Fig. 7: Direct inhibition of DNA methylation causes loss of Cdx2 binding and remethylation at binding loci allows its recruitment.

a Experimental schematic showing treatment of HCT116 cells with DNA methylation inhibitor GSK-3484862 or DMSO (control) for 6 days in order to eliminate CpG methylation, followed by 3 days of recovery without treatment to allow remethylation. b Fluorescence micrographs showing loss of DNA methylation in cells upon treatment with inhibitor GSK-3484862 for 6 days, (4 independent experiments). c Representative genomic track showing fractional methylation of CpGs within ACTA1 gene body in HCT116 cells. Lollipop plots show 14 consecutive CpGs within 10 random representative amplicon sequences (from > 100× coverage on nanopore amplicon bisulfite sequencing) and their methylation status upon DMSO or GSK-3484862 treatment and 3 days of recovery; average methylation of all sequenced amplicons (>100× coverage) is represented for all treatment conditions in the right panel. d Heatmap showing loss of CDX2 binding at loci with CpG-containing CDX2 motif in HCT116 cells treated with DNA methylation inhibitor as compared to DMSO (control); upon removal of the inhibitor, CDX2 is recruited back to these loci within 3 days. e Representative genomic tracks showing loss of CDX2 binding upon elimination of DNA methylation in HCT116 cells through treatment with GSK-3484862 as opposed to the stable binding upon treatment with DMSO (control); removing the inhibitor from the cell culture media causes recovery of the CDX2 binding. Lollipop plots (right panel) represent methylation level at the CpG within CDX2 motif in HCT116 cells (WGBS, 7 reads from 2 replicates) and cells treated with DMSO or GSK-3484862 treated and after recovery (average methylation from >900 reads, nanopore amplicon bisulfite sequencing). f Box and whisker plots showing significant loss of CDX2 binding at 471 loci with CpG-containing CDX2 binding motif upon treatment with DNA methylation inhibitor and recovery of this binding upon removal of the inhibitor (right-sided Wilcoxon test; ns, p > 0.05); there is no significant change in CDX2 binding at 500 randomly chosen loci without the CpG-containing motif (g) (right-sided Wilcoxon test; ns, p > 0.05). The horizontal bars within the box plots represent medians, the tops and bottoms of the boxes represent the 75th and 25th percentiles, respectively, and the upper and lower whiskers extend to the maximum and minimum values, respectively. The outliers are indicated by dots. Source data are provided as a Source Data file.