Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 294, Issue 5, Pages 1451-1463Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.006620
Keywords
Polycomb; epigenetics; heterochromatin; chromatin; gene regulation; histone methylation; histone; chromatin modification; chromatin structure; chromatin regulation; CBX2; liquid-liquid phase separation; PcG; phase separation; PRC1
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Funding
- NCI, National Institutes of Health [R03CA191443]
- National Science Foundation [CHE-1500285]
- NIGMS, National Institutes of Health [R01GM098401]
- Office of Research Services (ORS) at the University of Colorado Denver
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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.
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