A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation.

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.

The zebrafish genes encoding the Polycomb repressive complex (PRC) 1.

Polycomb repression controls regulation of hundreds of genes involved in development, signalling or cancer and is mediated by essentially two classes of chromatin-associated protein complexes, the Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). PRC2 trimethylates histone H3 at Lysine 27 and this H3K27me3 epigenetic mark serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates. In particular, mammalian genomes encode five Pc family members (CBX2, 4, 6, 7 and 8), six Psc family members (BMI1, PCGF1, 2, 3, 5, and 6), three Ph family members (PHC1, 2 and 3) and two Sce family members (RING1 and RNF2) generating an enormous scope for potential combinatorial diversity. In order to identify the corresponding PRC1 genes in zebrafish, homology searches were undertaken and allowed the identification of a total of 19 genes. Using phylogenetic, gene organization and gene location analyses, these genes were classified. The zebrafish genes encoding the PRC1 protein complex include 8 Pc orthologs (cbx2, cbx4, cbx6a, cbx6b, cbx7a, cbx7b, cbx8a and cbx8b), 6 Psc orthologs (bmi1a, bmi1b, pcgf1, pcgf5a, pcgf5b and pcgf6), 4 Ph orthologs (phc1, phc2a, phc2b and phc3) and a single Sce ortholog (rnf2). Our results indicate that the potentially high number of distinct PRC1 protein complexes generated by the components combinatorial appeared early in the vertebrate evolution. In addition to conserved gene organization and syntenies, transcript analyses revealed that transcriptional regulation leading to various isoforms syntheses is also conserved at genes encoding the PRC1 components, highlighting a possible important biological role of these isoforms.

Interaction proteomics: characterization of protein complexes using tandem affinity purification-mass spectrometry.

Most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. Thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. Advances in MS have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the TAP (tandem affinity purification) methodology efficiently isolates native protein complexes from cells for proteomics analysis. TAP is a generic method based on the sequential utilization of two affinity tags to purify protein assemblies. During the first purification step, the Protein A moiety of the TAP tag is bound to IgG beads, and protein components associated with the TAP-tagged protein are retrieved by TEV (tobacco etch virus) protease cleavage. This enzyme is a sequence-specific protease cleaving a seven-amino-acid recognition site located between the first and second tags. In the second affinity step, the protein complex is immobilized to calmodulin-coated beads via the CBP (calmodulin-binding peptide) of the TAP tag. The CBP-calmodulin interaction is calcium-dependent and calcium-chelating agents are used in the second elution step to release the final protein complex preparation used for protein identification by MS. The TAP-MS approach has proven to efficiently permit the characterization of protein complexes from bacteria, yeast and mammalian cells, as well as from multicellular organisms such as Caenorhabditis elegans, Drosophila and mice.