Loops and the activity of loop extrusion factors constrain chromatin dynamics

Abstract

The chromosomes-DNA polymers and their binding proteins-are compacted into a spatially organized, yet dynamic, three-dimensional structure. Recent genome-wide chromatin conformation capture experiments reveal a hierarchical organization of the DNA structure that is imposed, at least in part, by looping interactions arising from the activity of loop extrusion factors. The dynamics of chromatin reflects the response of the polymer to a combination of thermal fluctuations and active processes. However, how chromosome structure and enzymes acting on chromatin together define its dynamics remains poorly understood. To gain insight into the structure-dynamics relationship of chromatin, we combine high-precision microscopy in living Schizosaccharomyces pombe cells with systematic genetic perturbations and Rouse model polymer simulations. We first investigated how the activity of two loop extrusion factors, the cohesin and condensin complexes, influences chromatin dynamics. We observed that deactivating cohesin, or to a lesser extent condensin, increased chromatin mobility, suggesting that loop extrusion constrains rather than agitates chromatin motion. Our corresponding simulations reveal that the introduction of loops is sufficient to explain the constraining activity of loop extrusion factors, highlighting that the conformation adopted by the polymer plays a key role in defining its dynamics. Moreover, we find that the number of loops or residence times of loop extrusion factors influence the dynamic behavior of the chromatin polymer. Last, we observe that the activity of the INO80 chromatin remodeler, but not the SWI/SNF or RSC complexes, is critical for ATP-dependent chromatin mobility in fission yeast. Taking the data together, we suggest that thermal and INO80-dependent activities exert forces that drive chromatin fluctuations, which are constrained by the organization of the chromosome into loops.

FIGURE 1:

Visualization and tracking of DNA loci over time reveals characteristic, ATP-dependent chromatin dynamics in fission yeast. (a) Cells labeled with a lacO array inserted adjacent to the gene of interest or at random are visualized using GFP-LacI fluorescence. DNA loci are tracked using a custom two-dimensional single particle tracking algorithm (Bailey et al., 2021). (b) Chromatin diffusivity is nearly identical across six different genomic locations as shown by the MSD of each genetic locus as a function of the time. Dashed line marks “window of observation,” along with its calculated diffusion coefficient, D. (c) Cells depleted of ATP by treatment with sodium azide show much slower chromatin dynamics. For comparison, cells fixed with formaldehyde were imaged and analyzed to estimate systematic error in our image acquisition and analysis system. Error bars in panels b and c designate standard errors of the mean. Lines are the best fit of Eq. 1 with the indicated D values.

FIGURE 2:

Microtubule dynamics actively drive chromatin motion at the centromeres and, to a lesser extent, the chromosome arms in fission yeast. In S. pombe, large-scale chromatin dynamics are influenced by movement of the SPB through its attachment to the centromeres (Funabiki et al., 1993). (a) The centromeres (cen2) demonstrate actively driven motion at the tens-of-seconds timescale as revealed by MSD analysis. Upon addition of the microtubule-depolymerizing agent carbendazim (MBC), this mobility is greatly depressed. The effect of microtubule dynamics is far less prominent for a locus in the chromosome arm (mmf1). (b) On the seconds time scale, the mobility of cen2 is far more constrained than the mmf1 locus and microtubule dynamics play a more muted effect. Error bars indicate standard errors of the mean.

FIGURE 3:

Loss of cohesin or condensin activity increases chromatin mobility. (a) MSD analysis of G2 cells harboring temperature-sensitive alleles of the cohesin-loading protein Mis4 and condensin complex subunit 2 (Cut14). The fluorescently labeled mmf1 locus was imaged at the nonpermissive temperature (36°C) to inhibit G2 cohesin and condensin function, respectively. (b) The same enhancement of chromatin dynamics is observed at a separate genomic locus, pfl5. Error bars indicate standard errors of the mean. Lines are the best fit of Eq. 1 with the indicated D values.

FIGURE 4:

LEF activity constrains chromatin motion as revealed by Rouse-type polymer simulations. (a) The formation of time-dependent loops driven by loop extrusion according to the LEF-CTCF model with semipermissible boundaries at CTCF binding sites (orange) or LEF-only model lacking explicit boundaries (blue) applied to the mouse genome leads to decreased MSD compared with the polymer without loops (red). MSDs arise from a Rouse model with beads of friction coefficient ζ, connected by springs of spring constant κ; τ_p = ζ /(4κ) is the characteristic time of polymer relaxation. (b) Examples of the instantaneous polymer configurations from simulations for the mouse genome. (c) The same analysis as described in panel a but applied to the fission yeast genome for the LEF-only model (blue) compared with the polymer without loops (red). In magenta is the LEF-only model in which only half of the LEFs are present. (d) Examples of the instantaneous polymer configurations from fission yeast simulations. (e) Loop size distribution for simulations of the mouse chromatin with LEF-CTCF and LEF-only models. (f) Loop size distribution for simulations of the fission yeast chromatin with LEF-only models at “full” or 1/2 numbers of LEFs.

FIGURE 5:

Impact of loop extrusion on chromatin mobility depends on the LEF lifetime. (a) MSD results of the Rouse-type polymer simulations combined with loop extrusion simulations that considered two types of LEF complexes (in equal amounts) with different lifetimes. Blue, simulations with both types of LEFs present; green, only long-lived LEF present; dark green, only short-lived LEF present; red, no LEFs. Lines represent the average over all beads and all simulations. See also Supplemental Figure S3. (b) Experimental MSDs for WT cells at 36°C (red) and cells harboring a temperature-sensitive allele of the condensin complex subunit 2 (Cut14) imaged at the nonpermissive temperature (36°C; pink) and cells harboring the temperature-sensitive allele of Cut14 imaged after depletion of the Rad21 subunit of the cohesin complex at the permissive temperature (30°C; green) and at the nonpermissive temperature (36°C; blue). Lines are the best fit of Eq. 1 with the indicated D values.

FIGURE 6:

Loop extrusion decreases the apparent subdiffusive exponent. (a, b) Effective “instant” exponent, α, vs. time for the mouse and fission yeast models presented in Figure 4. Thin lines correspond to individual beads; thick lines represent the average over all beads and simulations.

FIGURE 7:

Loss of the nucleosomal remodeling complex protein Arp8 decreases chromatin mobility likely through a mechanism distinct from loop extrusion. (a) Loss of Arp8, a component of the INO80 complex, reduces ATP-dependent chromatin mobility by ∼40%, while loss of Arp9, a component of both the SWR1 and RSC complexes, has a minimal effect. For comparison, cells depleted of ATP (treated with sodium azide) are shown (replotted from Figure 1c). (b) Loss of Arp8 dampens the elevated chromatin dynamics in the cut14-208 background at the nonpermissive temperature to a slightly lesser extent than in WT cells (an ∼30% decrease) but remains much more diffusive than at the permissive temperature. Error bars indicate standard errors of the mean. Lines are the best fit of Eq. 1 with the indicated D values.