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

Manuscript number: GBIO-D-19-00822R1 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. Results Both imprinted domains are located within overarching TADs (‘Topologically Associating Domains’) that are similar on both parental chromosomes. At each domain, a single differentially-methylated region (DMR) is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq (Circular Chromosome Conformation Capture) 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 DMR adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organisation. Perturbation of maternal-specific CTCF binding site at the Dlk1-Dio3 locus, using genome-editing, results in perturbed sub-TAD organisation and biallelic 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 organisation, on top of an existing 3D 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.