Phase separation of Polycomb-repressive complex 1 is governed by a charged disordered region of CBX2

Abstract

Mammalian development requires effective mechanisms to repress genes whose expression would generate inappropriately specified cells. The Polycomb-repressive complex 1 (PRC1) family complexes are central to maintaining this repression. These include a set of canonical PRC1 complexes, each of which contains four core proteins, including one from the CBX family. These complexes have been shown previously to reside in membraneless organelles called Polycomb bodies, leading to speculation that canonical PRC1 might be found in a separate phase from the rest of the nucleus. We show here that reconstituted PRC1 readily phase-separates into droplets in vitro at low concentrations and physiological salt conditions. This behavior is driven by the CBX2 subunit. Point mutations in an internal domain of Cbx2 eliminate phase separation. These same point mutations eliminate the formation of puncta in cells and have been shown previously to eliminate nucleosome compaction in vitro and generate axial patterning defects in mice. Thus, the domain of CBX2 that is important for phase separation is the same domain shown previously to be important for chromatin compaction and proper development, raising the possibility of a mechanistic or evolutionary link between these activities.

Keywords: PRC1; Polycomb-repressive complex; chromatin; development; gene repression; nucleosome compaction; phase separation.

Figure 1.

PRC1 phase-separates in vitro. (A) Schematic of canonical PRC1 subunits. (B) Turbid solutions of individual PRC1 subunits at increasing protein concentrations were measured at absorbance 405 nm. All proteins are wild type except PHC2-L307R, which contains a point mutation in the SAM domain required for expression and purification. (C) Spin-down assay of monomeric enhanced GFP (mEGFP) and mEGFP-CBX2 + RING1b to visualize separation of high-concentration condensates at increasing protein concentrations. (D) Micrographs of full PRC1 complex containing mEGFP-CBX2, RING1b, BMI1, PHC2-L307R, mEGFP-CBX2 + RING1b, or individual mEGFP-tagged PRC1 subunits at 6.3 µM protein concentration in buffer containing 20 mM HEPES (pH 7.9), 100 mM KCl, and 1 mM MgSO₄. For each experiment, a representative micrograph from two independent protein preparations is shown. (E) Micrographs of mEGFP-CBX2 + RING1b at the indicated micromolar protein concentration. Scale bars, 10 µm. Black circles common across images in all figures are the result of permanent impurities in the epifluorescence microscope used in data acquisition.

Figure 2.

The LCDR of CBX2 mediates phase separation in vitro. (A) Schematic of CBX2 mutants and CBX7 protein domains. (Chromo) Chromodomain; (AT) AT hook; (HD) homology domain; (Cbox) Polycomb box. Point-mutated residues in CBX2-23KRA and CBX2-DEA are highlighted in red and blue, respectively. (B) Schematic of CBX2 protein domains. (C) Graph plotting intrinsic disorder with predictor of natural disordered regions (PONDR) using the VSL2 algorithm for CBX2. The purple bar designates the LCDR in CBX2. (D) Heat map indicating charge distribution across CBX2 for wild type and the indicated charge mutants. (E) Spin-down assay of mEGFP-tagged CBX2 mutants and CBX7 + RING1b heterodimers as well as full PRC1 complexes to visualize separation of high-concentration condensates at increasing protein concentrations. (F) Micrographs of mEGFP-tagged CBX2 mutants and CBX7 + RING1b heterodimers as well as full PRC1 complexes (containing mEGFP-CBX2 or mEGFP-CBX2-23KRA), all at 6.3 µM. For each experiment, a representative micrograph from two independent protein preparations is shown. Scale bar, 10 µm.

Figure 3.

CBX2 condensate formation is promoted by phosphorylation. (A) Phase diagram of mEGFP-CBX2 + RING1b at the indicated salt and protein concentrations. (B) Micrographs of 12.5 µM mEGFP-CBX2ΔCbox alone (unphosphorylated) or from cells coexpressing catalytic subunits of CK2 (phosphorylated). (C) Micrographs of salt-dependent reversibility assay. The top panels show 12.5 µM phosphorylated mEGFP-CBX2ΔCbox in buffer containing 100 mM KCl followed by buffer containing 500 mM KCl and then diluted back to 100 mM KCl and 2.2 µM protein concentration. The bottom panel shows phosphorylated mEGFP-CBX2ΔCbox diluted to the same final volume and protein concentration as above without transition through 500 mM KCl. Scale bars, 10 µm.

Figure 4.

Mutations in the CBX2 LCDR disrupt condensate formation in vivo. (A) Representative micrographs of 3T3 fibroblasts after 500 ng/mL doxycycline induction. The mEGFP construct in each cell line is indicated. Column panels from left to right are the GFP channel, DAPI channel, and merged images. Scale bar, 10 µm. All cell images are of z-stacks using formaldehyde-fixed cells. (B) Zoomed-in images of representative nuclei from A showing puncta or diffuse signal pattern of mEGFP fusion constructs. Scale bar, 5 µm. (C,D) High-content imaging quantification from the indicated 3T3 cell line expressing mEGFP-tagged CBX2 variants at the indicated doxycycline concentration. (C) Mean number of spots per nucleus from three replicates. (D) Distribution of the number of spots per nucleus from three pooled replicates. P-value thresholds for statistically significant differences in the distributions of puncta per cell for each doxycycline treatment, as assessed using a two-tailed Mann-Whitney U-test, are indicated with asterisks. (*) P-value ≤ 0.01; (**) P-value ≤ 0.0001. All other combinations (not shown) have a P-value of ≤0.0001 when doxycycline is present.

Figure 5.

CBX2 puncta specifically incorporate PRC1 factors in cells. (A–C) Coimmunofluorescence in 3T3 fibroblasts after 500 ng/mL doxycycline induction of mEGFP-CBX2. Column panels from left to right are the Cy3 channel, GFP channel, DAPI channel, and merged images of Cy3 and GFP for RING1b using the indicated commercially available antibodies (A), H3K27me3 (B), and H3K27ac or Rpb1 CTD (C).

Figure 6.

PRC1 ligands partition into condensates with PRC1. (A) Micrographs of 6.3 µM mEGFP-CBX2 + RING1b with 0.3 µM indicated Cy5-labeled substrate. (Left panels) GFP channel. (Right panels) Cy5 channel. (B) Micrographs of 6.3 µM mEGFP-PRC1 with 0.3 µM indicated Cy5-labeled substrates. Panels are the same as for A. (C) Micrographs of 6.3 µM unphosphorylated mEGFP-CBX2ΔCbox alone (top) or with 0.3 µM indicated Cy5-labeled heterogeneously modified (mixed) or H3K27me3-modified polynucleosomes. (D) Same as in C with a higher concentration (12.5 µM) of unphosphorylated mEGFP-CBX2ΔCbox. Scale bar, 10 µm. (E) Model of PRC1 nucleosome compaction and phase separation.