3D Chromatin organization of signal-induced specific enhancers safeguards early ESC differentiation by promoting cluster gene expression [CUT&Tag]

Cells undergo fate transformation in response to external signaling stimuli, a process that depends on the precise expression of specific genes, but the intrinsic regulatory mechanisms remain largely unclear. Here, by integrating bulk RNA sequencing, single-cell RNA sequencing, 4D-SmartDIA quantitative proteomics data, and a CRISPR/Cas9-mediated RA receptor deletion model, we find that RA signaling-induced early ESC differentiation is multilineage. During early ESC differentiation, in the context of attenuated and enhanced RA signaling, we identified Cyp26 and HoxB cluster genes as its direct downstream targets. Upon activation of RA signaling, RA receptors induce new specific enhancers in the proximal/distal regions of cluster gene loci, whereas inhibition of RA signaling significantly reduces proximal/distal enhancer activity, chromatin accessibility and cluster gene expression. Mechanistically, we found that proximal/distal enhancers form specific chromatin loops with cluster gene loci under RA induction, and RA receptor deletion significantly reduces these chromatin interactions. Knockout of the proximal/distal enhancers inhibits cluster gene expression, thereby further disrupting RA-induced early ESC differentiation. In summary, our findings reveal an insightful mechanism underlying the signal-induced specific 3D genomic structure and cluster gene expression critical for early ESC fate transitions.