The solid and liquid states of chromatin

Abstract

The review begins with a concise description of the principles of phase separation. This is followed by a comprehensive section on phase separation of chromatin, in which we recount the 60 years history of chromatin aggregation studies, discuss the evidence that chromatin aggregation intrinsically is a physiologically relevant liquid-solid phase separation (LSPS) process driven by chromatin self-interaction, and highlight the recent findings that under specific solution conditions chromatin can undergo liquid-liquid phase separation (LLPS) rather than LSPS. In the next section of the review, we discuss how certain chromatin-associated proteins undergo LLPS in vitro and in vivo. Some chromatin-binding proteins undergo LLPS in purified form in near-physiological ionic strength buffers while others will do so only in the presence of DNA, nucleosomes, or chromatin. The final section of the review evaluates the solid and liquid states of chromatin in the nucleus. While chromatin behaves as an immobile solid on the mesoscale, nucleosomes are mobile on the nanoscale. We discuss how this dual nature of chromatin, which fits well the concept of viscoelasticity, contributes to genome structure, emphasizing the dominant role of chromatin self-interaction.

Fig. 1

Schematic illustration of the different conformational states of an array of nucleosomes in solution. Depending on the extent of cation-dependent DNA charge neutralization, nucleosomal arrays can exist in an extended 10-nm conformation, a folded 30-nm conformation, or as a phase-separated condensate

Fig. 2

Information obtained from the differential centrifugation assay. In this assay, chromatin is combined with MgCl₂ to the indicated value, pelleted in a microfuge for 10 min, and A₂₆₀ of the supernatant measured. Data are expressed as the fraction of the chromatin in the supernatant as a function of MgCl₂ concentration. Results are shown for native 12-mer nucleosomal arrays (red points and line). Factors that shift the native curve to the left or right are indicated

Fig. 3

Visualization of chromatin condensates formed in MgCl₂ by fluorescence and transmission electron microscopy. A Condensates were formed from Alexa 488-labeled 12-mer nucleosomal arrays in 4 mM MgCl₂ and analyzed by fluorescence microscopy as described in reference [34]. Taken from Fig. 1D of reference [34] with permission. B Condensates were formed from 12-mer nucleosomal arrays in 5 mM MgCl₂ and analyzed by transmission electron microscopy with negative staining as described in reference [34]

Fig. 4

Two-color mixing assay for determination of the material state of chromatin condensates. 12-mer nucleosomal arrays (60 bp linkers) were reconstituted with recombinant Xenopus histone octamers in which histone H4 was labeled with Alexa 488 or Alexa 649. Labeled arrays were incubated in either 4 mM MgCl₂ (A) or 150 mM K Acetate/1 mM Mg Acetate plus 0.1 mg/ml BSA, 5 mM DTT, and 5% glycerol (B) to form condensates. The green and red labeled condensates were then mixed for 20 min, followed by fluorescence microscopy. Shown are the images obtained in the green channel (left), red channel (center), and overlay (right) after the 20 min incubation. Data courtesy of Dr. Thomas Tolsma

Fig. 5

Nuclear bodies visualized by transmission electron microscopy. A K562 cell nucleus obtained by fixation with 4% paraformaldehyde and embedding in Epon 812 was imaged by electron spectroscopic imaging at 250 eV energy loss. This generates high contrast images of the biological specimen where contrast is related to the mass density. The image shows examples of condensed chromatin, the nucleolus, a Cajal body, and an interchromatin granule cluster (splicing factor compartment). The brightness was increased in the regions containing these structures to highlight their locations. The classification is based upon their morphologies in the transmission electron microscope. The scale bar represents 1 µm

