The Role of Phase Separation in Heterochromatin Formation, Function, and Regulation

Abstract

In eukaryotic cells, structures called heterochromatin play critical roles in nuclear processes ranging from gene repression to chromosome segregation. Biochemical and in vivo studies over the past several decades have implied that the diverse functions of heterochromatin rely on the ability of these structures to spread across large regions of the genome, to compact the underlying DNA, and to recruit different types of activities. Recent observations have suggested that heterochromatin may possess liquid droplet-like properties. Here, we discuss how these observations provide a new perspective on the mechanisms for the assembly, regulation, and functions of heterochromatin.

Figure 1.

Model for the different physical states of chromatin. (a) Physical states accessible to HP1–chromatin complexes in vitro may inform on the organization of functional chromatin states. By altering the physical state of chromatin via tuning local conditions and binding interactions, the chromatin can be made accessible to transcription or made more condensed and refractive to gene activation. (inset) Domain architecture of HP1 proteins (green); the N-terminal extension (NTE), chromodomain (CD), Hinge (H), chromoshadow domain (CSD), and the C-terminal extension (CTE) are shown. Also shown are two pathways of altering HP1α phase separation behavior by a ligand that can bind at the CSD–CSD dimer interface (yellow molecule) and by phosphorylation on the NTE (red P). (1) Active euchromatin is highly acetylated (acetyltransferase in green) and less compact than soluble heterochromatin, resulting in increased accessibility of the underlying chromatin to the transcription machinery (RNA polymerase in orange and transcription factor in pink). (2) Soluble HP1α heterochromatin consists of a complex between HP1α molecules and chromatin with H3K9 methyl marks. The H3K9 methylase is shown in purple. In this state, spreading can occur via oligomerization of HP1α molecules across chromatin coupled to the action of the H3K9 methylase (purple). (3) Increase in HP1α concentration above the critical concentration for phase separation may drive soluble heterochromatin into a phase-separated droplet state. This process can be regulated by ligands that bind the CSD–CSD interface and by changes in phosphorylation of HP1α. The droplet state may promote gene silencing by inhibiting accumulation of the transcription machinery inside the droplets, by inhibiting access to the DNA template, or by inhibiting the activity of the transcription machinery. In contrast, accumulation of heterochromatin components such as the H3K9 methylase may be promoted through a combination of specific and nonspecific interactions with other components of the HP1α phase. (4) Certain heterochromatin components, solutes, and counterions may stabilize a gel-like state of HP1α heterochromatin, which may be less permeable than the droplet state and may serve a more structural role. (b) Model for some specific types of interactions affecting HP1α phase separation behavior. (1) The positive residues within the disordered hinge region of HP1 interact with the negatively charged DNA polyanion to form a specific type of coacervate that promotes compaction of the underlying chromatin. (2) Factors such as kinases and methylases that both interact and modify HP1 encourage spreading. In addition to linear spreading, distal chromatin regions with like qualities may partition into the silenced HP1α-rich phase while noninteracting components or those displaying qualities not permissive for solvation in the heterochromatin phase may be excluded. (3) Inhibition of HP1α spreading with HP1 paralogs such as HP1β (orange) that are not competent to phase separate or with factors that dissolve HP1α assembly (green factor bound to HP1α) could serve as boundaries to heterochromatin spread.

Figure 2.

Regulated dynamics of phase separation. Multiple physical states of heterochromatin are represented by the dial that can be moved via multiple inputs. The critical concentrations of HP1α required for transitions between soluble to liquid-liquid demixing to gel-like states are proposed to be sensitive to environmental conditions that tune the biophysical characteristics of the protein and binding partners that directly affect protein assembly and solvent conditions.