69 / 2026-04-05 11:06:07

Hyper Chromatin Hubs as Compact HighPower Regulatory Nodes Orchestrate Long-Range Regulatory Interactions Link to 3D Genome Organization and Cell Differentiation

HChubs, Chromatin interactions, Similarity of binding TF motifs, TADs

Coordinated binding of multiple transcription factors (TFs) at accessible chromatin is a defining feature of cell identity, yet how such combinatorial occupancy relates to three-dimensional genome organization remains incompletely resolved. Here, by integrating human, mouse, and pig ATAC-seq, ChIA-PET, and Hi-C datasets with machine learning approaches, we identify compact regulatory elements (150 – ~5,000 bp) termed Hyper Chromatin-hubs (HChubs). HChubs concentrate multiple lineage-dominant TFs within single enhancers or promoters and are frequently associated with long-range chromatin interactions extending up to 600 kb. Genome-wide analyses reveal that HChubs coincide with reinforced topologically associating domains (TADs), enhanced chromatin looping, and increased lineage-specific gene expression. The similarity of binding TF motifs between loop anchors strongly correlates with interaction strength and genomic span, providing a mechanistic basis for the role of HChubs as chromatin hubs. Specifically, this similarity is largely attributable to lineage-specific TF families, not the canonical architectural protein CTCF (CCCTC-binding factor). During myoblast differentiation, acquisition of dominant TFs coincides with strengthening of HChub-associated TAD boundaries and loops near myogenic genes. Furthermore, along with the known MyoD cooperator MyoG, binding of Cebpb (CCAAT/enhancer-binding protein beta) coincides with enhanced occupancy of the master regulator MyoD. Together, these findings identify HChubs as conserved TF-dense regulatory elements associated with lineage-specific chromatin architecture.