Multiscale structure of chromatin condensates explains phase separation and material properties

Abstract

The structure and interaction networks of molecules within biomolecular condensates are poorly understood. Using cryo-electron tomography and molecular dynamics simulations, we elucidated the structure of phase-separated chromatin condensates across scales, from individual amino acids to network architecture. We found that internucleosomal DNA linker length controls nucleosome arrangement and histone tail interactions, shaping the structure of individual chromatin molecules within and outside condensates. This structural modulation determines the balance between intra- and intermolecular interactions, which governs the molecular network, thermodynamic stability, and material properties of chromatin condensates. Mammalian nuclei contain dense clusters of nucleosomes whose nonrandom organization is mirrored by the reconstituted condensates. Our work explains how the structure of individual chromatin molecules determines physical properties of chromatin condensates and cellular chromatin organization.

Figure 1.. Linker length determines condensate properties and chromatin fiber structure.

(A) Turbidity (absorption at 345 nm) of chromatin solutions at the indicated nucleosome concentrations. Error bars indicate ± standard deviation of three measurements. (B) Time-lapse imaging of droplet fusion in 25 bp and 30 bp chromatin immediately after inducing phase separation. Scale bar is 3 μm. (C) Timescale of fusion for 25 bp and 30 bp chromatin droplets of different sizes. (D) Fluorescence Recovery After Photobleaching (FRAP) results for 25 bp and 30 bp chromatin. Error bars indicate ± standard deviation of 20 measurements. (E) Schematic of the compaction and phase separation of chromatin fibers as salt concentration increases (left and middle panels) and visualized by cryo-ET (right panel), where scale bar is 100 nm. Boxed array derives from dilute phase cryo-ET image in panel F. (F) Schematic of the computational approach used to reconstruct histone tails on a single chromatin array visualized by cryo-ET in low salt. From left to right, images show: maximum projection of chromatin density in a denoised cryo-ET tomogram; nucleosome models extracted from the density (corresponding to fiber boxed in panel E; scale bars are 20 nm); higher resolution fiber computationally reconstructed using molecular dynamics simulations steered according to nucleosome positions in the cryo-ET data.(G-H) Models based on average structural parameters of chromatin with 25 bp (G) and 30 bp (H) linkers in the dilute phase at low (left) and high (middle) salt concentrations. Right panels show overlays of the two conditions. (I) Schematic representation of the conformational changes induced by salt in 25 bp and 30 bp chromatin.

Figure 2.. Different histone tail interactions in 25 bp and 30 bp chromatin in the dilute phase.

(A-B) Schematic representations 25 bp (A) and 30 bp (B) chromatin arrays in low salt derived from cryo-ET and computationally extended to the chemical-specific model. DNA beads are represented in dark grey (A) and dark blue (B), histone protein beads are in light grey (A) and light blue (B) (C-D) As in (A-B) for chromatin in high-salt. In (C) DNA beads are dark pink (C) and dark green (D), histone protein beads are light pink (C) and light green (D). Insets show a pair of nucleosomes interacting face-to–side (C) and face-to-face ((D) via histone tails. Histone core beads are white and DNA beads are light grey. Histone tail beads are colored as: H3 green, H4 blue, H2A-N terminal red, and H2B cyan. (E) Schematic representation of inter-nucleosome histone tail interactions. (F-G). Representative structures of a nucleosome from 25 bp (F) and 30 bp (G) chromatin simulations illustrating average contact frequency of each residue; histone core and tails are represented by larger and smaller beads, respectively. Residues color-coded by the total number of inter-nucleosome contacts (averaged over 400 simulation frames, see Table S1 for n) made by each amino acid in histones (H3, H4, H2A(N), and H2B) of one nucleosome with DNA (nucleosomal and linker) and histones (core and tails) of neighboring nucleosomes. Acidic patch in G shown by dashed orange oval. (H) Inter-nucleosome contacts (average per residue per simulation frame) made by each amino acid in the histone of one nucleosome and the DNA and histones of neighboring nucleosomes (simulation details in F-G legend above) 25 bp fibers pink, 30 bp fibers green). Blue, cyan and orange vertical lines show positions of lysines, arginines and acidic patch residues, respectively. Shading indicates standard deviation from the mean. (I) Representative nucleosome arrays assigned in cryo-ET tomograms of 25 bp and 30 bp arrays (medium salt, dilute phase) assembled with either wild-type histone octamers (25_med,dil,WT, 30_med,dil,WT) or H4 tail deletion octamers (25_med,dil,H4TL, 30_med,dil,H4TL). (J) Radius of gyration of indicated nucleosome arrays. (K) Intramolecular interactions per 12-mer array, classified as face-to-face, face-to-side, side-to-side.

