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...
NH emerges as a clear-cut clinical picture. It is a noninfrequent primary headache. The particular topography suggests the pain has a probable epicranial source conveyed by, or originated in, one/a few terminal branch(es) of the cutaneous nerves of the scalp.
Ten patients (one man and nine women, mean age 48.8 +/- 20.1) presented with a stereotypical and undescribed type of head pain. They complained of strictly unilateral, shooting pain paroxysms starting in a focal area of the posterior parietal or temporal region and rapidly spreading forward to the ipsilateral eye (n = 7) or nose (n = 3) along a lineal or zigzag trajectory, the complete sequence lasting 1-10 s. Two patients had ipsilateral lacrimation, and one had rhinorrhoea at the end of the attacks. The attacks could be either spontaneous or triggered by touch on the stemming area (n = 2), which could otherwise remain tender or slightly painful between the paroxysms (n = 5). The frequency ranged from two attacks per month to countless attacks per day, and the temporal pattern was either remitting (n = 5) or chronic (n = 5). This clinical picture might be a variant of an established headache or represent a novel syndrome.
e Polycomb repressive complexes (PRCs) are important chromatin regulators of embryonic stem (ES) cell function. RYBP binds Polycomb H2A monoubiquitin ligases Ring1A and Ring1B and has been suggested to assist PRC localization to their targets. Moreover, constitutive inactivation of RYBP precludes ES cell formation. Using ES cells conditionally deficient in RYBP, we found that RYBP is not required for maintenance of the ES cell state, although mutant cells differentiate abnormally. Genomewide chromatin association studies showed RYBP binding to promoters of Polycomb targets, although its presence is dispensable for gene repression. We discovered, using Eed-knockout (KO) ES cells, that RYBP binding to promoters was independent of H3K27me3. However, recruiting of PRC1 subunits Ring1B and Mel18 to their targets was not altered in the absence of RYBP. In contrast, we have found that RYBP efficiently represses endogenous retroviruses (murine endogenous retrovirus [MuERV] class) and preimplantation (including zygotic genome activation stage)-and germ line-specific genes. These observations support a selective repressor activity for RYBP that is dispensable for Polycomb function in the ES cell state. Also, they suggest a role for RYBP in epigenetic resetting during preimplantation development through repression of germ line genes and PcG targets before formation of pluripotent epiblast cells.
Polycomb group (PcG) proteins participate in the maintenance of transcriptionally repressed state of genes relevant to cell differentiation. Here, we show anterior homeotic transformations of the axial skeleton of YY1(+/-) mice. We find that the penetrance of some of these alterations was reduced in mice that are deficient in the class II PcG gene Ring1/Ring1A, indicating a genetic interaction between those two genes. Further support for this interaction is an abnormal anterior eye formation in Ring1-deficient mice, which is enhanced in compound YY1(+/-)Ring1(-/-) mice. In addition, YY1 forms complexes with Ring1 and other class II PcG proteins such as Rnf2 and Bmi1 in GST pull down experiments in transfected cells. These findings provide evidence for a PcG function for YY1 in vertebrates.
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