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.

Polycomb group (PcG) transcription regulatory proteins maintain cell identity by sustained repression of numerous genes. The differentiation of embryonic stem (ES) cells induces a genome-wide shift in PcG target gene expression. We investigated the effects of differentiation and protein interactions on CBX family PcG protein localization and dynamics by using fluorescence imaging. In mouse ES cells, different CBX proteins exhibited distinct distributions and mobilities. Most CBX proteins were enriched in foci known as Polycomb bodies. Focus formation did not affect CBX protein mobilities, and the foci dispersed during ES cell differentiation. The mobilities of CBX proteins increased upon the induction of differentiation and decreased as differentiation progressed. The deletion of the chromobox, which mediates interactions with RING1B, prevented the immobilization of CBX proteins. In contrast, the deletion of the chromodomain, which can bind trimethylated lysine 27 of histone H3, had little effect on CBX protein dynamics. The distributions and mobilities of most CBX proteins corresponded to those of CBX-RING1B complexes detected by using bimolecular fluorescence complementation analysis. Epigenetic reprogramming during ES cell differentiation is therefore associated with global changes in the subnuclear distributions and dynamics of CBX protein complexes.

Polycomb group (PcG) proteins control the epigenetic inheritance of transcription regulatory states during development. Progression from pluripotency to differentiation requires the concurrent activation and repression of different PcG target genes. We found that REST and nine REST-associated proteins copurified with Cbx family PcG proteins from mouse embryonic stem (ES) cells. REST interacted with Cbx proteins in live cells and coprecipitated with endogenous Ring1b. Endogenous PRC1 subunits occupied all sites tested that were bound by REST in ES cells. Antibodies directed against different PRC1 subunits precipitated proximal versus distal RE1 elements with opposite relative efficiencies, suggesting that PRC1 bound different sites in distinct configurations. Deletion of the amino-terminal region of REST (Rest(ΔN) knockout) as well as short hairpin RNA depletion of REST (REST knockdown) in ES cells reduced PRC1 binding at distal RE1 elements and increased PRC1 binding at proximal RE1 elements. Rest(ΔN) and PRC1 subunit knockout as well as REST and PRC1 subunit knockdown had similar relative effects on transcription of neuronal genes in ES cells, derepressing genes with distal, but not genes with proximal, RE1 elements. In differentiating neurons, Rest(ΔN) knockout reduced PRC1 occupancy and derepressed transcription at distal RE1 elements but increased PRC1 occupancy and repressed transcription at proximal RE1 elements. The opposite effects of REST on PRC1 occupancy at different RE1 elements contributed to the gene-specific control of PRC1 functions during ES cell differentiation.

Compartmentalization by liquid-liquid phase separation is implicated in transcription. It remains unclear whether and how transcriptional condensates accelerate the search of transcriptional regulatory factors for their target sites. Furthermore, the molecular mechanisms by which regulatory factors nucleate on chromatin to assemble transcriptional condensates remain incompletely understood. The CBX-PRC1 complexes compartmentalize key developmental regulators for repression through phase-separated condensates driven by the chromobox 2 (CBX2) protein. Here, by using live-cell single-molecule imaging, we show that CBX2 nucleates on chromatin independently of H3K27me3 and CBX-PRC1. The interactions between CBX2 and DNA are essential for nucleating CBX-PRC1 on chromatin to assemble condensates. The assembled condensates shorten 3D diffusion time and reduce trials for finding specific sites through revisiting the same or adjacent sites repetitively, thereby accelerating CBX2 in searching for target sites. Overall, our data suggest a generic mechanism by which transcriptional regulatory factors nucleate to assemble condensates that accelerate their target-search process.

The Polycomb PRC1 plays essential roles in development and disease pathogenesis. Targeting of PRC1 to chromatin is thought to be mediated by the Cbx family proteins (Cbx2/4/6/7/8) binding to histone H3 with a K27me3 modification (H3K27me3). Despite this prevailing view, the molecular mechanisms of targeting remain poorly understood. Here, by combining live-cell single-molecule tracking (SMT) and genetic engineering, we reveal that H3K27me3 contributes significantly to the targeting of Cbx7 and Cbx8 to chromatin, but less to Cbx2, Cbx4, and Cbx6. Genetic disruption of the complex formation of PRC1 facilitates the targeting of Cbx7 to chromatin. Biochemical analyses uncover that the CD and AT-hook-like (ATL) motif of Cbx7 constitute a functional DNA-binding unit. Live-cell SMT of Cbx7 mutants demonstrates that Cbx7 is targeted to chromatin by co-recognizing of H3K27me3 and DNA. Our data suggest a novel hierarchical cooperation mechanism by which histone modifications and DNA coordinate to target chromatin regulatory complexes.

This work presents a general method for determining single-molecule intramolecular dynamics in biomolecules by using a reporter fluorophore, whose fluorescence is quenched or partially quenched as a result of intramolecular motion, and a remote observer fluorophore. These fluorophores were excited independently with two different lasers, and the ratio of the two fluorophores' fluorescence was calculated. The time-varying ratio was then filtered to reduce contributions from molecules outside the overlapped laser volume and then correlated. The rates of opening and closing of a DNA hairpin were measured by using both fluorescence correlation spectroscopy and this method for comparison. We found at 50 pM, where molecules were studied one by one as they diffused through the probe volume, we obtained accurate opening and closing rates and could also measure dynamic heterogeneity. To demonstrate applicability to a more complex biological molecule we then probed intramolecular motions in the dimer of a human telomerase RNA fragment (hTR_380-444), in the presence of an excess of monomer. The motion was found to occur on the time scale of 180-750 μs and slowed with increasing magnesium ion concentration. Blocking experiments using complementary oligonucleotides suggested that the motion involves substantial changes in dimer tertiary structure. This method appears to be a general method for selectively studying intramolecular motion in large biomolecules or complexes.