Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography

Abstract

It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) in vitro is responsible for chromatin domain organization in vivo. However, understanding nucleosomal LLPS has been hindered by the challenge to characterize the structure of the resulting heterogeneous condensates. We used cryo-electron tomography and deep-learning-based 3D reconstruction/segmentation to determine the molecular organization of condensates at various stages of LLPS. We show that nucleosomal LLPS involves a two-step process: a spinodal decomposition process yielding irregular condensates, followed by their unfavorable conversion into more compact, spherical nuclei that grow into larger spherical aggregates through accretion of spinodal materials or by fusion with other spherical condensates. Histone H1 catalyzes more than 10-fold the spinodal-to-spherical conversion. We propose that this transition involves exposure of nucleosome hydrophobic surfaces causing modified inter-nucleosome interactions. These results suggest a physical mechanism by which chromatin may transition from interphase to metaphase structures.

Keywords: chromatin; condensates; cryo-electron tomography; linker histone H1; liquid-liquid phase separation; nucleation and growth; nucleosome; nucleosome arrays; spinodal decomposition.

Figure 1.. Cryo-ET workflow of deposition, reconstruction, and modeling of tetranucleosome phase condensation.

(A) Optimized negative staining EM and AFM images of tetranucleosomes. (B) Deposition of 30 nM (25% biotinylated; red sphere) tetranucleosome incubation solution onto the SA-crystal. (C) Representative tilt series after deep learning-based denoising. White dashed contours depict two tracked clusters of condensates. (D) Initial 3D reconstruction of denoised and aligned tilt series with z-dimensional height encoded in color. (E) Central slice of the initial map (thickness of 7.02 nm, left) and corresponding deep learning-based segmentation showing predicted NCPs (cyan, right). (F) Missing wedge-corrected final denoised map density (NCPs in cyan, SA-crystal in pink). This final map was used to generate the fitted model (bottom) (NCPs in blue, 40 bp linker DNA in yellow). (G) Comparison of the final 3D map (left) and the fitted model (right) of the dash-line area in C-F. (H) Local areas showing tetranucleosomes in extended (top panels) and stacked (bottom panels) conformations.

Figure 2.. Time evolution of irregular condensates.

Final denoised maps (thickness of ~90-130 nm) with fitted models displaying irregular tetranucleosome condensates obtained in low and physiological salt at 4°C. (A; top) 2 min incubation at low salt (15 mM Na⁺, t2-LS); (B; top) 2 min at physiological salt (150 mM Na⁺ and 5 mM Mg²⁺, t2-PS); and (C; top) 10 min at physiological salt (t10-PS). SA-crystal and nucleosome densities are pink and transparent gray, respectively. Fitted NCPs and flanking DNA colored in cyan and yellow, respectively. Bottom panels show NCP clusters of four representative tomograms for each incubation condition, grouped by the DBSCAM algorithm. Each color represents a unique NCP cluster and black depicts free, unclustered NCPs. (D) Condensates’ dimensions along PC1 and PC3 axes derived from PCA. (E) Number of NCP per condensate. (F) Concentration of free NCPs surrounding a condensate within a 20 nm shell. (G) NND between condensates plotted as mean ± SEM and are compared using a two-tailed unpaired t-test, where *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; and ns, not significant. Schematic representation of the spinodal decomposition process as a function of time obtained from the Cahn-Hilliard equation: top, image modified from (Findik, 2013); bottom, evolution of irregular condensates in terms of their NND (wavelength), condensate internal concentration (amplitude), and surrounding free NCP concentration (sharpness) represented by a Squdel sine function.

Figure 3.. 3D reconstructions of initial stages and growth of Spherical Condensates.

Spinodal condensates (SpnCs) and spherical condensates (SphCs) of different size after 10 min incubation of tetranucleosome at 20°C in physiological salt. (A) Enhanced cryo-ET zero-tilt images from tomograms labeled I-IV, ordered by increasing SphCs size. (B) Corresponding tomogram slices (1.17 nm thickness) depicting NCPs (cyan) after deep learning-based segmentation. (C) Final 3D map of tomograms in (B) showing the NCP (transparent gray) and SA-crystal (pink) densities (tomograms thickness ~90-140 nm). NCP component of the fitted models shown in cyan and the flanking DNA segment in yellow. I-IV illustrate a possible mechanism of growth of SphCs: (I) formation of small, near-spherical condensates (~35 nm, yellow arrow) and merging of three larger SphCs (~100 nm, white arrow); (II) accretion of SpnC by a ~200 nm SphC (red arrow); (III) a ~300 nm SphC resulting from the apparent fusion (purple arrows) of smaller condensates; (IV) a larger (~400 nm) SphC resulting from growth processes.

