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

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. Molecular & Cellular

Chromatin regulation: How complex does it get?

“…Accordingly, paradigmatic complexes such as PRC1, which were originally viewed as a single entity, have now been revealed to exist in many different versions and to constitute a protein complex family. 13,100 Furthermore, variations in the stoichiometry of subunit composition of several of the newly identified complexes indicates that they can be further sub-divided 13,18,19,56 (Table 1). Metaphorically speaking, multisubunit protein complexes function as "toolboxes" which in order to accomplish specific tasks on chromatin combine different "tools" or up-grade their standard equipment by adding specific "tools."…”

BMI1–RING1B is an autoinhibited RING E3 ubiquitin ligase

“…Recently, it has been appreciated through work in many labs that, in addition to canonical PRC1, several variant PRC1 complexes are present in mammalian cells [18][19][20][21][22][23][24][25][26][27][28][29] . These are defined by the PCGF family member present 23 , and the copurifying subunits also differ by complex subtype 22,23,25 . Opinions currently differ on which type of complex, canonical 25 or non-canonical 23,29 , may contribute more strongly to H2A K119 ubiquitination.…”

From Flies to Mice: The Emerging Role of Non-Canonical PRC1 Members in Mammalian Development

“…Based on genetic interaction studies, it has long been postulated that PcG proteins interact with each other in Drosophila, form complexes and contribute to a common repressor mechanism. This hypothesis was proven later in mammalian systems as well [50][51][52].…”

“…Later, similar PRC1 complexes were purified from mammalian cells, all containing the homologs of Drosophila PC, PSC and PH (see Table 1), but lacking most non PcG proteins. Mammalians PRC1 complexes may contain different homologs of the Drosophila PRC1 subunits [51]. The presence of the different paralog PRC1 subunits increases the complexity of the possible repressor mechanisms in mammals.…”