Folding of mammalian genomes into spatial domains is critical for gene regulation. The insulator protein CTCF and cohesin control domain location by folding domains into loop structures, which are widely thought to be stable. Combining genomic and biochemical approaches we show that CTCF and cohesin co-occupy the same sites and physically interact as a biochemically stable complex. However, using single-molecule imaging we find that CTCF binds chromatin much more dynamically than cohesin (~1–2 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin for which the search process is long (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging 'dynamic complex' rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops are dynamic and frequently break and reform throughout the cell cycle.DOI: http://dx.doi.org/10.7554/eLife.25776.001

R.T. conceived the project. C.C., A.S.H., and I.P. performed genome editing and generated cell lines. A.S.H. characterized the C59D2 line (growth rate, cell cycle, and protein abundance). C.C. and I.P. performed and analyzed biochemistry experiments including western blots and coIPs. C.C. performed in vitro RNA-binding assays. R.S.-M. performed PAR-CLIP. D.R. supervised PAR-CLIP. A.S.H. performed and analyzed imaging experiments. T.-H.S.H. performed and analyzed Micro-C experiments. C.C. performed and analyzed ChIP-seq and RNA-seq experiments. T.-H.S.H. led and performed most bioinformatic analyses with input from A.

Abstract/summaryFolding of mammalian genomes into spatial domains is critical for gene regulation. CTCF and cohesin control domain location by folding domains into loop structures, which are thought to be highly stable. Combining genomic, biochemical and single-molecule imaging approaches, we show that although CTCF and cohesin can physically interact, CTCF binds chromatin much more dynamically than cohesin (~1 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging "dynamic complex" rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops constantly break and reform throughout the cell cycle.