DNA (de)methylation in embryonic stem cells controls CTCF-dependent chromatin boundaries

Coordinated changes of DNA (de)methylation, nucleosome positioning and chromatin binding of the architectural protein CTCF play an important role for establishing cell type specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the TET1 and TET2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC) or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation and gene expression in DKO ESCs, and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, while unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild type cells but not from 5fC enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, e.g. at promoters, boundaries of chromatin loops and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to downregulation of neighbouring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions and DNA sequence that determines differential CTCF binding and regulates gene expression.