Supplementary Figure 9: Dnmt3a^W326R/+ mice are small with reduced brain weight, cortical surface area, and exhibit hypermethylation at DMRs/DMVs.

a, Schematic of the CRISPR–Cas9-mediated homology-directed repair strategy for generation of Dnmt3a^W326R mice. Alignment of antisense repair template (ODN) to the genomic DNA (gDNA) sequence of Dnmt3a. The targeting sequence of gRNA is shaded in yellow, the PAM site in blue. The triplet coding for tryptophan 326 (TGG) is mutated to arginine (AGA), simultaneously destroying the PAM site. b, cDNA sequencing of Dnmt3a^W326R/+ and wild-type littermate (8 week old, female) confirms that the Trp326Arg mutant allele is present and expressed in mice. Sequence electropherograms of cDNA samples with the site of mutation indicated (c.TGG>AGA, p.Trp326Arg). c, Brain weight of female Dnmt3a^W326R/+ mice compared to wild-type littermates at 8 weeks of age. n = 3, each group. d, Body weight and cortical area for 5-month-old Dnmt3a^W326R/+ mice compared to wild-type littermates. n = 7 wild-type and n = 9 Dnmt3a^W326R/+ animals. (The body weight of these animals is also plotted in Fig. 6b at a time point corresponding to 6 weeks of age.) c,d, P value, two-tailed t test. Horizontal bar, mean per group. e, Bisulfite sequencing of the Sox1 locus for cortex samples from Dnmt3a^W326R/+ and wild-type littermates (n = 3/group; female, age 8 weeks). Blocks, individual mice; blue and red squares, unmethylated and methylated CpGs, respectively; white squares, undetermined methylation status; columns, CpG sites; rows individual sequences. f–h, RRBS confirms gain of DNA methylation at NPC DMRs in adult mouse cortexes. f, Heatmap of hypermethylated DMRs identified in in vitro NPC differentiation showing gains in Dnmt3a^W326R/+ cerebral cortex. Each column in the heatmap represents an individual mouse. g, Quantification of the data shown in f. Plotted is the median percent change in methylation (∆%mCpG) relative to the mean for controls for all hypermethylated regions per sample. P value, two-tailed t test. Horizontal red bar, mean per group. n = 3, each group. h, Genome browser view of a region that gains DNA methylation in Dnmt3a^W326R/+ mouse cortex. Scale 0–100%. M1–M6, individual mice.