DNMT3L (DNA methyltransferase 3-like) that is unable to bind unmethylated H3K4 is deficient in rescue of the de novo methylation defect in Dnmt3L-/- embryonic stem (ES) cells. The D90A and I107W substitutions were previously shown to prevent binding of human DNMT3L to histone H3 peptide unmethylated at lysine position 4 (H3K4) . The equivalent residues in mouse DNMT3L are D124A and I141W, respectively. (a) The mutant proteins were stable and were expressed at higher levels than the wild-type protein; two independent stable transfectants are shown for each. (b) Failure of mutant DNMT3L to rescue the de novo methylation defect. The long terminal repeat (LTR) of the retrovirus shown in Figure 1(a) was tested for de novo methylation at 12 days post-infection. These data demonstrate that de novo methylation in ES cells requires the interaction of DNMT3L and unmethylated H3K4.