found that in Ring1b-deficient ES cells key developmental genes were derepressed, including a subset of genes bound by stem cell regulators Oct4 and Nanog, suggesting that Ring1b deficiency results in activation of the differentiation program. Accordingly, specific differentiation-related pathways were differentially dereg- ulated, including TGFbeta signaling, cellular communication and cell cycle pathways. Importantly, retained expression of stem cell regulators Oct4 and Nanog indicates that Ring1b-deficient ES cells still have important ES cell specific characteristics. Based on re-analysis of previously published global binding studies per- formed in mouse ES cells, we found that Ring1b predominantly binds and represses genes with CpG-‘rich’ promoters. Further- more, these genes are associated with bivalent domains or the H3K4me3 histone mark alone. We suggest that Ring1b contributes to stable maintenance of pluripotency of ES cells by repressing bivalently or actively marked genes. We found that Ring1b is required to repress the differentiation program in ES cells, which is consistent with the observation that Ring1b-deficient ES cells are prone for differentiation [34]. Comparative analysis with genome-wide Ring1b binding data of mouse ES cells [21], indicated that Ring1b is involved in direct transcriptional regulation of key developmental genes, such as Bmp7, Gata3 and Msx. This is in agreement with other genome- wide and candidate-based PcG binding studies performed in mouse and human ES cells [21,24–28,34]. Notably, by directly repressing these extracellular and down-stream effector compo- nents of differentiation-related pathways, such as Bmp7 and Id3 of the Bmp/TGFbeta signaling pathway, and Col4a2, Gja1 and Ccnd2 of the cellular communication pathways, Ring1b may have an important role in influencing lineage choice and other develop- mental events. For example, expression of ECM protein Col4a2 was shown to induce mesodermal differentiation of ES cells in vitro, while repression of Bmp7 at the dorsal site of zebrafish gastrula was shown to allow cellular migration as is required for limb development. [52,53]. In Ring1b knockout embryos, several developmental markers are misexpressed at early gastrulation stages, extraembryonal mesoderm is over-represented, epiblast has expanded improperly, and embryonic growth is delayed [17]. This is in line with the aberrant expression of developmental markers and induction of apoptosis observed in Ring1b knockout ES cells. The early embryonic lethality is partially rescued in a Cdkn2a- knockout background, one of the PcG transcriptional targets [54,55], resulting in the delay of embryonic death of about 4 days [17]. Since relief of the Ink4a/Arf-mediated antiproliferative block only bypasses the proliferative defects induced by loss of Ring1b, this underscores the importance for Ring1b in regulating developmental genes during early embryogenesis. The observation that only a subset of Ring1b bound genes is derepressed following deletion of Ring1b in ES cells, suggests that additional mechanisms mediate silencing of the other Ring1b target genes. This would be in correspondence with observations by others, who show that only part of the PcG bound genes are deregulated after RNAi-mediated depletion of PcGs in human embryonic fibroblasts and in ES cells deficient for Eed or Suz12 [20,21,26]. Partial derepression could be also be explained by functional redundancy by related Ring1a, which possess ubiquitin E3 ligase activity towards histone H2A, alike Ring1b, although with less efficiency [56–58]. In addition, the composition of PRCs can vary per tissue and developmental stage [20,27,56,59]. Therefore, Ring1b might not be essential in every promoter- bound PcG repressive complex. We found that Ring1b represses genes that are co-occupied by Oct4 and Nanog. This is consistent with earlier reports, which show a role for PcG proteins in repressing a set of developmental Ring1b Regulates ES Cell Fate genes that are bound by Oct4 and Nanog in ES cells [25,51]. A possible link between these proteins is provided by Wang and colleagues, who co-purified Oct4, Yy1 and Ring1b with stem cell specific protein Rex-1 from ES cells [60]. In Drosophila, PcG- specific sequences (named polycomb group responsive elements or PREs) mediate PcG repression [61]. However, in mammalians, PREs or sequence-specific DNA binding PcG proteins have not been identified. Therefore, one might speculate that recruitment of PcG-silencing complexes to specific genes involves interactions with site-specific DNA-binding factors. This could be the recruitment of the PcG silencing complex to key developmental genes through interaction with stem cell specific factors Rex-1 and/or Oct4 in order to maintain an undifferentiated state of ES cells. This suggestion is supported by the observation that Suz12 dissociates from PcG/Oct4 co-occupied promoters upon down- regulation of Oct4 [20]. Interestingly, others have found that histone H2A E3 ligase 2A-HUB, but not RING1B, is selectively required for H2A mono-ubiquitination and subsequent repression of chemokine genes following recruitment by co-repressor N-CoR in human monocytes [32]. Together, this suggests that different site-specific factors are involved in the recruitment of distinct H2A E3 ligases to mediate repression of a specific set of genes. Ring1b-deficient ES cells retain expression of important stem cell regulators Oct4, Nanog and alkaline phosphatase, which was also observed in other PcG-deficient ES cells [20,31,34]. Derepression of developmental genes and downregulation of other stem cell markers, such as Rex-1, Sox2 and Dppa3, suggests activation of the differentiation program. During ES cell differentiation, repression of Oct4 and Nanog is essential, considering that ectopic expression of Oct4 can block differenti- ation, while expression of Nanog is enough to maintain ES cell self-renewal and pluripotency, even in the absence of extrinsic factor LIF or Oct4 expression [44,45,62]. Retained expression of Oct4 and Nanog could indicate that PcG proteins are either directly or indirectly involved in regulation of Oct4 and Nanog. A direct role for PcG proteins in regulating the expression of Oct4 and Nanog has been suggested by Pasini and co-workers, who show that PRC2 proteins bind to the Oct4 and Nanog promoters in ES cells [20]. Moreover, the H3K27me3 histone mark has been found at the Oct4 promoter in more committed cells, suggesting that PcG proteins are involved in direct repression of stem cell regulators in differentiated cells [28]. Comparative analysis of our expression profiling data with recently published global chromatin-state maps generated of mouse ES cells showed that Ring1b almost exclusively binds to genes with CpG-rich promoters. This is in consistent with a report showing that a strong correlation exists between promoters with highly conserved large CpG islands and Suz12 binding domains in undifferentiated human ES cells [63]. Furthermore, our data confirm that PcG proteins are involved in repressing bivalently marked developmental genes in ES cells [28–31]. This compar- ative analysis also revealed that a considerable number of actively transcribed H3K4me3 marked Ring1b bound genes are regulated (mainly repressed) in ES cells. The absence of H3K27me3 at these promoters suggests that Ring1b is recruited independently from PRC2. This is in agreement with other observations indicating that Xist RNA, but not PRC2, is required for PRC1 recruitment during X inactivation in differentiating ES cells [10]. The association of Ring1b to actively transcribed genes is consistent with global and candidate-based binding studies that show overlap between PcG and RNAPII occupancy or association of PcG proteins with expressed genes in mouse and human ES cells, neural progenitor cells and embryonic fibroblasts [20,25–27,64]. This suggests that Ring1b does not function as a silencer of these PLoS ONE | www.plosone.org 10 May 2008 | Volume 3 | Issue 5 | e2235