Stress-induced loss of CTCF reveals an alternative, promoter-based mode of cohesin looping.

Cells continually encounter environmental stressors that challenge homeostasis. How three-dimensional (3D) chromatin structure contributes to these stress responses, particularly under hyperosmotic conditions, remains poorly understood. Here, using time-resolved Hi-C, CUT&Tag, auxin-inducible depletion, and RNA-seq, we map 3D chromatin structure, its molecular drivers, and transcriptional outcomes during the hyperosmotic stress response. Within 1 hour of sorbitol treatment, pre-existing loops and domains undergo genome-wide collapse, accompanied by the emergence of several hundred de novo, sorbitol-induced loops that are more punctate, longer-range, and transient. These newly formed loops weaken over time and largely dissipate by 24 hours, coincident with recovery of pre-existing chromatin structure. Loop reorganization is consistent across human cell types and hyperosmotic stimuli. CUT&Tag and degron experiments reveal that sorbitol-induced loops require cohesin but not CTCF. Newly formed loop anchors are enriched at active promoters containing SP and KLF family motifs. Genes located at these anchors show little immediate transcriptional change but are activated several hours after loop formation, consistent with loops functioning upstream of gene activation. Together, our findings show that hyperosmotic stress triggers a rapid, reversible, and CTCF-independent reorganization of 3D chromatin interactions that helps coordinate transcriptional adaptation.