The tridimensional (3D) organization of mammalian genomes combines structures from different length scales. Within this organization, Topologically Associating Domains (TADs) are visible in Hi-C heat maps at the sub-megabase scale. The integrity of TADs is important for correct gene expression, but in a context-dependent and variable manner. The correct structure and function of TADs require the binding of the CTCF protein at both borders, which appears to block an active and dynamic mechanism of 'Cohesin-mediated loop extrusion'. As a result, mammalian TADs appear as so-called 'loop domains' in Hi-C data, which are the focus of this review. Moreover, we present a reanalysis of TADs from three "golden-standard" mammalian Hi-C data sets. Despite the prominent presence of TADs in Hi-C heat maps from all studies, we find consistently that regions within these domains are only moderately insulated from their surroundings. Moreover, single-cell Hi-C and superresolution microscopy have revealed that the structure of TADs and the position of their borders can vary from cell-to-cell. The function of TADs as units of gene regulation may thus require additional aspects, potentially incorporating the mechanism of loop extrusion as well. Recent developments in single-cell and multi-contact genomics and super-resolution microscopy assays will be instrumental to link TAD formation and structure to their function in transcriptional regulation.

Topologically Associating Domains (TADs) compartmentalize vertebrate genomes into sub-Megabase functional neighbourhoods for gene regulation, DNA replication, recombination and repair. TADs are formed by Cohesin-mediated loop extrusion, which compacts the DNA within the domain, followed by blocking of loop extrusion by the CTCF insulator protein at their boundaries. CTCF blocks loop extrusion in an orientation dependent manner, with both experimental and in-silico studies assuming that a single site of static CTCF binding is sufficient to create a stable TAD boundary. Here, we report that most TAD boundaries in mouse cells are modular entities where CTCF binding clusters within extended genomic intervals. Optimized ChIP-seq analysis reveals that this clustering of CTCF binding does not only occur among peaks but also frequently within those peaks. Using a newly developed multi-contact Nano-C assay, we confirm that individual CTCF binding sites additively contribute to TAD separation. This clustering of CTCF binding may counter against the dynamic DNA-binding kinetics of CTCF, which urges a re-evaluation of current models for the blocking of loop extrusion. Our work thus reveals an unanticipatedly complex code of CTCF binding at TAD boundaries that expands the regulatory potential for TAD structure and function and can help to explain how distant non-coding structural variation influences gene regulation, DNA replication, recombination and repair.