Principles of assembly and regulation of condensates of Polycomb repressive complex 1 through phase separation

Abstract

PRC1 (Polycomb repressive complex 1) plays a significant role in cellular differentiation and development by repressing lineage-inappropriate genes. PRC1 proteins phase separate to form Polycomb condensates (bodies) that are multi-component hubs for silencing Polycomb target genes; however, the molecular principles that underpin the condensate assembly and biophysical properties remain unknown. Here, by using biochemical reconstitution, cellular imaging, and multiscale molecular simulations, we show that PRC1 condensates are assembled via a scaffold-client liquid-liquid phase separation (LLPS) model by which Chromobox 2 (CBX2) is the scaffold and other subunits of the CBX2-PRC1 complex act as clients. The clients induce a reentrant phase transition of CBX2 condensates in a concentration-dependent manner. The composition of the multi-component, heterotypic LLPS systems directs the assembly and biophysical properties of CBX2-PRC1 condensates and selectively promotes the formation of CBX4-PRC1 condensates, but specifically dissolves condensates of CBX6-, CBX7-, and CBX8-PRC1. Additionally, the composition of CBX2-PRC1 condensates controls the enrichment of CBX4-, CBX7-, and CBX8-PRC1 into condensates but the exclusion of CBX6-PRC1 from condensates. Our results show the composition- and stoichiometry-dependent scaffold-client assembly of multi-component PRC1 condensates and supply a conceptual framework underlying the molecular basis and dynamics of Polycomb condensate assembly.

How a disordered linker in the Polycomb protein Polyhomeotic tunes phase separation and oligomerization

“…The ability of CBX proteins to drive phase separation of mammalian PRC1 and chromatin has also been investigated 60,62 . An IDR in CBX2 undergoes phase separation in vitro 36,60,64 , and Cbx2 forms condensates with properties consistent with phase separation in vivo 60,62,66 and is implicated in chromatin architecture and gene regulation in vivo 68,70 . PHC and CBX activities must be coordinated in PRC1, and recent work indicates a prominent role for Cbx2 in controlling mammalian PRC1 condensates 64 .…”

“…An IDR in CBX2 undergoes phase separation in vitro 36,60,64 , and Cbx2 forms condensates with properties consistent with phase separation in vivo 60,62,66 and is implicated in chromatin architecture and gene regulation in vivo 68,70 . PHC and CBX activities must be coordinated in PRC1, and recent work indicates a prominent role for Cbx2 in controlling mammalian PRC1 condensates 64 . In Drosophila embryos, Ph condensates form in mutants lacking Pc, while Pc condensates do not form in mutants lacking Ph 44 .…”

Sparse CBX2 nucleates many Polycomb proteins to promote facultative heterochromatinization of Polycomb target genes

“…The composition-dependent control of condensate assembly by phase separation is an emerging concept underlying the biophysical properties of Polycomb condensates 39,47 . CBX2 is an intrinsically disordered protein with alternating positively and negatively charged regions that drive the formation of homo-typic and hetero-typic CBX-PRC1 condensates 36,38,39,47 . We show that PRC2, which does not form condensates in vitro, is partitioned into CBX2-PRC1 condensates in a composition-dependent manner, suggesting that CBX2-PRC1 controls the compartmentalization of PRC2.…”

“…To create the positive to alanine (P2A) mutations within the ATH, ATL, and HPCR motifs for the CBX2 TM protein expression vector, PCR primers were developed to change alanine residues, and individual fragments were assembled to give the complete vector. The PRC1 expression vectors containing RING1B, MEL18, and PHC1 without a fluorophore were generated in a previous study 39 .…”

Dynamic PRC1-CBX8 stabilizes a porous structure of chromatin condensates

“…A scaffold-client based phase separation model has recently been proposed for PRC1-CBX2 complexes 28,35 , where CBX2 35 or chromatin 28 act as a scaffold that induces phase separation of PRC1 proteins. Since PRC1 C8 is insufficient to phase-separate without chromatin (Fig 1e ), we wished to test if chromatin might act as a scaffold that concentrates PRC1 C8 and induces phase separation.…”