PEhub resolves the hierarchical regulatory architecture of multi-way enhancer hubs in the human brain

Abstract Chromatin interaction assays capture regulatory architecture as stochastic pairwise contacts, limiting the ability to resolve how multiple enhancers cooperatively regulate transcription. Here we introduce a promoter-centric quantitative framework, termed PEhub, that resolves multi-way enhancer hubs as higher-order regulatory units from chromatin interaction data. By reparameterizing stochastic pairwise ligation events into promoter-conditioned enhancer networks, our approach explicitly models synergistic enhancer cooperation while accounting for distance-dependent interaction decay through a statistically principled null model. Using H3K27ac HiChIP data, we identify promoter-anchored enhancer hubs and validate their physical existence with single-molecule Pore-C, demonstrating that inferred hubs correspond to bona fide multi-way chromatin assemblies. Application to six human brain regions reveals that enhancer hubs are associated with elevated transcriptional output and exhibit a hierarchical organization spanning shared, circuit-specific, and region-restricted regulatory programs. This architecture hierarchically stratifies genetic risk and transcription factor deployment, linking three-dimensional genome organization to transcriptional control and disease-associated variation. Together, this promoter-centric framework provides a generalizable strategy for resolving higher-order regulatory architecture from 3D genome data and establishes multi-way enhancer hubs as a functionally and genetically meaningful layer of transcriptional regulation in complex tissues.