Chromatin organization by an interplay of loop extrusion and compartmental segregation

Abstract

Mammalian chromatin is spatially organized at many scales showing two prominent features in interphase: (i) alternating regions (1-10 Mb) of active and inactive chromatin that spatially segregate into different compartments, and (ii) domains (<1 Mb), that is, regions that preferentially interact internally [topologically associating domains (TADs)] and are central to gene regulation. There is growing evidence that TADs are formed by active extrusion of chromatin loops by cohesin, whereas compartmentalization is established according to local chromatin states. Here, we use polymer simulations to examine how loop extrusion and compartmental segregation work collectively and potentially interfere in shaping global chromosome organization. A model with differential attraction between euchromatin and heterochromatin leads to phase separation and reproduces compartmentalization as observed in Hi-C. Loop extrusion, essential for TAD formation, in turn, interferes with compartmentalization. Our integrated model faithfully reproduces Hi-C data from puzzling experimental observations where altering loop extrusion also led to changes in compartmentalization. Specifically, depletion of chromatin-associated cohesin reduced TADs and revealed finer compartments, while increased processivity of cohesin strengthened large TADs and reduced compartmentalization; and depletion of the TAD boundary protein CTCF weakened TADs while leaving compartments unaffected. We reveal that these experimental perturbations are special cases of a general polymer phenomenon of active mixing by loop extrusion. Our results suggest that chromatin organization on the megabase scale emerges from competition of nonequilibrium active loop extrusion and epigenetically defined compartment structure.

Keywords: Hi-C; active matter; chromatin; genome architecture; polymer physics.

Fig. 1.

Model of loop extrusion competing with compartmental phase separation. (A) Cartoon of typical Hi-C signatures of interphase chromatin organization: Topologically associating domains (TADs) are squares of increased contact frequency along the diagonal, while compartmentalization is a checkerboard pattern indicating spatial segregation. Upon removal of the cohesin loader Nipbl, Schwarzer et al. (11) observed that TADs disappear and a fine-scale compartmentalization emerges (indicated in red/blue; see Fig. 2A for a data example). (B) Sketch of our mechanistic model: Loop extrusion factors (LEFs) (yellow) counteract segregation of A (red)- and B (blue)-type chromatin. (C) Simulations. (Left) Example conformations from polymer simulations showing phase separation of A and B regions (here in periodic boundary conditions). (Right) The emergence of an A-rich and a B-rich phase in our simulations is quantified by the normalized number difference of A and B particles in small boxes, which becomes bimodal as the compartmental interaction E_attr is increased (colors from blue to red; the arrow indicates the value used throughout the text; E_attr = 0.12 k_BT).

Fig. 2.

Experiments and simulations show the interplay of loop extrusion and compartmentalization. (A) Removal of chromatin-associated cohesin (by knockout of the cohesin loader Nipbl)/removal of loop extrusion. (Left) Cohesin removal leads to stronger and fragmented compartmentalization and loss of TADs. Data from ref. . (Right) The same is observed in simulated Hi-C maps upon removal of loop extrusion. The loss of loop extrusion leads the loss of a characteristic hump in P(s), the contact probability as a function of genomic separation. The fragmentation is apparent in compartment profiles as the faster decay of their autocorrelation. The degree of compartmentalization (COMP score) is reduced by a similar factor upon removal of Nipbl/loop extrusion in experiments/simulation. (B) Removal of CTCF/removal of extrusion barriers. (Left) CTCF depletion strongly suppresses TADs but leaves compartmentalization almost unaffected. Data from ref. . (Right) The same is observed in simulations when loop extrusion barriers are removed (barrier permeability increased from 10 to 100%). LEF processivity λ and average separation d are as in A. The decay of the contact probability with genomic distance barely changes both in experiments and simulations. (C) Increased activity of cohesin (by knockout of the cohesin unloader WAPL)/more and longer loops. (Left) Removal of WAPL reduces compartmentalization and strengthens TADs, in particular secondary corner peaks. Data from ref. . (Right) The same is observed in simulations with a 10-fold increase in LEF processivity and a 1.5-fold increase in LEF density. The secondary corner peaks arise when the chromatin between barriers is fully extruded, forming contacts between several consecutive barriers (Lower cartoon). The characteristic hump in contact probability scaling extends to significantly larger distances, reflecting larger loops (14).

Fig. 3.

Impact of loop extrusion on compartments of different size. (A) Contact frequency maps without/with loop extrusion (upper/lower triangles). The lengths of A/B segments are indicated above the maps. (B and C) Example conformations of 50-Mb fibers without/with loop extrusion, in periodic boundary conditions (see SI Appendix, Fig. S10 for a comparison with spherical confinement). The approximate segment length where phase separation (PS) occurs is indicated in gray. (D) The degree of phase separation as a function of segment length is measured from contact frequency maps (COMP) and from spatial configurations (N; see text for details). The impact of loop extrusion on compartmentalization (Right) is measured by dividing each of the above order parameters in the absence of loop extrusion by their value with loop extrusion. The impact is maximal for segment lengths that exceed the segregation transition but not the mixing length, which is of the order of several LEF processivities (∼1 Mb).

Fig. 4.

The nonequilibrium nature of loop extrusion. (A–C) Effects of the speed of loop extrusion relative to thermal polymer dynamics. (A) Contact frequency maps. (B) Averages of TADs of sizes 675–825 kb rescaled to fixed size (see SI Appendix, Fig. S5 for other TAD sizes). (C) Strength of compartmentalization and of TADs score as a function of LEF speed: Compartmentalization decreases while TAD strength increases from no loops over static loops to extruding loops of increasing speed (the polymer dynamics due to thermal motion are kept constant). (D) Importance of chain passing: The impact of loop extrusion on compartmentalization, measured by the ratio of compartmentalization strength without/with loop extrusion, increases for reduced topoisomerase II activity, that is, reduced chain passing (implemented by increasing E_rep, the repulsive part of the monomer interaction potential; SI Appendix, Fig. S1). (E) Length scales relevant for equilibration of a loop: radius of gyration of an extruded loop R_g and diffusional displacement during loop growth. (F) R_g follows equilibrium theory (gray) for static loops, while with increasing LEF speed loops are more compact. R_g is measured in units of one monomer diameter of ≈50 nm. (G) The root-mean-square displacement of chromatin with/without loop extrusion differs on the LEF residence timescale, but not globally, indicating that loop extrusion cannot be described as an elevated effective temperature.

Fig. 5.

Effects of different mechanisms on TAD and compartment strength. Three main classes of responses to perturbations are identified: A trade-off between compartmentalization and TADs is observed for parameter changes related to cohesin dynamics and for the frequency of chain passing (topoisomerase II activity). Compartmental interaction and nuclear volume mainly affect compartmentalization. The permeability of extrusion barriers mainly affects TADs. The black dots indicate our simulations of WT interphase cells and removal of cohesin (by Nipbl deletion), of CTCF, and of WAPL.