Phase separation induced by cohesin SMC protein complexes

Abstract

Cohesin is a key protein complex that organizes the spatial structure of chromosomes during interphase. Here, we show that yeast cohesin shows pronounced clustering on DNA in an ATPindependent manner, exhibiting all the hallmarks of phase separation. In vitro visualization of cohesin on DNA shows DNA-cohesin clusters that exhibit liquid-like behavior. This includes mutual fusion and reversible dissociation upon depleting the cohesin concentration, increasing the ionic strength, or adding 1,6-hexanediol, conditions that disrupt weak interactions. We discuss how bridging-induced phase separation can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in vivo, a fraction of cohesin associates with chromatin in yeast cells in a manner consistent with phase separation. Our findings establish that SMC proteins can exhibit phase separation, which has potential to clarify previously unexplained aspects of in vivo SMC behavior and constitute an additional principle by which SMC complexes impact genome organization. One sentence summary:Yeast cohesin complex is observed to phase separate with DNA into liquid droplets, which it accomplishes by ATP-independent DNA bridging.Members of the structural maintenance of chromosome (SMC) protein family such as condensin, cohesin, and the Smc5/6 complex are key proteins for the spatial and temporal organization of chromosomes (1)(2)(3)(4). Recent in vitro experiments visualized real-time DNA loop extrusion mediated by condensin and cohesin (5-7). While loop extrusion by SMC proteins constitutes a fundamental building block in the organization of chromosomes, other factors may also contribute. In the last decade, it has become abundantly clear that phase separation plays a role in many processes in biological cells (8), including chromosome organization (9, 10). Thus far, SMC proteins have not been implied in phase separation. While ATP-independent clustering of DNA and SMC proteins has been reported (11)(12)(13)(14), such observations were attributed to imperfect protein purification or non-physiological buffer conditions. For example, Davidson et al. (7) reported in vitro DNA loop extrusion by the human cohesin complex when the complex concentration was limited to very low values (<0.8 nM, i.e., much lower that physiological concentrations of ~333 nM (15,16), and mentioned that the cohesin complexes were prone to aggregation at higher concentrations. Such findings raise the question whether such aggregate formation may be intrinsic and may have a physiological meaning.Here we report that interactions between the yeast cohesin SMC complex and DNA lead to pronounced phase separation. This clustering behavior is ATP independent but depends on DNA length. We find that single cohesin complexes are able to bridge distant points along DNA that act as nucleation points for recruiting further cohesin complexes -a behavior indicating bridging-induced phase separation (BIPS) (17,18), also known as polymer-polymer phase separation (PPPS) (19), a type of phase co...

Computational and experimental analyses of mitotic chromosome formation pathways in fission yeast

“…Alternatively, two condensin complexes that each embrace one DNA might engage with each other. A tendency of SMC complexes to form clusters on DNA in vitro (13)(14)(15) is consistent with the latter possibility. In the diffusion capture scenario, condensin establishes contacts both within chromosomes and between chromosomes, consistent with experimental observations in yeasts (16)(17)(18).…”

Cohesin architecture and clustering in vivo

“…Second, the pattern of trans -biotinylation for different AviTag reporters is more restricted than the patterns of biotinylation by freely diffusible BirA or cis -biotinylation within cohesin. Importantly, the restricted trans -biotinylation between cohesin, on and off DNA, could not be inferred from a model in which cohesin clustering is mediated by disordered aggregation or DNA-dependent phase separation ( Ryu et al, 2020 ). Rather, it fits with the model that cohesin molecules in butterfly conformation form oligomers (dimers or multimers), with the head domain of one cohesin molecule near the head and hinge domains of the other cohesin molecules of the same oligomer.…”

A Brownian ratchet model for DNA loop extrusion by the cohesin complex

“…Alternatively, cohesin and condensin could generate loops by sequentially topologically embracing two DNAs that come into proximity by Brownian motion, a mechanism that we refer to as diffusion capture ( Cheng et al, 2015; Gerguri et al, 2021 ). Interactions between more than one cohesin binding site in the diffusion capture model could also arise from bridging-induced phase separation ( Ryu et al, 2020a ). When cohesin is depleted and re-supplied to human cells, small and large loops form with similar kinetics, a behavior that is more readily explained by a diffusion-mediated process than by gradual loop growth ( Suhas et al, 2017 ).…”