Mammalian chromosomes are organized into megabase-sized compartments that are further subdivided into topologically associating domains (TADs). While the formation of TADs is dependent on cohesin, the mechanism behind compartmentalization remains enigmatic. Here, we show that the bromodomain and extraterminal (BET) family scaffold protein BRD2 promotes spatial mixing and compartmentalization of active chromatin after cohesin loss. This activity is independent of transcription but requires BRD2 to recognize acetylated targets through its double bromodomain and interact with binding partners with its low-complexity domain. Notably, genome compartmentalization mediated by BRD2 is antagonized on the one hand by cohesin and on the other hand by the BET homolog protein BRD4, both of which inhibit BRD2 binding to chromatin. Polymer simulation of our data supports a BRD2-cohesin interplay model of nuclear topology, in which genome compartmentalization results from a competition between loop extrusion and chromatin-state-specific affinity interactions.

To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA-fluorescence in situ hybridization (FISH), RNA-FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.