Fig. 3

Despite similar levels of methylation, imprints can be restored only in 3abKO cells. a Methylation level assessed by pyrosequencing for parental (WT), DNMT1 rescued (1KO + 1) and 3abKO rescued (3abKO + 3a2) cells. Compared to WT, the methylation level remains significantly lower for all gDMRs in 1KO + 1 cells. All gDMRs show significant increase in methylation in 3abKO + 3a2 cells as compared to 1KO + 1. b Global DNA methylation in WT cells, 1KO, 3abKO and 3abKO + 3a2 rescued cells estimated by LUMA. c COBRA for two representative imprinted gDMRs, H19 and Snrpn. Smaller fragments represent methylated DNA (me) and can be clearly seen in the WT and 3abKO + 3a2 lanes, but not in the 1KO + 1 lanes: un, unmethylated (D) clonal methylation analysis for Snrpn and H19 showing methylation restoration in 3abKO + 3a2 cells and no recovery for 1KO + 1 cells. e Graphical summary of clonal data in d. f RT-qPCR for H19, Igf2 and Peg1 in the different ES lines. For H19, a significant increase in expression was observed in 1KO and 3abKO cells compared to WT (p value <0.05 for all). Rescue with Dnmt3A2 (3abKO + 3a2), but not Dnmt1 (1KO + 1), caused a significant decrease in transcription again (p value <0.05 3abKO + 3a2 vs. 3abKO cells). Igf2 transcription is inversely linked to H19 [Fig. 1a(i)] and as expected decreases significantly (p value <0.001 WT vs. 1KO and 3abKO) on loss of methylation in 1KO and 3abKO lines. Of the two rescue lines, 3abKO + 3a2 shows a greater recovery of transcription, though it fails to reach statistical significance. At the Peg1 locus, there is an increase in transcription on loss of methylation (n.s.) and a significant decrease on reintroduction of DNMT3A2. Error bars indicate s.e.m in all panels: only significant changes are shown