Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo.
While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.
Liu
Kui
K
Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210, USA.
Wang
Yun Fei
YF
Cantemir
Carmen
C
Muller
Mark T
MT
D000818:Animals; D002460:Cell Line; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis* / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D029721:Drosophila Proteins; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D007700:Kinetics; D051379:Mice
C000717967:DNMT3A protein, human; C000715535:Dnmt3a protein, mouse; D029721:Drosophila Proteins; C491786:Dnmt2 protein, mouse; C491784:Mt2 protein, Drosophila; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse; C112218:TRDMT1 protein, human