Fig. 4: The BAH1–H4K20me3 binding regulates DNMT1-mediated methylation at the H4K20me3-enriched genomic regions.

a Immunoblots of the indicated Flag-tagged DNMT1 after its stable reconstitution into 1KO-ESCs among the independently derived cell lines. The experiment was repeated three times with consistent results. b CoIP (right panels) detecting association of the indicated Flag-tagged DNMT1 with H4K20me3 or histone H3. Left panels are immunoblots of input. The experiment was repeated three times with consistent results. c Representative confocal immunofluorescence images revealing localization of the indicated DNMT1 (Flag-tagged, red), H4K20me3-dense loci (green), and chromatin (stained by DAPI, blue) in the 1KO-ESC stable expression lines, synchronized in S phase. Scale bar, 5 µm. The experiment was repeated three times with consistent results. d, e Box plots showing the levels of H4K20me3 (d) or H3K9me3 (e) in ESCs, genome-wide (left) or within DMRs found associated with the W796A mutation of DNMT1 relative to WT, either hypo (middle) or hyper-methylated (right) (for panels d, e, n = 422 for Hypo, 377 for Hyper and 422 for randomized genome regions as a control). DMR calling is performed using DSS. Box-and whisker plots depict 25–75% in the box, whiskers are 10–90%, and median is indicated. Statistical analysis used two-tailed Student’s t test. f Pie chart showing distribution of the DMRs associated with the W796A-mutated DNMT1 relative to WT, either hypo (left) or hyper-methylated (right), among the indicated genomic feature. DMR calling is performed using DSS. g, h Representative IGV views show CpG methylation levels at the called W796A-asociated hypo-DMRs (green bar), either located in the chromosome 3 (g) or 13 (h), among three replicated 1KO-ESC lines with stable expression of WT (black) or the BAH1-mutated (blue) DNMT1. Cytosines covered by at least five reads according to eRRBS data are shown, with each site designated by a vertical line. The bottom panels show IGV view of H4K20me3 (purple) and H3K9me3 (red) in WT ESCs, and position of LINE-1. i Bisulfite sequencing analysis of CpG methylation at the regulatory region of L1 in cells transfected with DNMT1^W796A or DNMT1^WT. j Immunoblotting of H4K20 methylation (H4K20me1/2/3; with H3 as control) in E14 ESCs treated with 10 µM A-196, vs. DMSO, for three days. The experiment was repeated twice with consistent results. k Site-targeted bisulfite sequencing results (bottom) revealed hypomethylation of a LINE-1 element (L1Md_T) in E14 ESCs treated with A-196 vs. DMSO. Top panel shows an IGV view of eRRBS data at this L1Md_T site, with a red box marking the CpG-containing region examined by site-targeted bisulfite sequencing. Source data are provided as a Source Data file.