Multiplexed Super-Resolution Imaging of Chromatin Domains in Single Cells

The spatial organization of chromatin is essential for regulating many crucial genome functions such as transcription, replication and DNA damage response. Here we report an imaging method for tracing chromatin organization with kilobase- and nanometer-scale resolution, unveiling the chromatin conformation across topologically associating domains (TADs) in thousands of individual cells. At the population-average level we observed a striking correlation between the imaging distance measurements and the aggregate Hi-C contact probabilities, providing validation between the two orthogonal technologies. At the single-cell level, imaging revealed TAD-like structures with spatially-segregated globular conformation and sharp domain boundaries in single cells. The boundaries varied from cell to cell, occurring stochastically at all genomic positions, but preferentially at CTCF/cohesin sites. Moreover, we observed wide-spread, cooperative, multi-way chromatin interactions occurring ubiquitously throughout the chromatin regions imaged. Cohesin depletion, using auxin induced degradation, provided crucial insights into the mechanisms behind these single-cell TAD-like domains and multi-way chromatin hubs.