Motif distribution and DNA methylation underlie distinct Cdx2 binding during development and homeostasis

Precise spatiotemporal and cell type-specific gene expression is essential for proper tissue development and function. Transcription factors (TFs) guide this process by binding to developmental stage-specific targets and establishing an appropriate enhancer landscape. DNA and chromatin modifications form barriers to the genomic binding of TFs, however, the details of how TFs navigate various chromatin features and selectively bind a small portion of the millions of possible genomic target loci remain unknown. Here we show that Cdx2, a pioneer TF that binds distinct targets in developing and adult intestinal epithelial cells, has a preferential affinity to a non-canonical CpG-containing motif in vivo. A higher frequency of this motif at the embryonic and fetal target loci of Cdx2 and the methylated state of the CpG particularly during development allows its selective binding and activation of developmental enhancers and linked genes. Conversely, demethylation at these enhancers prohibits ectopic Cdx2 binding in adult cells, where Cdx2 binds the canonical motif without a CpG. This distinct Cdx2 binding allows corecruitment of Ctcf and Hnf4 facilitating the establishment of intestinal superenhancers during development and enhancers activating adult tissue functions, respectively. Induced gain of DNA methylation in the adult epithelium in vivo or in cultured cells causes recruitment of Cdx2 to the developmental target loci and cobinding of the partner TFs. Our results demonstrate that the distinct propensities of Cdx2 to bind motifs with and without CpGs and the divergent distribution of these motifs at developmental and adult target sites allow Cdx2 to navigate distinct DNA methylation profiles and activate cell type-specific enhancers and genes.