Current Understanding of Molecular Phase Separation in Chromosomes

Abstract

Biomolecular phase separation denotes the demixing of a specific set of intracellular components without membrane encapsulation. Recent studies have found that biomolecular phase separation is involved in a wide range of cellular processes. In particular, phase separation is involved in the formation and regulation of chromosome structures at various levels. Here, we review the current understanding of biomolecular phase separation related to chromosomes. First, we discuss the fundamental principles of phase separation and introduce several examples of nuclear/chromosomal biomolecular assemblies formed by phase separation. We also briefly explain the experimental and computational methods used to study phase separation in chromosomes. Finally, we discuss a recent phase separation model, termed bridging-induced phase separation (BIPS), which can explain the formation of local chromosome structures.

Keywords: biomolecular phase separation; bridging-induced phase separation; chromosomes; cohesin; compartments; intrinsically disordered proteins; multivalent DNA-binding proteins; stickers-and-spacers framework.

Figure 1

Biomolecular condensates in the nucleus: A and B compartments, nucleolus, paraspeckles, and transcriptional condensates. Chromosomes are largely segregated via phase separation into two compartments: euchromatin (A, red) and heterochromatin (B, blue). Phase separation is also involved in the formation and regulation of membraneless organelles such as the nucleolus (gray), transcription condensates (magenta), and paraspeckles (green) in the nucleus.

Figure 2

Phase diagrams of prototypical two-component systems. Phase diagrams for (A) the monomer-monomer system and (B) the polymer-monomer system. Blue and green dots represent different types of unit molecules. The x-axis indicates the concentration of unit molecules of the blue species, and the y-axis indicates the system temperature. In panel B, the valence of a multimer, M, is set to three. Multimerization results in the expansion of the two-phase regime. (C) Anatomy of a phase diagram (see text for the definitions of different concentrations). The x-axis shows the multimer concentration, and has a different scale from panels A and B. The multimer concentration, however, is proportional to the unit molecule concentration, and the two can be interchangeably used.

Figure 3

Techniques to observe phase separation in chromosomes. (A–D) Reconstitution of biomolecular condensates in vitro. (A) Fluorescence microscopy image showing liquid droplets of fluorescently labeled PRM-SH3-6His in the presence of Ni²⁺ ions obtained with a wide-field microscope (mCherry was fused with the protein) [38]. (B) DIC microscopy image showing unlabeled droplets of PRM-SH3-6His. (C) Single-molecule DNA tethered assay with fluorescent-stained DNA (green) and labeled cohesin proteins (red) forming condensates with DNA [20]. (D) AFM image of an unlabeled cohesin/DNA condensate [20]. (E–H) Live-cell imaging of biomolecular condensates. (E) Confocal microscopy image showing HP1α in the nucleus of a HCT116 cell. The condensates of Dendra2-tagged HP1α are clearly visible. (F) Super-resolution images of the Dendra2-Pol II cluster in a HCT116 cell obtained using a PALM microscope [19]. (G) Construct for “optoDroplet,” combining optogenetic-induced oligomerization with IDR-driven phase separation. The IDR (magenta) driving phase separation is fused with a fluorescent protein (red) and Cry2, a protein domain that forms oligomers upon blue-light activation [116]. (H) Blue-light activation induced the oligomerization of Cry2, which controls the interactions between IDRs to induce phase separation. (I,J) Genomic analysis via Hi-C. (I) Experimental scheme for Hi-C experiments. (J) Example genome-wide contact map (Hi-C map of compartmentalization). The X and Y axes denote the genomic positions in a chromosome. A and B compartments are shown in the Hi-C map by the “checkerboard” pattern. High frequencies of contacts are colored red, and low frequencies, white.

Figure 4

Criteria for liquidity. (A) The spherical shape of a liquid droplet. To quantify how much the droplet is close to a spherical shape, circularity is calculated by measuring the area (magenta regions) and the perimeter of a droplet (blue boundaries). The circularity is defined by 4πA/P² and it ranges from 0 (very different from a circular shape) to 1 (a circular shape). (B) Merging of two distinct droplets. (C) FRAP experiment showing exchangeability of molecules between background solution and a droplet (top) and diffusability in a single droplet (bottom). (D) Reversibility test. If background proteins are depleted, a liquid droplet is dissolved into a solute phase.

Figure 5

BIPS versus SIPS. (A) Cartoon of a multivalent DNA-binding protein that has at least two DNA-binding sites. DNA-binding sites of the protein are depicted as orange circles, and the protein is denoted as a blue circle. (B) Schematic of the BIPS model. Two DNA-binding sites per protein are sufficient for condensation, and a long DNA molecule is irreplaceable in this mechanism. (C) Cartoon of a multivalent protein-binding protein that induces typical phase separation. Yellow circles on the protein (blue circle) depict protein binding sites. (D) Typical phase separation mechanism (SIPS), which uses multivalent protein-protein interactions. At least three binding sites are necessary for phase separation, and DNA plays an auxiliary role in this process. (E,F) Dependence of the protein-DNA cluster size on the length of DNA shown in the previous study of cohesin-mediated BIPS [20]. (E) Cartoons of possible protein-DNA complex topologies for a range of DNA lengths and (F) a plot showing cluster size versus DNA length [20]. With <3 kbp of DNA, a single protein binds to DNA with no cooperativity (blue line). With ~3 kbp DNA, multivalent DNA-binding proteins can bridge a DNA to form a loop. For longer DNA (>3 kbp), a larger cluster can be formed, and the cluster size scales as a power law with the DNA length (red line).