Extended Data Fig. 10: DNA demethylation is limiting for K36M-dependent enhancer rewiring.

(a) Distribution of DNA methylation (WGBS, n = 2 biologically independent replicates were integrated for this analysis) at differentially active enhancers in H3K36me2 domains (n = 4,939) in day-4 reprogramming intermediates of WT, K36M and K36M + DMOG samples. (b) Distribution of DNA methylation (WGBS, n = 2 biologically independent replicates were integrated for this analysis) at ectopic (n = 45,095) and ESC-exclusive (n = 27,708) Sox2 binding sites in day-4 reprogramming intermediates of WT, K36M and K36M + DMOG samples. (c) Representative histogram plots from flow cytometric analysis for Epcam of K36M cells with knockdown of the indicated Tet demethylases. (d) Fraction of Epcam⁺ cells in K36M cells with Tet knockdown on day 4 of reprogramming, error bars indicate mean ± SD (n = 3 biologically independent experiments). (e) qPCR of miRNAs miR-200b-3p, miR-205-5p, and miR-290a-5p in untreated (K36M Ctrl) and DMOG-treated K36M cells (K36M DMOG). P values were determined by unpaired Student’s t test, error bars indicate mean ± SD (n = 3 independent biological experiments). (f) Fraction of Epcam⁺ cells in K36M cultures transduced with either an empty vector or dox-inducible overexpression vectors for Dnmt3a and Dnmt3b, error bars indicate mean ± SD (n = 3 independent biological experiments). (g) Bisulfite-seq of a Cdh1 enhancer in K36M cells transduced with either empty vector (left), or overexpression of Dnmt3a (middle) or Dnmt3b (right). (h) H3K36me2 levels within H3K36me2 domains (n = 7,610) on day 4 of reprogramming in WT, untreated K36M cells (K36M Ctrl), and DMOG-treated K36M cells (K36M DMOG). Crossbars indicate median (n = 2 biologically independent replicates were integrated for this analysis). (i-k) Representative gene tracks of Krt8, Pou5f1, and the miR-290 cluster (n = 2 biologically independent replicates). Putative regulatory elements highlighted in grey.

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