regulatory and catalytic functions of the enzyme in vivo, we have tested various deletion mutations by mean s 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 express ion, the truncated MTase molecules exhibited neither de novo nor maint enance methylation activity. These findings might indicate that in viv o, an efficient control mechanism prevents the ectopic activity of the DNA MTase that is structurally compromised in its N-terminal regulato ry region.