A bottleneck in understanding the principles of 3D chromatin structures is caused by the paucity of known regulators. Cohesin is essential for 3D chromatin organization, and its interacting partners are candidate regulators. Here, we performed Cohesin chromatin proteomic profiling and identified transcription factors, RNA-binding proteins, and chromatin regulators associated with Cohesin. Acute protein degradation followed by time-series genomic binding quantitation and BAT Hi-C analysis were conducted, and the results showed that the transcription factor ZBTB21 insulates local chromatin interactions by facilitating the chromatin occupancy of Cohesin. Strikingly, multiomic analyses revealed that the other four ZBTB factors colocalized and interacted with Cohesin, and ZBTB21 and ZBTB7B double degradation led to a further decrease in Cohesin chromatin occupancy. We propose that multiple ZBTB transcription factors orchestrate the chromatin binding of Cohesin to insulate 3D chromatin interactions, and we provide a catalog of many additional proteins associated with Cohesin that warrant further investigation.

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