Fig. 6

Larger nuclear compartments of the interphase nucleus. The image highlights the three large nuclear compartments present in the interphase nucleus. A living mouse C3H10T1/2 cell nucleus expressing SC35-GFP and counterstained with Hoechst 33,342 is shown following deconvolution. The SC35 (green) contrasts the splicing factor compartments and negatively contrasts the chromatin and nucleoli. The Hoechst contrasts the chromatin and negatively contrasts the splicing factor compartments and nucleoli. Circles highlight examples of large chromatin structures (pericentric heterochromatin) and splicing factor compartments in the respective images. No in the SC35 image set represents the location of the nucleoli. The scale bar represents 1 µm

Fig. 7

Phase-separated compartments formed from overexpression of histone deacetylase 4. SK-N-SH neuroblastoma cells were transfected with histone deacetylase 4-green fluorescent protein (HDAC4-GFP) expression vector and counterstained with Hoechst 33342 (DNA). The circles highlight condensates that form spontaneously upon overexpression of HDAC4-GFP. These are found in chromatin-poor regions of the nucleus outside of the nucleolus. The box highlights a heterochromatic region of the nucleus. Note the inverse relationship between HDAC4-GFP concentration and chromatin concentration, further highlighting that the condensates are forming outside of dense regions of chromatin. The scale bar represents 5 µm

Fig. 8

Hierarchical chromatin organization in the nucleus—a simplified view. The negatively charged 10-nm fiber is compacted into chromatin domains (e.g., topologically associating domain [TAD]/contact domain/loop domain) [–139]. The domains are clustered over long distances to form chromatin compartments [147]. Compartments generally represent a transcriptionally active chromatin state (compartment A) and an inactive chromatin state (compartment B). A single interphase chromosome is occupied in a chromosome territory (highlighted as different colors) [219]. This illustration was reproduced with modifications from [220].

Fig. 9

Visualization of dynamic chromatin motion. Schemes for LacO/LacI-GFP (A) and CRISPR-based chromatin labeling (B). C Constrained diffusion motion of chromatin: mean square displacement (MSD) plots (± SD among cells) of single nucleosomes in living (black) and formaldehyde-fixed (red) human RPE-1 cells over time (0.05 to 3 s) (data from [185]). D Scheme for single-nucleosome tracking. A small number of nucleosomes are labeled with photoactivatable GFP or other fluorescent tag to get sparse labeling. E Multiscale model of chromatin integrating three resolutions: atomistic (left), amino acid/base pair (center), and nucleosome (right) [128]. These models allow the exploration into how atomistic and residue level variations affect the structure and dynamics of chromatin fibers and domains. Illustration was reproduced from [189]. F (left) Scheme of chromatin heat map. In the heat map, small movements for 50 ms are shown in blue, and large movements are shown in red. (center) PALM (photo-activated localization microscopy) super resolution image and the chromatin heat map of a living mouse ES (embryonic stem) cell (right). Heterochromatin regions (nuclear periphery and pericentromeric heterochromatin) show dark blue. Data reproduced from [133]

Fig. 10

Local chromatin motion in the cell. A (left) Decondensed chromatin in cells treated with histone deacetylase (HDAC) inhibitor TSA showed increased chromatin movements because of weakening nucleosome–nucleosome interactions and subsequently less local chromatin constraint. (center) The typical state of chromatin: chromatin domains are organized by local nucleosome–nucleosome interactions and global folding by cohesin. (right) Cohesin loss leads to less constraint and a resultant increase in chromatin motion. B (left and center) Cluster/condensate of active RNAPII and transcription factors (blue sphere) can work as a transient hub (green sphere) to weakly connect multiple chromatin domains and to globally constrain chromatin motion. (right) RNAPII inhibition or its rapid depletion releases the chromatin constraints and increases chromatin motion. C–F Cartoons showing how various nuclear condensates are organized with different chromatin substrates. The orange line represents chromatin. Chromatin seems solid at the mesoscale and behaves like a liquid at the nanoscale, which is consistent with its viscoelastic property [216] (G). Black arrow designates active transcription start site, and green squiggled lines show RNA. Schemes in Panels A and B were reproduced from [220] with modifications