Fig. 6.

Function of Dnmt3L's PHD in de novo methylation at the Dnmt3L promoter in differentiating mouse embryonic stem cells. (A) Western detection of the Dnmt3L PHD mutant proteins expressed in Dnmt3L-knockout ES cells. Flag-tagged Dnmt3L wild-type and mutants with substitutions D124A, I141W and PHD deletion were introduced into Dnmt3L-knockout ES cells to generate stable cell lines. Wild-type (WT) and Dnmt3L knockout (KO) ES cell lines were used as controls. GAPDH was used for loading normalization. (B) COBRA analysis of the methylation level at the Dnmt3L promoter upon in vitro differentiation of ES cells. PCR fragments amplified from bisulfite-treated genomic DNA were digested with TaqI. The relative amount of DNA sensitive and resistant to the digestion reflects methylated (me) and unmethylated (un) states at the TaqI sites examined. (C) Bisulfite sequencing profiles of DNA methylation of the Dnmt3L promoter in differentiated ES cells. CpG sequences are shown with filled (methylated) and open (unmethylated) circles. The numbers above the profiles show the percentages of methylated CpGs. Arrows below indicate the CpG dinucleotides within the TaqI recognition sequence.