Characterization of the human DNA methyltransferase splice variant Dnmt1b

Tissue- and gene-specific patterns of cytosine-DNA methylation are characte ristic features of vertebrate genomes, The generation and proper maintenanc e of DNA methylation patterns are essential for embryonic development, as d emonstrated by the lethal phenotypes of mice with either a targeted disrupt ion of Dnmt1, the gene responsible for the maintenance of DNA methylation, or targeted disruption of Dnmt3a or Dnmt3b, the genes involved in generatio n of newly formed methylation patterns. Recently, a novel mRNA, Dnmt1b, res ulting from alternative splicing of Dnmt1 was identified (Hsu, D. W., Lin, M. J., Lee, T. L., Wen, S. C., Chen, X., and Shen, C. K., (1999) Proc. Natl . Acad Sci. U. S. A. 96, 9751-9756). The abundance of Dnmt1b mRNA was estim ated by semiquantitative reverse transcription polymerase chain reaction an d was suggested to encode a major C-5 DNA methyltransferase isoform, Here w e report characterization of this novel DNA methyltransferase transcript, D nmt1b, and its protein product in human cell lines and in freshly isolated human peripheral blood mononuclear cells. The abundance of Dnmt1b transcrip t, as determined by quantitative RNase protection analysis, was determined to range from 6% to 25% of Dnmt1 in human cells. Second generation antisens e inhibitors targeted to the 5'- and 3'-ends of Dnmt1 inhibited the accumul ation of both Dnmt1 and Dnmt1b in cells. Dnmt1b protein purified from a bac ulovirus expression system was demonstrated to be a functional DNA methyltr ansferase, and to have Michaelis constants for both DNA and S-adenosyl-L-me thionine similar to baculovirus-expressed Dnmt1, However, antibodies raised against Dnmt1b epitopes demonstrated that Dnmt1b protein was present at ap proximately 2-5% of the level of Dnmt1 and therefore represents only a mino r DNA methyltransferase isoform in human cells.