Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

Abstract

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

Compartment-dependent chromatin interaction dynamics revealed by liquid chromatin Hi-C

“…The compacted states of polycomb and HP1α bound chromatin appear to form via a similar phase-separation mechanism mediated by multivalent interactions between specific CBX homologs. In vitro and in vivo, both CBX2 (polycomb subunit) and CBX5 (HP1α) are capable of forming condensates of polycomb bodies and constitutive heterochromatin, respectively (Larson et al, 2017;Plys et al, 2019;Tatavosian et al, 2019). Our data indicate that these different structures or condensates and associated chromatin have very different properties.…”

Temporal scaling in developmental gene networks by epigenetic timing control

“…H3K27me3 marks repress gene expression by promoting the association of nucleosomes to form condensed, polymerase-inaccessible assemblies. For instance, H3K27me3 recruits Polycomb Repressive Complex (PRC1), which can self-interact to form compacted, phase-separated chromatin domains (Plys et al, 2018;Tatavosian et al, 2018) . Therefore, we considered a second model, where H3K27me3 levels do not determine gene synthesis rates per se , but influence chromatin compaction dynamics to modulate promoter accessibility and gene transcription ( Figure 4D).…”

“…In standard models of epigenetic switching Zhang et al, 2014) , where activation is safeguarded purely by histone methylation/demethylation dynamics, activation time constants are extremely sensitive to small changes in epigenetic-modifying enzyme activity ( Figure 4A-C, left); these models cannot explain how activation times can be robustly set and tunably controlled over long timescales, as observed ( Figure 3D). It is well established that H3K27me3 modifications repress gene expression by promoting chromatin compaction; furthermore, emerging evidence indicates that they do so by recruiting protein complexes that self-associate to form phase-separated condensates (Plys et al, 2018;Tatavosian et al, 2018) , a physical process that can generate the cooperativity needed for all-or-none transitions in the methylation-compaction model ( Figure 4E). Importantly, for activation times to be tunable in this model, nucleosomes must retain some self-interaction affinity in their demethylated states, such that methylation promotes but is not strictly necessary for nucleosomal association.…”

On the relation of phase separation and Hi-C maps to epigenetics

“…The similarity between HP1-and Pc-domains/complexes extends to their ability to phase separate and to generate B-type heterochromatic compartmental domains in Hi-C maps. For one, the mammalian Pc homologue CBX2, like HP1 proteins, can promote phase separation that is dependent upon amino-acids in CBX2 necessary for nucleosome fibre compaction (197,198). Second, the histone modifications H3K9me2/3 and H3K27me3 are diagnostic for HP1-and Pc-dependent domains/complexes respectively (Table 1) (199) and are used, inter alia, to define B-type compartmental domains (170).…”