Abstract
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.
Highlights
From the ‡Chromatinomics, Interdisciplinary Research Institute, Univ
Antibodies directed against two components of the PRC1, RING1, and PCGF3 were used to identify PRC1-like-containing fractions
Gel filtration analyses of nuclear-cell extracts derived from TAPtagged cell lines showed that most CBX proteins are present in fractions of molecular weight higher than 1 MDa, and smaller protein amounts are present in fractions of ϳ200 to 500 kDa (Fig. 1A)
Summary
PcG, Polycomb group; TAP, Tandem affinity purification; LC, liquid chromatography; MS/MS, Tandem mass spectrometry; HA, Hemagglutinin; GST, Glutathione S-transferase; PRC, Polycomb repressive complex; Pc box, Polycomb box; Pc, Polycomb; Sce, Sex combs extra; Ph, Polyhomeotic; Psc, Posterior sex combs; GFP, Green fluorescent protein; DBD, DNA binding domain; UAS, Upstream activating sequence; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ORF, Open reading frame; PBS, Phosphate buffer saline; TEV, Tobacco etch virus; BiFC, Bimolecular fluorescence complementation; WB, Western blot; IP, immunoprecipitation; CBX, Chromobox homolog; PCGF, Polycomb group ring finger; BMI, B lymphoma Mo-MLV insertion; RNF, Ring Finger protein; PHC, Polyhomeotic homolog. Null mutations of Eed and Suz PRC2 subunits abolish H3K27 trimethylation, but do not prevent the recruitment of PRC1 proteins to either the inactive X chromosome or to many of their target genes [39, 40]. It is unclear whether H3K27me recognition is necessary or sufficient for stable chromatin association by CBX proteins or whether other interactions, potentially mediated by additional components of the PRC1 complex, are involved. We show that protein kinase 2 binds to PRC1 protein complexes conferring to Polycomb complexes an additional kinase activity
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