from the inner cell mass of pre-implantation blastocysts [22]. ES can self-renew and maintain a pluripotent, undifferentiated state under the right conditions in vitro, while retaining the capacity to differentiate into every cell type required during development in vitro and in vivo when reintroduced back into a host blastocyst [23]. Genome-wide and candidate-based studies revealed that PcG proteins maintain this undifferentiated state of ES cells through direct repression of developmental genes [21,24–27]. Develop- mental genes are largely associated with bivalent (i.e. both repressive H3K27me3 and active H3K4me3) histone marks and are silent or expressed at very low levels in ES cells [28–30]. Bivalent domains tend to resolve during development [28–30]. Therefore, it is suggested that this bivalent chromatin state poises genes for transcriptional activation (loss of H3K27me3) or prolonged repression (loss of H3K4me3 or both marks) during later developmental stages. Only recently, it was shown that Ring1b-mediated uH2A deposition at repressed bivalent genes restrains a poised RNA polymerase II (RNAPII) configuration [31]. Conditional deletion of Ring1b and subsequent loss of uH2A from the promoter and coding region results in release of poised RNAPII and gene derepression. More evidence for a direct role of uH2A in negative transcriptional regulation was found by another group, which investigated the role of histone H2A E3 ligase 2A- HUB in repressing chemokine genes in human monocytes [32]. They show that the presence of uH2A in the promoter-proximal region physically blocks recruitment of FACT, thereby causing RNAPII-dependent transcription elongation to pause. To explore the role of Ring1b in regulation of gene expression during early development, we analyzed genome-wide changes in gene transcription following deletion of Ring1b in ES cells. Hereto, we employed an inducible knockout system and performed a genome-wide screen using microarrays to identify genes governed by Ring1b. We identify several processes and pathways differentially regulated in Ring1b-deficient ES cells, which are implicated in differentiation and embryogenesis. Importantly, we find that Ring1b-deficient ES cells retain expression of key stem cell regulators Oct4 and Nanog. Finally, by comparing our expression data with previously published global binding studies, we find that Ring1b preferentially represses genes with high CpG-content promoters and bivalent or H3K4me3 histone marks. This suggests that Ring1b has an important role in maintaining an undifferentiated state of ES cells. Furthermore, this study provides more insight in the characteristics of the genes that are under direct transcriptional control of Ring1b in ES cells. Results Generating Ring1b conditional knockout mouse embryonic stem cells To gain insight in the role of Ring1b in ES cells, we generated Ring1b conditional knockout mouse ES cells. Previously, we have shown that deletion of the RING finger domain of Ring1b, encoded by exon 3 and 4, generates a functional allele [17]. Ring1b conditional knockout (Ring1b2/Lox) ES cells were then generated by a second targeting round in Ring1b+/2 ES cells [17] using a targeting vector, which introduced loxP sequences flanking these exons (Figure 1A; details in Materials and Methods). To generate inducible Ring1b2/Lox ES cells, we targeted a 4-hydroxytamoxifen (4-OHT) inducible R26CreERT2 construct (Cre-recombinase fused to a mutated ligand binding domain of the human estrogen receptor) to the ROSA26 locus of Ring1b2/Lox ES cells, hereafter named Ring1b2/Lox;CreERT2 ES cells. The presence of loxP sequences in the genomic locus of Ring1b did not alter the expression of Ring1b in ES cells. Accordingly, Ring1b2/Lox and Ring1b Regulates ES Cell Fate Ring1b2/Lox;CreERT2 mice developed indistinguishable from wild type mice (E.B, M.v.L, unpublished results). Addition of 4-OHT to the culture medium resulted in recombi- nation of the Ring1b conditional allele in Ring1b2/Lox;CreERT2 ES cells and subsequent depletion of Ring1b protein as was shown by Southern and Western blot analysis (Figure 1B, 1C). We observed cellular detachment and apoptosis concurrent with an increase in cells with a differentiated phenotype after deleting Ring1b in Ring1b2/Lox;CreERT2 ES cells, but not in similarly treated Ring1b+/+;CreERT2 control ES cells. Based on Annexin V staining followed by flow cytometric analysis, about 83% of all Ring1b2/Lox;CreERT2 ES cells were undergoing apoptosis four days following 4-OHT treatment (data not shown). Of the adherent population only 15% was Annexin V positive, compared to 5% of the untreated population (data not shown). We therefore focused our studies on the immediate early effects of the first days following Cre- mediated deletion of Ring1b in the viable, adherent ES cells. Ring1b is required for mono-ubiquitination of H2A in ES cells Since Ring1b is a ubiquitin E3 ligase for histone H2A at lysine 119, we studied the effect of Ring1b deletion on the levels of uH2A. Western blot analysis showed that uH2A levels decreased to almost undetectable levels after deletion of Ring1b from Ring1b2/Lox;CreERT2 ES cells (Figure 1C). In addition, immunofluorescence stainings confirmed that uH2A was lost in ES cells that had lost Ring1b expression (Figure 2A). The same held true for mouse embryonic fibroblasts (MEFs) derived from Ring1b2/Lox;CreERT2 mouse embryos, which showed no uH2A immunofluorescence staining in Ring1b- negative cells (Figure S1). Immunofluorescence stainings for H3K27me3, the polycomb repressive mark set by PRC2 member Ezh2, showed that this mark was present in all cells (Figure 2B). These results confirm that Ring1b is the main E3 ligase for mono- ubiquitination of H2A in both mouse ES cells and MEFs and suggest that deletion of Ring1b does not affect H3K27 trimethylation. Ring1b deficiency affects expression of other PRC1 core members Studies in Drosophila, mouse and human cells have shown that PcG genes themselves are PcG protein binding targets, suggesting auto-regulation within the PcG family [21,25,33]. We therefore examined the effect of Ring1b deletion on other PRC1 and PRC2 members in ES cells. Microarray analysis (described in more detail in the following section) showed that the transcript levels of Ring1b and Phc1/Mph1 were significantly downregulated over time, and correspondingly decreased in protein levels (Figure 3A, Table S1). Interestingly, PRC1 member Bmi1 was derepressed transcription- ally in the absence of Ring1b, but its protein levels were down- regulated, suggesting that Bmi1 is post-transcriptionally regulated by Ring1b (Figure 3B). This is consistent with observations by others [34], and may be explained by the fact that interaction between Bmi1 and Ring1b protects both proteins from ubiquitin- mediated degradation [35]. The effect of loss of Ring1b on other PRC1 core members may explain the severity of the Ring1b mouse knockout phenotype compared to knockout models of other PRC1 members [14–18]. Other polycomb group genes of which the transcription had increased significantly were Epc1, Phf1, and Phc2 (Table S1). No effects were observed on the protein or transcript levels of PRC2 members Ezh2 and Eed, or the levels of H3K27me3, which is in agreement with the immunofluorescence analysis (Figure 3A, 3B, Table S1). The effect on PcG protein and transcript levels after loss of Ring1b is in agreement with an earlier study performed in established Ring1b-deficient ES cells, with the PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2235