Fig. 4

Deletion of Dnmt1 in adult ISCs alters crypt homeostasis and postnatal epigenetic regulation. a mRNA expression analysis confirms decreased expression of Dnmt1 in Lgr5-EGFP⁺ ISCs isolated from Dnmt1 ^ISCKO mice, in both small intestine (SI) and colon. TAM tamoxifen, WT wild type. b Immunofluorescent analysis of ISCs (EGFP, green), Dnmt1 (red), and nuclei (Topro-3, blue) in small intestine crypts. In tamoxifen-treated control (Dnmt-wild type; Lgr5-EGFP-CreER) mice (top panel), dotted lines mark crypts, and ISCs (green) show strong Dnmt1 staining (indicated by white arrowheads). In Dnmt1 ^ISCKO mice (bottom panel), the Lgr5-EGFP⁺ mutant crypt (outlined by solid line) shows decreased Dnmt1 staining and aggregation of CBCs, whereas an adjacent EGFP-negative crypt (outlined by dashed line) contains CBCs with wild-type Dnmt1 and high expression of Dnmt1 (white arrowheads). Scale bars, 20 μm. c Immunofluorescent analysis of ISCs and Dnmt1 in control (top panel) and Dnmt1 ^ISCKO (bottom panel) colons. Similar mosaic patterns of Dnmt1 deletion are observed in colon. Scale bars, 20 μm. d Immunofluorescent analysis of Paneth cells in Dnmt3a ^ISCKO and Dnmt1 ^ISCKO mice. Paneth cells staining positive for lysozyme (red) are exclusively located at crypt bottoms in Dnmt3a ^ISCKO mice (top panel), whereas lysozyme⁺ cells (arrowheads) were detected at villus epithelia in Dnmt1 ^ISCKO mice (bottom panel). Scale bars, 50 μm. Far right panels show a magnified view of Lgr5-EGFP⁺ ISCs for each genotype; the clear segregation between CBCs and Paneth cells is lost in the Dnmt1 ^ISCKO mice. DNA methylation (e) and gene expression (f) analysis of 3′ CGI-associated genes in the colonic Lgr5⁺ ISCs collected from wild-type (wt) control, Dnmt3a ^f/f; Lgr5-EGFP-CreER and Dnmt1 ^f/f; Lgr5-EGFP-CreER mice, with or without tamoxifen (TAM) administration. Error bars represent standard error of the mean of at least three replicate experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control (two-sided t test)