Figure 3.. Molecular structures of 25 bp and 30 bp chromatin in the condensed phase.

(A) Cross-section of a chromatin condensate in denoised cryo-ET tomogram (left, scale bar 100 nm; middle, magnified view with scale bar 20 nm). Right panel shows nucleosome model assignments from densities in the center panel (scale bar 20 nm). (B) Nucleosome structure (8.0 Å resolution) from 25 bp chromatin obtained by sub-tomogram averaging 104,438 particles in 10 tomograms. Density shown as grey surface, fitted model (PDB: 6pwe) shown as red and green ribbons. (C,D) Classification of mono- and di-nucleosome structures in 25 bp chromatin. Classes colored differently. (E) Overview of 30 bp chromatin condensate, analogous to panel A. (F) Nucleosome structure (5.8 Å resolution) from 30 bp chromatin obtained by sub-tomogram averaging 111, 909 particles in 10 tomograms. Density shown as grey surface, fitted model (PDB: 6L4A) shown as red and green ribbons. (G) Classification of mono- and tri-nucleosome structures in 30 bp chromatin. Classes colored differently and are highly overlapping. (H) Two classes of tri-nucleosome structures in 30 bp chromatin. (I) Structural modeling of a tetra-nucleosome in 30 bp chromatin. Density shown as grey surface, fitted four individual mononucleosome models (PDB: 6pwe) shown as red and green spheres.

Figure 4.. Linker length determines balance of intra- and inter-molecular interactions in the condensed phase.

(A) Radial distribution functions, g(r), of 25 bp (magenta), 30 bp (green) chromatin, and a bath of randomly oriented nucleosomes at equivalent density (grey). Peaks at 6, ~12, and ~16 nm in 30 bp chromatin reflect pair-wise and higher-order nucleosome stacking. (B) Distributions of nearest di-nucleosome orientations for 25 bp (magenta) and 30 bp (green) chromatin. Zero and 90 degrees represent parallel and perpendicular orientations, respectively. (C, D) Representative manually traced individual nucleosome arrays (25 bp magenta in C, 30 bp green in D) and their immediate neighboring nucleosomes (grey) within chromatin condensates. See Figures S5E–J and Movies S4, S5 for nucleosome tracing. (E) Intra and inter-nucleosome interactions of 25 bp (magenta) and 30 bp (green) nucleosome arrays within chromatin condensates, Statistical analysis was performed using a t-test; **** indicates p < 0.0001, n = 11. (F) Intermolecular contacts between traced arrays and their immediate neighboring nucleosomes with face-to-face, face-to-side and side-to-side geometries (25 bp magenta, 30 bp green). (G, H) Representative high-resolution snapshots of reconstructed clusters of chromatin arrays in the 25 bp (G, magenta/orange) and 30 bp (H, green/cyan) chromatin condensates. In each panel the array most similar to a traced array in a cryo-ET tomogram (see Methods) is highlighted. All arrays interacting with this central array are shown, with one colored orange (25 bp) or cyan (30 bp). Inset expands image of the two interacting arrays, to show face–to-face (G) or side-to-side (H) inter-array stacking. (I) Number of inter-nucleosome contacts (average per residue per simulation frame) between histone tails of one nucleosome and the DNA and histones of a neighboring nucleosome (intra-array top, inter-array bottom) within simulated clusters (25 bp magenta, 30 bp green, averaged over 400 simulation frames, Table S1.) The blue and cyan vertical lines show positions of lysines and arginines, respectively. Shading indicates one standard deviation from the mean.

