The role of loop extrusion in the dynamics of chromosome folding

In mammalian genomes, physical interactions between chromosomal loci and notably between enhancers and promoters, enable control of gene expression across large genomic distances. However, it remains unknown how frequent and stable contacts between chromosomal sequences are over the course of the cell cycle and how they depend on the underlying dynamics of chromosome folding and on the loop extrusion activity of cohesin.
To address these questions, we engineered mouse embryonic stem cells (mESCs) to carry tens of random integrations of bacterial operator arrays that can be visualized using live cell fluorescence microscopy. By imaging these in the context of auxin-inducible degron systems for factors such as CCCTC-binding factor (CTCF) and RAD21, a subunit of the cohesin complex, we observed that on a global scale cohesin serves to constrain chromosome motion, while the loss of CTCF does not impact global chromosome dynamics.
However, by imaging two distinguishable chromosomal locations separated by 150 kb in cis within a topologically associating domain (TAD), we discovered that their interactions are transient events occurring frequently during the course of a cell cycle and that these interactions become substantially more frequent and longer (∼16 minutes) in the presence of convergent CTCF sites. By contrast, cohesin depletion results in shorter and less frequent cis-contacts. These observations thereby indicate a role for cohesin and CTCF in the suppression of variability in chromosome folding across time. Comparison of the experimental results to physical models of chromosome dynamics additionally suggests that individual CTCF anchored, cohesin-mediated loops last around 10 minutes. These measurements therefore indicate that long-range transcriptional regulation might rely on transient physical proximity, and show that cohesin and CTCF stabilize otherwise highly dynamic chromosome structures to facilitate selected subsets of chromosomal interactions.
In a complimentary approach, we further investigated what function of cohesin leads to this effect: its role in sister-chromatid cohesion or in loop extrusion. We therefore employed a single-particle tracking (SPT) approach following individual RAD21 molecules bound to DNA at the same time as the motion of DNA loci. We found that cohesin motion on DNA is characterized by a more sub-diffusive behavior than that of DNA itself. Furthermore, by simultaneously depleting regulators of cohesin in loop extrusion (NIPBL) or sister-chromatid cohesion (Sororin), we showed that both functions contribute to imposing constraints on the dynamics of chromosomes.