Cohesin prevents local mixing of condensed euchromatic domains in living human cells

The human genome is folded into chromatin loops by the cohesin complex, forming functional chromatin domains that underlie transcription and DNA replication/repair. However, the physical properties of these domains in living cells, especially in active euchromatin, and how they are regulated by cohesin remain elusive. To address these questions, we combined single-nucleosome imaging/tracking and super-resolution 3D-structured illumination microscopy (3D-SIM) with euchromatin-specific labeling of histone H3.3. Using this nanoscopic approach, we revealed that euchromatin is organized into condensed domains constrained by cohesin-mediated loops, challenging the established textbook view of euchromatin as largely open. Transcription machinery appear to be located near the condensed domain surfaces. Cohesin loss increased nucleosome fluidity of these domains without altering their compaction, leading to local mixing of domains and compromising transcriptional insulation. These findings uncover an unexpected physical role of cohesin in maintaining the integrity of condensed euchromatic domains and ensuring proper higher-order regulation of gene expression.