Fig. 5.. Distinct dynamics of 25 bp and 30 bp chromatin condensate on molecular- and meso-scales.

(A-B) Cross sections of interaction networks derived from coarse-grained simulations of 25 bp (A) and 30 bp (B) chromatin condensates. Node diameter is scaled by number of molecules contacted by each array; edge thickness scaled by energy of association between each pair of molecules. (C) Normalized turbidity (A_{345 nm}) of solutions containing chromatin droplets after introduction of trypsin. (D) Schematic illustrating Passive Microrheology with Optical Traps (PMOT). 1 μm bead is optically trapped within chromatin condensate, and its trapped motion tracked over time using brightfield camera or quadrant photodetector. (E) Average elastic/storage (G′) and viscous/loss (G″) moduli of 25 bp (magenta; n=12) and 30 bp (green; n=11) chromatin condensates. Representative individual plots shown in Fig. S11. Thick lines indicate average, thin lines represent standard error from the mean. (F) Viscosity of 25 bp (magenta; n=12) and 30 bp (green; n=11) chromatin condensates, calculated from G” data in panel (E); plotted as in (E). Thick lines: average; thin lines: standard error. (G) Schematic illustrating single-molecule tracking experiments. Sparse labelling of nucleosome arrays with Atto647N dye enables tracking of individual molecules. (H) Trajectories of single nucleosome array in 25 bp (magenta) and 30 bp (green) chromatin condensates. Scale bar = 1 μm. (I) Diffusion coefficients calculated for nucleosome arrays in 25 bp (total 7670 trajectories) and 30 bp (total 3167 trajectories) chromatin condensates. (J) Schematic illustration of trajectory angular analysis, where the angle between every pair of steps in a single-molecule trajectory is measured. Zero degrees corresponds to directed motion and 180 degrees represents back-and-forth movement. (K) Trajectory angular analysis distributions for nucleosome arrays in 25 bp (left) and 30 bp (right) chromatin condensates. Asymmetry coefficient (AC) describes the ratio of forward to backward movement on a log₂ scale.

Fig. 6.. Native chromatin forms discrete domains organized similarly to 25 bp chromatin condensates.

(A-F) Tomographic slices and 3D reconstructions illustrating nuclear nucleosome organization. (A-C) Overview slice (scale bar = 100 nm), expanded regions (scale bar = 20 nm) and 3D nucleosome model assignments from a purified Hela cell nucleus. (D-F) Analogous images from the nucleus of an intact NIH3T3 cell. Note that panels B and E are not expansions of regions from A and D, but are derived from different planes of the tomograms. Different colors in panel C, F represent distinct chromatin nanodomains by manual segmentation. (G) Radial distribution functions, g(r), for randomly oriented nucleosomes (grey), nucleosomes in 25 bp chromatin condensates (magenta), purified Hela cell nuclei (cyan), and nuclei of intact NIH3T3 cells (black). (H) Distributions of nearest di-nucleosome orientations for 25 bp chromatin condensates (magenta), purified Hela cell nuclei (cyan), and nuclei of intact NIH3T3 cells (black). Zero and 90 degrees represent parallel and perpendicular orientations, respectively. (I) Fractions of face-to-face, face-to-side and side-to-side pairwise nucleosome contacts in a random nucleosome distribution, 25 bp reconstituted chromatin condensates, nucleosomes in purified Hela cell nuclei, and nuclei of intact NIH3T3 cells.