Supplementary Figure 3: Substrate/enzyme ratio may affect post-replication dynamics.

(a) Genome browser tracks for bulk wild-type (WT) and double knock out (DKO; DNMT3A^−/− and DNMT3B^−/−) human ESCs for a 165 kb region on chromosome 17. Globally, DKO cells are hypomethylated in comparison to WT (mean methylation shown on top left). Only CpGs with ≥10X coverage are displayed. (b) Mean methylation levels for WT and DKO nascent and bulk DNA is shown. Samples were pulsed with BrdU for 1 h then chased for variable lengths of time (x-axis). For WT, n = 24.5 M, 8.9 M, 7.6 M; for DKO, n = 2.5 M, 459 K, 511 K for chase times of 0, 1 and 4 h respectively. Bulk levels for WT and DKO cells are displayed as dashed lines. DKO cells show a higher proportion of methylated CpGs for nascent DNA with respect to bulk and reach their respective bulk methylation level more quickly than WT cells. (c) Histograms display the distribution of CpG methylation for bulk (top) and nascent (bottom) WT and DKO DNA at 0 h chase time (n = 2,422,761 matched CpGs). After a 1 h BrdU pulse, the majority of nascent CpGs in WT cells display methylation levels of around 0.7 while DKO cells display a more bimodal distribution. This suggests that, with less CpGs to methylate, DKO cells are able to fully methylate a greater number of CpGs within the same time frame. (d) The proportion of cells in different cell cycle phases is displayed for WT and DKO cells based on single cell RNA-seq data (see Methods). Also note that expression of DNMT1 in both cell lines is similar, as shown in Liao, J. et al. Nat Genet 47, 469–478, 2015.