CTCF modulates allele-specific sub-TAD organization and imprinted gene activity at the mouse Dlk1-Dio3 and Igf2-H19 domains

BACKGROUND: Genomic imprinting is essential for mammalian development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. So far, the detailed allele-specific chromatin organization of imprinted gene domains has mostly been lacking. Here, we explored the chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints—the Igf2-H19 and Dlk1-Dio3 domains—and assessed the involvement of the insulator protein CTCF in mouse cells. RESULTS: Both imprinted domains are located within overarching topologically associating domains (TADs) that are similar on both parental chromosomes. At each domain, a single differentially methylated region is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq and DNA-FISH revealed that bi-allelic CTCF binding alone, on the paternal chromosome, correlates with a first level of sub-TAD structure. On the maternal chromosome, additional CTCF binding at the differentially methylated region adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organization. Perturbation of maternal-specific CTCF binding site at the Dlk1-Dio3 locus, using genome editing, results in perturbed sub-TAD organization and bi-allelic Dlk1 activation during differentiation. CONCLUSIONS: Maternal allele-specific CTCF binding at the imprinted Igf2-H19 and the Dlk1-Dio3 domains adds an additional layer of sub-TAD organization, on top of an existing three-dimensional configuration and prior to imprinted activation of protein-coding genes. We speculate that this allele-specific sub-TAD organization provides an instructive or permissive context for imprinted gene activation during development.