Insulation between adjacent TADs is controlled by the width of their boundaries through distinct mechanisms

Abstract Topologically Associating Domains (TADs) are sub-Megabase regions in vertebrate genomes with enriched intra-domain interactions that restrict enhancer-promoter contacts across their boundaries. However, the mechanisms that separate TADs remain incompletely understood. Most boundaries between TADs contain CTCF binding sites (CBSs), which individually contribute to the blocking of Cohesin-mediated loop extrusion. Using genome-wide classification, we show here that TAD boundary width forms a continuum from narrow to highly extended and correlates with CBS distribution, chromatin features, and gene regulatory elements. To investigate how these boundary widths emerge, we modified the Random Cross-Linker (RCL) polymer model to incorporate specific boundary configurations, enabling us to evaluate the differential impact of boundary composition on TAD insulation. Our analysis identifies three generic boundary categories, each influencing TAD insulation differently, with varying local and distal effects on neighboring domains. Notably, we find that increasing boundary width reduces long-range inter-TAD contacts, as confirmed by Hi-C data. While blocking loop extrusion at boundaries indirectly promotes spurious intermingling of neighboring TADs, extended boundaries counteract this effect, emphasizing their role in maintaining genome structure. In conclusion, TAD boundary width not only enhances the efficiency of loop extrusion blocking but may also modulate enhancer-promoter contacts over long distances across TADs boundaries, providing a mechanism for transcriptional regulation. Significance statement Topologically Associating Domains (TADs) compartmentalize vertebrate genomes to limit cross-domain enhancer-promoter loops. Our study reveals that boundaries between TADs are diverse genomic entities that range from narrow to highly extended. Using an interdisciplinary approach that combines genome-wide data analysis with biophysical polymer modeling, we find that wider TAD boundaries reduce spurious long-range interactions between neighboring domains. Moreover, we reveal how different boundary components can create this difference in insulating capacity. Our identification and characterization of TAD boundary width and composition suggests they have the potential to regulate the formation of enhancer-promoter loops across TAD boundaries at close and long distance.