Figure 4.. Small spherical nuclei arise from SpnCs.

(A) Cryo-EM image showing single (dashed triangles) and multiple (dashed circles) small SphCs formed at 20°C, physiological salt and 20 min of incubation. (B) Representative particles of individual (left columns) and clustered (right columns) small SphCs. (C) Statistics from 12 low-magnification images indicating a similar probability of finding condensates in clusters or in isolation. Data plotted as mean ± SEM. ns, not significant. (D) High-magnification cryo-ET zero-tilt image (left), tomogram slice (middle), and 3D model (right) of a cluster of small SphCs (red dashed circle), SpnCs (yellow dashed circles), and small SphCs in proximity to SpnCs (magenta dashed circle). (E) Various stages of the nucleation and growth processes (I-IV) of SphCs shown by representative tomographic slices (top) and their corresponding 3D maps and models from ~70 nm thickness tomograms (bottom). The states include: (I) conversion of a SpnC into a small nucleus; (II) formation of multiple nuclei in side-by-side contact; (III) small nuclei organized in a cluster; (IV) cluster of nuclei with smeared boundaries suggesting a process of fusion. Yellow arrows identify cavities within the small nuclei.

Figure 5.. Linker histone H1 accelerates the transition from SpnCs to SphCs.

(A) Fluorescence microscopy images of 30 nM (left) and 300 nM (right) Cy3 labeled tetranucleosomes incubated at 20°C in physiological salt with and without H1. (B) Representative zero-tilt cryo-ET images of SphCs obtained in the presence of H1 at 20°C, physiological salt and 10 min of incubation ordered by increasing size and labeled I-IV. (C) Tomogram slices corresponding to B; (D) 3D maps (tomograms thickness, ~90-130 nm) and models of the corresponding condensates, illustrating various stages of SphCs growth. White arrows indicate the sparsely distributed SpnCs in the surrounding areas. Orange arrows indicate fusion events between condensates.

Figure 6.. Quantitative analysis of NCP concentration and organization throughout condensate growth.

(A) Schematic representation of a series of condensate-shaped concentric contours (2 nm apart, left), volume enclosed by the nth contour (orange, middle) and volume delimited by contours n and n-1 (green, right). Internal volume and shell concentrations are calculated as described in the text. (B) The shape of the condensate was defined by 10 nm low-pass maps shown in different colors. The numbering of the maps follows the order from early (I), intermediate (II), and late stage (III) SpnCs to the small nuclei (IV) and larger SphCs without (V) and with (VI) H1. (C) Quantification of the nth-internal volume (orange) and nth-shell (green) NCP concentration corresponding to stages I through VI. Horizontal dashed lines mark the highest volume concentration beyond n= 5 (vertical dashed lines). (D) Radial distribution function g(r) of NCPs’ centers of mass for stages I through VI. Blue and pink dashed lines indicate two characteristic nucleosome’s core-to-core distances at 8 nm and 21 nm, respectively. (E) Theoretical inter-nucleosome distances for side-to-side (11 nm) and stacked (5.5 nm) configurations (left), and intra-nucleosome distance between neighboring NCPs within an array (21 nm, right).

Figure 7.. Proposed two-step mechanism of nucleosome phase separation.

(A) Initial, dispersed phase of tetranucleosome arrays; (B) organization of tetranucleosome into irregular SpnCs; (C) spherical nuclei emerging inside the spinodal phase and growing by addition of neighboring spinodal material; (D) bundle of spherical nuclei; (E) fusion of nuclei bundles enables growth into larger, energetically stable SphCs; (F) fusion of SphCs; (G) growth of SphCs by accretion of surrounding spinodal material; and (H) large SphC corresponding to the early stage of liquid droplets. Light blue, pale brown, and purple background indicate the major steps of nucleosome condensation: spinodal decomposition, spherical nuclei formation, and growth of SphCs, respectively. H1 catalyzes steps C-H. SpnCs are in yellow, and SphCs are multicolored. All states are illustrated with experimentally observed condensates.