Ubiquitin E3 Ligase Ring1b/Rnf2 of Polycomb Repressive Complex 1 Contributes to Stable Maintenance of Mouse Embryonic Stem Cells Petra van der Stoop1., Erwin A. Boutsma1., Danielle Hulsman1, Sonja Noback1, Mike Heimerikx2, Ron M. Kerkhoven2, J. Willem Voncken4, Lodewyk F. A. Wessels3, Maarten van Lohuizen1,5* 1 Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands, 2 Center for Biomedical Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands, 3 Bioinformatics and Statistics, Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands, 4 Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, The Netherlands, 5 Department of Neurogenetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Abstract Background: Polycomb repressive complex 1 (PRC1) core member Ring1b/Rnf2, with ubiquitin E3 ligase activity towards histone H2A at lysine 119, is essential for early embryogenesis. To obtain more insight into the role of Ring1b in early development, we studied its function in mouse embryonic stem (ES) cells. Methodology/Principal Findings: We investigated the effects of Ring1b ablation on transcriptional regulation using Ring1b conditional knockout ES cells and large-scale gene expression analysis. The absence of Ring1b results in aberrant expression of key developmental genes and deregulation of specific differentiation-related pathways, including TGFbeta signaling, cell cycle regulation and cellular communication. Moreover, ES cell markers, including Zfp42/Rex-1 and Sox2, are downregulated. Importantly, retained expression of ES cell regulators Oct4, Nanog and alkaline phosphatase indicates that Ring1b-deficient ES cells retain important ES cell specific characteristics. Comparative analysis of our expression profiling data with previously published global binding studies shows that the genes that are bound by Ring1b in ES cells have bivalent histone marks, i.e. both active H3K4me3 and repressive H3K27me3, or the active H3K4me3 histone mark alone and are associated with CpG- ‘rich’ promoters. However, deletion of Ring1b results in deregulation, mainly derepression, of only a subset of these genes, suggesting that additional silencing mechanisms are involved in repression of the other Ring1b bound genes in ES cells. Conclusions: Ring1b is essential to stably maintain an undifferentiated state of mouse ES cells by repressing genes with important roles during differentiation and development. These genes are characterized by high CpG content promoters and bivalent histone marks or the active H3K4me3 histone mark alone. Citation: van der Stoop P, Boutsma EA, Hulsman D, Noback S, Heimerikx M, et al. (2008) Ubiquitin E3 Ligase Ring1b/Rnf2 of Polycomb Repressive Complex 1 Contributes to Stable Maintenance of Mouse Embryonic Stem Cells. PLoS ONE 3(5): e2235. doi:10.1371/journal.pone.0002235 Editor: Simon Williams, Texas Tech University Health Sciences Center, United States of America Received February 13, 2008; Accepted March 28, 2008; Published May 21, 2008 Copyright: 2008 van der Stoop et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Dutch Cancer Foundation (KWF) and the Dutch organization for Scientific Research (NWO). Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.v.lohuizen@nki.nl . These authors contributed equally to this work. Introduction PcG proteins have initially been identified in Drosophila as transcriptional repressors required for correct expression of homeotic (Hox) genes. By means of chromatin remodeling, PcG proteins maintain stable gene repression to ensure proper embryonic development. In mammals, two biochemically and functionally distinct PcG core complexes have been identified [1]. Mammalian polycomb repressive complex 1 (PRC1) consists of close homologs of the Drosophila PRC1 core members Ph, Pc, Psc and dRing [1,2]. Ring1b/Rnf2, the mouse homolog of dRing, acts as a ubiquitin E3 ligase towards histone H2A at lysine 119 resulting in mono-ubiquitinated H2A (uH2A) [3,4]. Ezh2/Kmt6, a methyltransferase that trimethylates histone H3 at lysine 27 (H3K27me3), acts in complex with Suz12 and Eed, constituting Polycomb repressive complex 2 (PRC2) [5–7]. PRC1 and PCR2 do not physically interact, but the Ezh2 catalyzed histone mark H3K27me3 is recognized by PRC1 member Pc, providing a mechanism of communication between the two complexes [8]. Furthermore, PRC1 binding to chromatin requires PRC2, although PRC2-independent recruitment of PRC1 is also reported [9,10]. The counteracting trithorax group (TrxG) proteins mediate an active transcriptional state by methylation of histone H3 at lysine 4 (H3K4me3) [2]. Proper expression of PRC2 proteins is essential during early embryonic development,, since mice lacking one of these proteins die due to gastrulation defects [11–13]. In contrast, PRC1 members appear to be more important during later stages of development, with the clear exception of Ring1b, which evokes an early embryonic lethal phenotype similar to PRC2 mice [14– 18]. PcG proteins were shown to be important for self-renewal and maintaining pluripotency of ES cells [19–21]. ES cells are derived PLoS ONE | www.plosone.org 1 May 2008 | Volume 3 | Issue 5 | e2235