Figure 4: DNMT3A mutants have significant changes to DNA methylation, with more extreme changes in the R878H mutant compared to the P900L.

(A-B) Average genome-wide methylation levels from brain regions measured using whole genome bisulfite sequencing for (A) percent mCA and (B) percent mCG for both P900L and R878H mutants and their WT littermates. (All groups n=4, 2 male, 2 female; Unpaired Student’s T-Test with Bonferroni Correction)

(C-D) Representative genome browser view showing percent mCA and mCG (C). (D) Zoomed in browser to show changes in CG at hypo-differentially methylated regions (DMRs).

(E) Heatmap of CG-DMRs identified in P900L and R878H mutants vs. their WT littermates. Log₂(Fold change mCG/CG) indicated between each littermate pair for each DMR.

(F) Average mCG in each genotype at DMRs called in both mutant strains. Both mutants show a consistent decrease at hypo-DMRs called in either mutant. Hyper-DMRs are only significant in the strain they were defined in.

(G) Overlap analysis of DMRs with genomic regions of interest. Adult DMRs are the regions that significantly gain mCG over postnatal development in neurons (Lister et al. 2013). No point indicated for R878H Hyper-DMRs CpG islands, due to 0 resampled DMRs overlapping. Significance assessed with a Chi-Squared test with expected proportions of overlapping and nonoverlapping measured by resampling DMRs.

(H) Average mCA level at regions of interest (top) and percent reduction of mCA between WT and mutants (bottom).

(I) Average mCG level at regions of interest (top) and percent reduction of mCG between WT and mutants (bottom). Promoters and CpG islands have low levels of mCG, and a trending, but not significant difference between P900L and R878H loss.

Bar graphs indicate mean ± SEM. Notched box and whisker plots indicate median, interquartile, and confidence interval of median. All groups n=4, 2 male, 2 female. Detailed statistics, and sample sizes in Supplemental Table 1. * p<0.05; ** p<0.01; *** p<0.001; # p<0.0001; ## p<2 e⁻¹⁰