Extended Data Fig. 2: Characterization of DKO-DMRs and TET rescue in PKO cells.

a, Bar chart showing the proportion of CpGs with varying methylation loss in DKO cells compared to WT. CpGs that lost >60% methylation were generally located in closer proximity to one another compared to CpGs that lost 10–20% methylation.b, The proportion of cDKO-DMRs with respective mean methylation levels in single DNMT3A^−/− (3AKO) or DNMT3B^−/− (3BKO) knockout ESCs. c, Composite plot showing methylation difference between passage (P) 20 and 6 in PKO cells as distance from the cDKO-DMR center. There is a small background loss (−0.045) with a slightly greater focal decrease (−0.07), but methylation levels stay generally high. d, The methylation level in HUES64 DKO-A, HUES64 WT and HUES8 TKO cells including 2 kb from each boundary. For regions where the neighboring 2 kb is hypomethylated in WT cells (class 1), almost every region shows an increase in methylation following TET loss. e, Violin plots showing methylation at TKO-DMRs across different HUES8 lines. We used stringent parameters to define TKO-DMRs that aberrantly gain methylation upon loss of TET expression (also described later in Extended Data Fig. 9a). Methylation levels were rescued by re-expression of TET. Violin plots extend from the data minima to the maxima, white dot indicates median, thick bar shows the interquartile range and thin bar shows 1.5x interquartile range. f, Methylation levels across 1 kb tiles for PKO ESCs rescued with either TET1s or TET2. Intensity of blue shading indicates the relative density of data points. Pearson correlation coefficient (cor) is displayed. g, ChIP-seq (fragments per bp, per peak) enrichment (from ref. ²¹) for TET1 and DNMT3B over CpG islands (CGI), H1-specific enhancers and cDKO-DMRs. h, Representative genome browser tracks displaying methylation levels in WT and DKO cells as well as 5hmC levels in WT ESCs. Increased 5hmC is observed over the cDKO-DMR and at the border of the hypomethylated CGI, where TETs are known to localize.