COMPLEMENTATION OF METHYLATION DEFICIENCY IN EMBRYONIC STEM-CELLS BY A DNA METHYLTRANSFERASE MINIGENE

Previous attempts to express functional DNA cytosine methyltransferase (EC 2.1.1.37) in cells transfected with the available Dnmt cDNAs have met with little or no success, We show that the published Dnmt sequen ce encodes an amino terminal-truncated protein that is tolerated only at very low levels when stably expressed in embryonic stem cells. Norm al expression levers were, however, obtained with constructs containin g a continuation of an ORF with a coding capacity of up to 171 amino a cids upstream of the previously defined start site. The protein encode d by these constructs comigrated in SDS/PAGE with the endogenous enzym e and restored methylation activity in transfected cells. This was sho wn by functional rescue of Dnmt mutant embryonic stem cells that conta in highly demethylated genomic DNA and fail to differentiate normally. When transfected with the minigene construct, the genomic DNA became remethylated and the cells regained the capacity to form teratomas tha t displayed a wide variety of differentiated tell types, Our results d efine an amino-terminal domain of the mammalian MTase that is crucial for stable expression and function in vivo.