Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains [Tri-C]

The genome is organized into self-interacting chromatin domains containing genes and the cis-regulatory elements controlling their expression. How these domains form and how elements within them interact is not fully understood. We have developed Tri-C, a sensitive, high-resolution Chromosome Conformation Capture (3C) approach to identify concurrent chromatin interactions in single cells. Combining Tri-C with conventional 3C and polymer modeling, we show that, rather than folding into stable pre-formed loops, self-interacting domains form dynamic compartmentalized chromatin structures, delimited by CTCF/Cohesin boundaries. Within these tissue-specific domains, all regions of chromatin contact each other, but preferential structures are formed in which multiple enhancers and promoters interact simultaneously. Flanking CTCF/Cohesin-bound elements are excluded from these interactions and form distinct structures. These observations are best explained by a dynamic loop extrusion mechanism and subsequent stabilization of enhancer-promoter interactions, rather than the current view of long-range interactions occurring via the formation of discrete pre-formed chromatin loops. Tri-C is a new 3C-based technique that can analyze multi-way interactions. It combines 3C library preparation (NlaIII restriction enzyme) with oligonucleotide capture enrichment for viewpoints of interest and the fragments they interact with. The capture step has been optimized to capture two or more interacting fragments with high efficiency. We have used this to analyze the 3D structure of the murine alpha and beta globin loci in active primary erythroid cells and inactive ES cells.