Fig. 5: Dnmt3L acetylation regulates naive pluripotency and germline gene expression via DNA methylation.

a Western blot analysis of DNA methylation-related enzymes and pluripotency factors in long-term cultured mouse ES cells stably expressing nontagged Dnmt3L WT, K238R or K412R proteins. β-actin was used as a loading control. b Heat map depicting gene expression profiles of DNA methylation regulators, naive pluripotency genes, germline markers and imprinted genes in the indicated stable mouse ES cell lines. Expression values represent the mean of three independent biological replicates. c RT–qPCR analysis of representative genes for naive pluripotency (Tfcp2l1), germline (Stra8), neural (Foxa1) and cardiac (Cer1) lineages, and genomic imprinting (Snrpn) in cells at the indicated passages (n = 3). Expression data for additional genes are shown in Supplementary Fig. 8d. d MeDIP–qPCR analysis of 5mC enrichment at the selected genomic loci in stable mouse ES cell lines expressing nontagged WT Dnmt3L or mutants (n = 4). e Bisulfite sequencing analysis of the indicated target regions. Methylated and unmethylated CpG sites are shown as filled and open circles, respectively. The percentage of methylated CpG is indicated below each bisulfite sequencing profile (n = 3). f Schematic representation of the murine Stra8 locus, showing the H3K27me3 histone modification mark and Dnmt3A2-binding sites based on published ChIP–seq datasets^26,39. Genomic features include CpG islands (green bar) and locations of primers used for MeDIP– and ChIP–qPCR assays in this study (red bar). Additional locus maps are shown in Supplementary Fig. 9. g ChIP–qPCR analysis of Flag–Dnmt3L WT, K238R and K412R proteins at the regulatory regions of the target genes affected by Dnmt3L K238Ac or K412Ac (n = 4). All quantitative data are presented as mean ± s.e.m. Statistical significance was assessed by a one-way ANOVA with the Bonferroni post hoc test; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 compared with control groups. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with WT Dnmt3L. The exact P values and replicate numbers are provided in the Source Data. See also Supplementary Figs. 8 and 9. ET empty vector, B Bound, UB Unbound.