Interaction Proteomics Analysis of Polycomb Proteins Defines Distinct PRC1 Complexes in Mammalian Cells

Polycomb group (PcG) proteins maintain transcriptional repression of hundreds of genes involved in development, signaling or cancer using chromatin-based epigenetic mechanisms. Biochemical studies in Drosophila have revealed that PcG proteins associate in at least two classes of protein complexes known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Sex combs extra (Sce), Polyhomeotic (Ph), and Posterior sex combs (Psc). Each of these proteins has multiple orthologs in vertebrates classified respectively as the CBX, RING1/RNF2, PHC, and BMI1/PCGF families. Mammalian genomes encode five CBX family members (CBX2, CBX4, CBX6, CBX7, and CBX8) that are believed to have distinct biological functions. Here, we applied a tandem affinity purification (TAP) approach coupled with tandem mass spectrometry (MS/MS) methodologies in order to identify interacting partners of CBX family proteins under the same experimental conditions. Our analysis identified with high confidence about 20 proteins co-eluted with CBX2 and CBX7 tagged proteins, about 40 with CBX4, and around 60 with CBX6 and CBX8. We provide evidences that the CBX family proteins are mutually exclusive and define distinct PRC1-like protein complexes. CBX proteins also interact with different efficiencies with the other PRC1 components. Among the novel CBX interacting partners, protein kinase 2 associates with all CBX-PRC1 protein complexes, whereas 14-3-3 proteins specifically bind to CBX4. 14-3-3 protein binding to CBX4 appears to modulate the interaction between CBX4 and the BMI1/PCGF components of PRC1, but has no effect on CBX4-RING1/RNF2 interaction. Finally, we suggest that differences in CBX protein interactions would account, at least in part, for distinct subnuclear localization of the CBX family members. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.002642, 1-23, 2011.