To explore how cohesin organizes chromatin domains in living human cells, we combined single-nucleosome imaging/tracking5,29,44,45,54 with rapid cohesin depletion using the auxin-inducible degron (AID2) system55. Single-nucleosome imaging/tracking can sensitively detect changes in the chromatin state in living cells. We used HCT116 cells expressing H2B-HaloTag (H2B-Halo)45 and RAD21-mAID-mClover (mAC55; Extended Data Fig. 1a,b). H2B-Halo was labeled with tetramethylrhodamine (TMR) to visualize nucleosomes genome wide, including those in both euchromatin and heterochromatin (Extended Data Fig. 1c). Oblique illumination (highly inclined and laminated optical sheet, HILO) microscopy56 enabled imaging of individual nucleosomes as TMR-labeled dots of H2B-Halo (Fig. 1a–c), which were precisely tracked in two-dimensional (2D) at 50 ms per frame for a total of 15–20 s, yielding ~1,400 trajectories per cell (Fig. 1b,d and Extended Data Fig. 1d,e; see Methods for further details). Position-determination accuracy is 12.5 nm (Extended Data Fig. 1d). Tracking was specific to nucleosome-incorporated H2B-Halo, as the small free H2B-Halo pool (3.3%) diffused too fast to be observed (Supplementary Video 1 and Extended Data Fig. 1f). The mean squared displacement (MSD) analysis revealed subdiffusive nucleosome motion with an anomalous exponent (α) of 0.43, suggesting constrained motion (Fig. 1e,g, black plots). Fixation nearly abolished this motion (gray plot, Fig. 1e,g)...

Intact full-length PR and CoRs were expressed and purified as recombinant proteins in Spodoptera frugiperda (Sf9) insect cells using the baculovirus system55. This is an ideal expression system for full-length PR, shown previously to retain native folding and post-translational modifications,...