Figure 4. Tet2 KO and Dnmt3a KO promote differential methylation of accessible transcription factor binding sites, favoring CpG rich erythroid motifs.

a) Schematic representation of modulation of transcription factor activity through mutation in Tet2 or Dnmt3a, as a function of the CpG enrichment of the binding motif. Filled circle = methylated CpGs, unfilled circles represent unmethylated CpGs. b) Fold change in transcription factor expression between Ery 1–3 and IMP 1–2 in WT (n = 7 mice) clusters. Erythroid and myelo-monocytic transcription factors with FDR < 0.05 and absolute log₂ fold change > 0.3 are highlighted in red and blue, respectively (permutation test followed by Benjamini-Hochberg (BH) correction). Inset: examples of CpG frequency per motif position are shown as grey bars. Mean CpG frequency per base for the motifs are shown as black bars. c) Mean CpG frequency per base of the DNA binding motifs of myelo-monocytic- (n = 8) and erythroid-associated (n = 11) transcription factors (two-sided Students t-test). d) Schematic representation of ATAC-Bseq experimental protocol. e) Mean CpG frequency per base for de novo discovered transcription factor binding motifs in peaks associated with erythroid (n = 20 motifs) or myelo-monocytic (n = 20 motifs) genes (two-sided Students t-test). f) Differential ATAC-Bseq accessibility between WT (n = 2 mice) and Tet2 KO (n = 2 mice) or WT and Dnmt3a KO (n = 2 mice). g) Differential methylation (FDR < 0.05 and absolute methylation difference higher than 5%) at accessible regions for Tet2 KO and Dnmt3a KO mice, as calculated with MethylKit⁶⁰ (Chi-squared with sliding linear model correction). h) Number of hyper-methylated CpGs (FDR < 0.25 and methylation difference > 5%) for Tet2 KO (n = 104,829 total CpG sites; left panel) or hypo-methylated CpGs (FDR < 0.25 and methylation difference < −5%) for Dnmt3a KO (250,353 total CpG sites; right panel) per 100 motifs in ATAC-Bseq peaks for erythroid, myelo-monocytic or other fates is shown in red, blue and grey, respectively (Pearson correlation; two-sided Students t-test; grey area represents the 95% confidence interval of the linear fit). i) Methylation values of accessible sites containing the DNA binding motif for Tal1 were divided into quartiles, and the distribution for WT (n = 1,669 motifs; n = 2 biologically independent mice), Tet2 KO (n = 880 motifs; n = 2 mice) and Dnmt3a KO (n = 1,226 motifs; n = 2 mice) is shown (two-sided Fisher exact test between first and fourth quartiles).