Abstract: The simultaneous engagement of multiple transcription factors (TFs) within open chromatin regions is a prevalent phenomenon in mammalian cells. However, the mechanisms by which TF cooperation modulates 3D genome architecture remain poorly understood. In this study, we performed ATAC-seq on different cells and tissues from humans, mice, and pigs to identify open chromatin regions (OCRs) and footprints, which represent transcription factor (TF) binding sites. We observed that a fraction of OCRs have numerous of TF binding, which possess a fine span length (300-5000bp) but can attract cis-regulatory elements to form more distant, stronger and structurally more complex chromatin interactions, we termed as "Hyper Chromatin-hubs (HChubs)". Interestingly, analysis of the motif characteristics of TFs binding in HChubs revealed that the strength of interactions between regulatory elements depends on the similarity of the TFs they bind, which enables HChubs with multiple TFs to act as attractors, thus forming more stable and complex interaction structures. Further joint analysis with RNA-seq showed that the HChubs are associated with higher gene transcriptional activity. Additionally, using differentiating myoblast cells as a model, we found that the gain or loss of footprints for muscle lineage-specific TFs (e.g., MyoD/MyoG, Cebpb, and Max) in HChub can affect chromatin looping and regulate cell differentiation. Moreover, Cebpb and MyoG were found to interact with MyoD and enhance its binding, as revealed by localization analysis among TFs within HChubs. Collectively, these insights enhance our comprehension of the role of TF interactions in 3D genome architecture and pivotal biological processes within mammalian systems.
 

Transcription factors, 3D genome, Transcription regulation, Cell differentiation