Overexpressing YFP-Cbx2 and HaloTag (HT)-Cbx2 (fusion proteins) in transgenic PGK12.1 mouse embryonic stem cells in vivo and examining them by fluorescence live-cell imaging showed that both YFP-CBX2 and HT-CBX2 formed condensates in living wild-type mES (particle size and count) cells and that the CBX2 condensates exhibit liquid-like properties (FRAP). Under the basal expression, the protein level of YFP-CBX2 was similar to that of the endogenous protein CBX2, the distribution of YFP-CBX2 and HT-CBX2 in Cbx2-/- mES cells was similar to that in wild-type mES cells (particle size and count by microscopy). YFP-CBX2 condensates colocalized with condensates of other PRC1 complex subunits RING1B and PHC1 in vivo. Immunofluorescence of H3K27me3 and YFP-CBX2 showed that CBX2 condensates colocalize with chromatin with the dense H3K27me3 mark suggesting that PcG-targeted genes are recruited to CBX2 condensates, or vice versa. CBX2 condensates were protein concentration-dependent. Recombinant GST-CBX2-FLAG (GST-CBX2) did not undergo LLPS in vitro at high salt concentration or in the presence of glutathione. After dialyzing the high salt of GST-CBX2 fusion to 140 mM NaCl at 4 °C overnight and transferring 10 μl of sample to coverslip CBX2 condensates with a size of a few hundred nanometers were observed by DIC microscopy. So did FLAG-CBX2. In vitro phase separation of CBX2 was protein concentration-dependent. In vitro treatment of CBX2 condensates with increasing concentrations of NaCl and Triton X-100 caused a reduction in the number of CBX2 condensates (particle size and count by microscopy). Fluorescent dye-labelled 24-bp double-stranded DNA and similarly labeled nucleosomes did not form condensates, however, in the presence of CBX2, DNA was concentrated and CBX2 condensates colocalized with the concentrated DNA or nucleosomes. Site-directed mutagenesis studies 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. Genetic engineering studies implied 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 (particle size and count) (PMID:30514760). EGFP-CBX2+ RING1b could form dense spherical droplets in the presence of volume excluder that increased in size as a function of concentration and could fuse with each other (particle size and count bz microscopy). Mutational studies suggested that the positive charges within the CBX2 LCDR are critical for phase separation in vitro in addition to the previously described roles in chromatin compaction and proper axial patterning in vivo, in mice. At higher salt concentration, the preformed droplets drastically reduced in number and size (particle size and count by microscopy). Reducing the salt concentration to 100 mM KCl resulted in reformation of droplets. In vivo results recapitulated the findings of in vitro assays and underscore the importance of positively charged residues in the CBX2 LCDR for PRC1 phase separation (PMID:31171700).