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.During differentiation, the pluripotency of embryonic stem (ES) cells is restricted by epigenetic changes (7, 11). Polycomb group (PcG) proteins contribute to the stable inheritance of both pluripotent and differentiated cell states (48, 50). These functions implicate PcG proteins in the control of the transition between pluripotency and differentiation. Genome-wide studies of PcG protein binding in mammalian cells have identified hundreds of genes that bind PcG proteins (8,9,31). The expression of many of these genes is altered during ES cell differentiation.Biochemical studies of PcG proteins have identified two Polycomb repressive complexes (PRCs), PRC1 and PRC2 (12,28,52). PRC2 has lysine methyltransferase activity for K27 of histone H3; PRC1 contains a subunit (Pc in Drosophila and CBX family proteins in mammals) that can bind trimethyl-K27 H3 in vitro (6, 12). Many genes that are bound by PRC1 are enriched in H3 K27 trimethylation (8, 9). These observations, in combination with epistatic relationships among mutations in Drosophila PcG genes (59), have given rise to the model that histone H3 K27 trimethylation by PRC2 is required for the recruitment of the PRC1 complex to specific genes. These results have also been interpreted to indicate that PRC2 initiates silencing and that PRC1 maintains the silenced state.Genetic studies of mice indicate that the functions of PcG proteins are at least in part nonoverlapping since the ablation of genes encoding different PcG proteins produces distinct phenotypes (2,14,17,25,32,38,39,49,(54)(55)(56)(57). Null mutations in the EED and Suz12 subunits of PRC2 eliminate histone H3 K27 trimethylation, but do not prevent the r...

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.DOI: http://dx.doi.org/10.7554/eLife.17667.001

The expression and spatial profile of multiple RNA species at high precision in single cells is key information for understanding cellular behaviors and functions. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinct fluorophores. Here, a DNA-sequence-encoded fluorescence barcoding method based on thermodynamically tuning DNA hybridization was applied for fluorescence tagging RNAs, which allows for highly multiplexed imaging of RNA in single cells with single-molecule and singlenucleotide resolution.