Fig 2: Dnmt3b represses Wnt9b and Shh in vivo and in vitro. a Methylation of Wnt9b locus in E11.5 embryos of indicated genotypes determined by RRBS. b Wnt9b expression by RNA-seq (n = 3; *p < 0.05 by DESeq; left) and real-time qRT-PCR (n = 3; normalized to Gapdh; right) in E11.5 embryos. Horizontal line represents median, bounds of box—likely range of variation and whiskers—min and max values. c Immunoblot of Wnt9b and Hsc70 levels in E11.5 embryos. d Wnt9b expression by real-time qRT-PCR in fetal brain of E11.5 embryos (n = 3), normalized to Gapdh. e GSEA using RNA-seq data shows positive enrichment in Beta-catenin–TCF complex assembly in E11.5 Dnmt3b-/- embryos (n = 3). Normalized enrichment scores (NES), false discovery rate (FDR) and p values are shown. f Left. Fgf8 expression by RNA-seq in Dnmt3b+/+, Dnmt3b-/-, and Dnmt3bCI/CI E11.5 embryos (n = 3), *p < 0.05 (DESeq). Horizontal line represents median, bounds of box—likely range of variation and whiskers—min and max values. Right. Immunoblot analysis of Fgf8 levels in E11.5 embryos. g GSEA shows negative enrichment in developmental pathways in E11.5 Dnmt3b-/- embryos relative to Dnmt3b+/+ (n = 3). h Dnmt3b and Hsc70 expression in mouse Dnmt3b-/- T cell lymphoma cells (-/-) 72 h after transduction with lentiviruses expressing control vector (empty), Dnmt3bWT (3b WT), Dnmt3bCI (3b CI) as analyzed by immunoblot. i Real-time qRT-PCR analysis of Wnt9b and Fgf8 expression in samples prepared as in h. Average of two independent experiments normalized to ß-actin. j Left: Schematic of mouse Wnt9b promoter with position of primers (arrows): P1: -763/-582 bp, P2: -603/-411 bp; P3: -342/-127 bp; P4: -143/ + 36 bp relative to TSS. Right: Real-time qRT-PCR on DNA immunoprecipitated with anti-FLAG antibody from Dnmt3b-/- lymphoma cells expressing FLAG tagged Dnmt3bWT or empty vector. Data are shown as averaged fold enrichment over empty vector (n = 2), *p < 0.001 (two-tailed Student’s t-test). Assay was performed in triplicates and normalized to the input DNA. k Shh expression by real-time qRT-PCR in samples described in h. Averaged data of two independent experiments were normalized to ß-actin, *p < 0.001 (two-tailed Student’s t-test). Data in figures b, d, i, j, and k are presented as means ± SEM. Source data are provided as a Source Data file

Fig 3: Dnmt3b’s CA is important in the prevention of hematologic malignancies.A, Disease spectrum of Dnmt3b+/+ (+/+; n = 30) Dnmt3b+/CI (+/CI; n = 67), and Dnmt3bCI/CI (CI/CI; n = 32) mice as determined by FACS. B, Representative image of the enlarged spleen of terminally ill Dnmt3b+/CI (+/CI) mouse that developed CLL and healthy spleen of age-matched Dnmt3b+/+ (+/+). C, Representative FACS diagram of CD5 and CD19 expression in cells isolated from the spleen of healthy Dnmt3b+/+ mice (+/+) and terminally sick Dnmt3b+/CI (+/CI) and Dnmt3bCI/CI (CI/CI) mice that developed MBL as analyzed by FACS. Quadrant statistics are indicated in red here and throughout whole figure. D, CD5, CD19, and B220 expression in cells isolated from the spleen of healthy Dnmt3b+/+ mice (+/+) and terminally sick Dnmt3b+/CI (+/CI) mice that developed CLL as analyzed by FACS. E, Time to tumor development for primary mice (primary CLL), primary (first), secondary (second), and tertiary (third) sublethally irradiated FVB-recipient mice serially transplanted with cells isolated from the spleen of terminally sick Dnmt3b+/CI mouse. Data are presented as mean ± SEM. F, CD5 and CD19 expression in cells isolated from the spleen of healthy Dnmt3b+/+ mice (+/+) and terminally sick Dnmt3b+/CI (+/CI) mice that developed BCL as analyzed by FACS. Expression of IgM and IgD on CD19+CD5-cells is shown on the bottom panel.

Fig 4: Methylation changes at E11.5 are partially mediated by Dnmt3b. a Pairwise comparison of CG methylation in Dnmt3b+/+ embryos at E8.5 and E10.5; E10.5 and E11.5 and Dnmt3b-/- embryos at E8.5 and E11.5. The density of points increases from green to red. R-values represent Pearson correlation coefficients. b Total number of differentially methylated CGs in Dnmt3b+/+ embryos at E11.5 relative to E10.5 (meth diff. =30%). c Violin plots showing CG methylation in Dnmt3b+/+ embryos at E8.5, E10.5, and E11.5, Dnmt3b-/- embryos at E8.5 and E11.5, and Dnmt3bCI/CI embryos at E11.5 in indicated genomic elements. d Visualization of single-CpG profiles at de novo methylated locus of Ppp1r2 that remains hypomethylated in Dnmt3b-/- and Dnmt3b CI/CI embryos at E11.5. Visualization of single-CpG profiles at Evc2 locus that maintains methylation across E8.5–E11.5 in Dnmt3b+/+ and loses methylation in Dnmt3b-/-. e The total number of hypermethylated (de novo) and hypomethylated (lost) DMRs identified in Dnmt3b+/+ embryos at E11.5 relative to E10.5 (n = 2 for each timepoint) (methylation diff. = 30%; p(MWU) <0.05). Out of de novo methylated DMRs, those which are hypomethylated in Dnmt3b-/- and Dnmt3bCI/CI relative to Dnmt3b+/+ embryos at E11.5 are marked yellow and green, respectively. f Total number of hypermethylated (de novo) DMRs identified in Dnmt3b+/+ embryos at E11.5 relative to E10.5 within indicated genomic elements (left panel). Out of these, DMRs which are hypomethylated in Dnmt3b-/- and Dnmt3b CI/CI embryos relative to Dnmt3b+/+ at E11.5 are marked red and green, respectively (right panel). g Heat map of genes de novo methylated within promoter or enhancers and downregulated (FC = 2; p < 0.05 by DESeq) in Dnmt3b+/+ embryos at E11.5 relative to E10.5. Source data are provided as a Source Data file

Fig 5: Dnmt3b controls developmental pathways involved in embryogenesis. a Ingenuity pathway analysis (IPA) of genes differentially expressed (FC = 2, p < 0.05) in E11.5 Dnmt3b-/- embryo. The top subcategories obtained in a category Physiological system development and function are displayed (p < 0.05 by right-tailed Fisher’s exact test). b Panther pathway analysis of upregulated genes (FC = 2, p < 0.05 by DESeq) identified in E11.5 Dnmt3b-/- embryo, which were found to be enriched in IPA analysis in a subcategory Embryonic development. c Gene ontology analysis of up-regulated genes (FC = 2, p < 0.05 by DESeq) identified in E11.5 Dnmt3b-/- embryo, which were found to be enriched in IPA analysis in a subcategory Embryonic development. Source data are provided as a Source Data file