Fig. 2: Heightened presence of CpG containing Cdx2 motif at its developmental binding sites.

a Heatmap showing relative prevalence of TF motifs at developmental (Dev 1–3) and adult (Adult 1–3) cell-specific Cdx2 binding sites as determined by HOMER. b Line plot showing number of Cdx2 binding loci containing Cdx2 motifs within an increasing distance (on X-axis) from center of the Cdx2 peak. c Line plot showing abundance of canonical and CpG-containing Cdx2 motifs at developmental (shades of blue) and adult (shades of red) cell-specific Cdx2 binding sites within an increasing distance (on X-axis) from center of the Cdx2 peak. d Density plots showing relative prevalence of CpG-containing motif near the center of developmental Cdx2 peaks (Dev 1–3) in comparison with the adult cell-specific Cdx2 peaks (Adult 1–3); canonical Cdx2 motif shows widespread presence near Adult 1–3 sites in contrast with the Dev 1–3 loci. e Higher percent of regions with development-specific Cdx2 binding contain CpG-containing motif as compared to regions with adult cell-specific Cdx2 binding near the center of Cdx2 peak (within ±50 bp); beyond 50 bp of the peak center, canonical motif is more prevalent. f Heatmap showing DNA methylation at Cdx2 binding loci (as in Fig. 1a) in early endoderm (E6.5)²⁵, and embryonic (E12.5), fetal (E16.5), and adult epithelial (villus) cells¹⁵; Cdx2 binding in embryonic epithelium is associated with early loss of DNA methylation (blue dotted box), while sites first bound by Cdx2 in the fetal epithelium show higher methylation levels (red dotted box). g Fractional methylation change at CpG (4th base position) within the Cdx2 motif (For Dev1–3 and Adult 1–3, as in Fig. 1a) shows relatively early reduction in methylation at loci with developmental binding of Cdx2 (blue arrowhead) compared to binding in adult epithelium (red arrowhead) (p < 2.2e⁻¹⁶, right-sided Wilcoxon test). The CpG remains hypomethylated in adult villus at both developmental and adult loci. 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. h A model for DNA methylation-based Cdx2 binding at developmental target loci; recruitment at adult target loci is driven by DNA methylation independent binding. While relative abundance of the CpG-containing motif and methylation at the CpG within the Cdx2 motif during development allows its early recruitment to these sites, demethylation at these loci protects them from ectopic Cdx2 binding in adult cells. Source data are provided as a Source Data file.