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. Author manuscript; available in PMC: 2020 Dec 8.
Published in final edited form as: Nat Genet. 2020 Jun 8;52(8):819–827. doi: 10.1038/s41588-020-0639-9

Figure 2: Re-expression of TET enzymes demethylated PKO cDKO-DMRs.

Figure 2:

a) TET1s, TET2 and TET3 were reintroduced into PKO cells using PiggyBac transposition. Control transfection was performed with only the transposase (PBase). A schematic diagram depicts the experimental design with the constructs shown below, w = weeks, Ins = insulator sequence.

b) WGBS-based mean methylation barplots for matched CpGs. P20 = passage 20.

c) Genome browser tracks across a 39-kb region of the LRP1 locus for HUES8 WT and seven of the modified HUES8 lines. Re-expression of TET1s, TET2 or TET3 in PKO cells causes global and specific loss of methylation at cDKO-DMRs (blue boxes).

d) Violin plots displaying methylation levels for CpGs within cDKO-DMRs (n = 176,669, 186,438, 155,912, 180,233, 128,709 CpGs respectively). Violin plots extend from the data minima to the data maxima with the white dot indicating median, thick bar showing the interquartile range and thin bar showing 1.5× interquartile range.

e) Composite plot of mean methylation levels for each class of cDKO-DMR, including 5 kb on either side for PKO cells at P20, control cells as well as the three TET rescue lines.

f) Hydroxymethylcytosine (5hmC) levels in WT HUES64 cells shown as distance from class 1 or class 2 cDKO-DMR center, CGIs or active ESC (H1) enhancers (enh).