A revised model for promoter competition based on multi-way chromatin interactions

Specific communication between gene promoters and enhancers is critical for accurate regulation of gene expression. However, it remains unclear how specific interactions between multiple regulatory elements and genes contained within a single chromatin domain are coordinated. Recent technological advances allow for the investigation of multi-way chromatin interactions at single alleles in individual nuclei. This can provide insights into how multiple regulatory elements cooperate or compete for transcriptional activation. We have used these techniques in a mouse model in which the alpha-globin domain is extended to include several additional genes. This allows us to determine how the interactions of the alpha-globin super-enhancer are distributed between multiple promoters in a single domain. Our data show that gene promoters do not form mutually exclusive interactions with the super-enhancer, but all interact simultaneously in a single complex. These finding show that promoters within the same domain do not structurally compete for interactions with enhancers, but form a regulatory hub structure, consistent with the recent model of transcriptional activation in phase-separated nuclear condensates. Overall design: Tri-C is a 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 active mouse alpha globin locus in primary erythroid cells.