Proteomics Analysis of Ring1B/Rnf2 Interactors Identifies a Novel Complex with the Fbxl10/Jhdm1B Histone Demethylase and the Bcl6 Interacting Corepressor

Abstract

Ring1B/Rnf2 is a RING finger protein member of the Polycomb group (PcG) of proteins, which form chromatinmodifying complexes essential for embryonic development and stem cell renewal and which are commonly deregulated in cancer. Ring1B/Rnf2 is a ubiquitin E3 ligase that catalyzes the monoubiquitylation of the histone H2A, one of the histone modifications needed for the transcriptional repression activity of the PcG of proteins. Ring1B/Rnf2 was shown to be part of two complexes, the PRC1 PcG complex and the E2F6.com-1 complex, which also contains non-PcG members, thus raising the prospect for additional Ring1B/Rnf2 partners and functions extending beyond the PcG. Here we used a high throughput proteomics approach based on the single step purification, using streptavidin beads, of in vivo biotinylated Ring1B/Rnf2 and associated proteins from a nuclear extract from erythroid cells and their identification by mass spectrometry. About 50 proteins were confidently identified of which 20 had not been identified previously as subunits of Ring1B/Rnf2 complexes. We found that histone demethylases LSD1/Aof2 and Fbxl10/Jhdm1B, casein kinase subunits, and the BcoR corepressor were among the new interactors identified. We also isolated an Fbxl10/Jhdm1B complex by biotinylation tagging to identify shared interacting partners with Ring1B/Rnf2. In this way we identified a novel Ring1B-Fbxl10 complex that also includes Bcl6 corepressor (BcoR), CK2␣, Skp1, and Nspc1/Pcgf1. The putative enzymatic activities and protein interaction and chromatin binding motifs present in this novel Ring1B-Fbxl10 complex potentially provide additional mechanisms for chromatin modification/recruitment to chromatin and more evidence for Ring1B/Rnf2 activities beyond those typically associated with PcG function. Lastly this work demonstrates the utility of biotinylation tagging for the rapid characterization of complex mixtures of multiprotein complexes achieved through the iterative use of this simple yet high throughput proteomics approach.Molecular & Cellular Proteomics 6:820 -834, 2007.In multicellular organisms, cell identity is controlled, at least in part, by epigenetic events, including DNA methylation and post-translational modifications of histones that lead to chromatin structure regulation (1). These modifications are carried out by protein complexes recruited through DNA sequences and/or specific recognition of modified histones. The Polycomb group (PcG) 1 of proteins, first identified genetically as regulators of Hox genes in the fly Drosophila melanogaster (for a review, see Ref. 2), is an example of such a complex. PcG functions cover many aspects of vertebrate development and tissue homeostasis by preventing the inappropriate activation of many transcription factor-coding genes and other genes involved in cell signaling and cell proliferation (3-7). Currently it is believed that PcG proteins play a role in setting the balance between proliferation and differentiation in normal development. Deregulation of PcG proteins disrupts such a ba...

BCL6promoter interacts with far upstream sequences with greatly enhanced activating histone modifications in germinal center B cells

“…An intriguing alternative possibility is that BCL6 itself may bind this region and lead to reduction in H3K4me3. In this regard, it is interesting that BCOR, a BCL6 corepressor, associates with FBXL10, a H3K4me3 demethylase (32,33). The BCL6 promoter region and intron 1 contain seven conserved predicted BCL6 binding sites (Fig.…”

Epigenetic regulation of normal and malignant hematopoiesis

“…A recent study by demonstrated the interaction of the JmjC HDM JARID1d with the polycomb-like protein RING6a/ MBLR and showed that RING6a/MBLR enhanced JARD1d-mediated H3K4 demethylation, associated with transcriptional repression. Jhdm1b has also been shown to interact with PcG proteins in a complex containing Ring1b/Rnf2 and the Bcl6 interacting corepressor, among other proteins (Sanchez et al, 2007). This co-repressor complex may be implicated in gene repression mediated by Bcl6, a transcription factor frequently dysregulated in B-cell lymphomas, and could provide the basis for studying the effect of HDM inhibitors in this disease.…”

The H3K4me3 Histone Demethylase Fbxl10 Is a Regulator of Chemokine Expression, Cellular Morphology, and the Metabolome of Fibroblasts

“…Fbxl10 was previously reported to confer specific changes in histone modification either directly via its JmjC domain or indirectly by its binding partners (10,12,23,24). Whereas H3K4me3 and H3K36me2 were discussed as direct targets, H3K27me3 and H2Aub were reported as indirect targets because Fbxl10 was found in Polycomb complexes with H3K27 methylation activity and H2A ubiquitination capability (23,24).…”