that Ring1b has a dual role in cell cycle regulation in ES cells, which is supported by the observation that Ring1b is involved in direct repression of cell cycle promoting gene Ccnd2 and cell cycle inhibitory genes Cdkn1c and Gadd45g. Various components of cellular communication pathways were deregulated in the absence of Ring1b. These comprise ECM genes (derepressed Col4a2 and Thbs1) involved in focal adhesion, gap junction genes (derepressed Gja1 and Csnk1d and downregulated Itpr1), and adherens junction pathways, which are linked to the actin cytoskeleton organization (downregulated Vcl and Actn3, derepressed Iqgap1). Several of these genes were reported to change expression during differentiation and development [39–41]. Summarizing, this suggests that Ring1b deficiency induces changes in cellular pathways, such as Bmp/TGFbeta signaling, cell cycle regulation, and cellular communication, which strongly suggests a link to differentiation programs in ES cells. Retained expression of stem cell regulators in Ring1b- deficient ES cells ES cells, derived from the epiblast of early blastocysts, can self- renew and maintain a pluripotent, undifferentiated state in vitro by extrinsic factors, such as LIF and Bmp, and intrinsic factors, including stem cell regulators Oct4 and Nanog [42–45]. Upon induction of lineage-specific genes, these stem cell regulators are downregulated through a negative-feedback-loop by not yet understood mechanisms [42,45,46]. In line with this, the transcript levels of stem cell specific genes Sox2, Dppa3/Stella and Zfp42/Rex-1 were strongly downregulated in Ring1b-deficient ES cells [43,47] (Figure 4D, Table S1). However, QPCR analysis showed that Nanog transcript levels had not altered and Oct4 transcript levels had reduced only to about 55% (Figure 4D). Importantly, Oct4 was not identified as an outlier on the microarray and Western blot analysis showed no significant changes in Oct4 protein levels (Figure 3A, Table S1). To examine Oct4 expression in more detail, we also quantified the immunofluorescence levels of Oct4 and Ring1b stainings in individual cells. We compared this to guided neural differentiation induced by retinoic acid (RA) treatment, which was demonstrated to result in significant downregulation of Oct4 [48]. As expected, we found that three days after RA induced neural differentiation Oct4 was completely downregulated, in about 65% of the cells (Figure 5A, 5B). However, whereas four days following Ring1b deletion, 81% of all cells were negative for Ring1b, only 5% had lost Oct4 expression (Figure 5A, 5B, 5C). To assess the extent of neural differentiation, we next examined the expression of neuronal- lineage marker Nestin [49]. We found that three days after RA treatment approximately 44% of the cells showed filamentous Nestin staining, compared to 32% of the cells after deletion of Ring1b (Figure 5D, 5E). This is remarkable considering the number of cells expressing Oct4. This suggests that expression of lineage markers co- exists with stem cell regulators in absence of Ring1b in ES cells. Finally, we examined the activity of alkaline phosphatase, which is highly expressed in undifferentiated, pluripotent ES cells [50]. We found that after deleting Ring1b a substantial number of cells still showed high activity of alkaline phosphatase compared to RA treated ES cells, which is indicative for a pluripotent, undifferenti- ated state of ES cells (Figure 5F). Combined, our findings suggest that Ring1b-deficient ES cells show features of differentiated cells, while retaining important ES cell characteristics. Ring1b represses genes that are co-occupied by stem cell regulators Oct4 and Nanog in ES cells The stem cell-specific transcription factors Oct4 and Nanog control ES cell pluripotency by regulating expression of stem cell Ring1b Regulates ES Cell Fate specific genes and repression of differentiation-specific genes, of which a subset is co-occupied by PcG proteins [25,43,51]. Considering the retained expression of Oct4 and Nanog, we were interested to see the effect on gene transcription of these co- occupied genes in absence of Ring1b. We therefore analyzed the changes in expression of genes that were bound by Ring1b [21] and by Oct4 and/or Nanog in mouse ES cells as was identified by Loh and co-workers [51]. We found that the expression of 25 of the 212 Nanog/Oct4/Ring1b bound genes represented on our microarray was altered in Ring1b-deficient ES cells (Figure 4D, Table S1). We found that 18 of these 25 genes were derepressed, including Fgf15, Bmp7, Gata3, Bmi1, Msx2, Podxl, Col4a2, Gadd45g, Gja1 and Eif4g3. This suggests that Ring1b is required to repress a specific subset of Oct4 and Nanog bound genes to maintain a pluripotent, undifferentiated ES cell state. Ring1b represses bivalent or H3K4me3 marked genes with CpG-rich promoters Ring1b-mediated H2A mono-ubiquitination was shown to restrain a poised RNAPII configuration specifically at the promoters of bivalent genes, which were derepressed upon loss of Ring1b and uH2A [31]. Therefore, we investigated the chromatin state of the genes that were deregulated in absence of Ring1b in ES cells. Hereto, we used a data set from Mikkelsen and colleagues, who generated genome-wide chromatin-state maps for mouse ES cells, neural progenitor cells (NPC) and MEFs [30]. Of 948 of the 1078 Ring1b bound genes [21] represented on our microarray chromatin-state maps were retrieved for the histone modifications H3K4me3 and H3K27me3 [30]. Notably, virtually all (99%) of the Ring1b bound genes that were mapped as marked with both H3K27me3 and H3K4me3 or H3K27me3 alone, according to Mikkelsen-data set [30], were associated with H3K27me3 according to the H3K27me3 mapping data by Boyer et al [21], indicating a strong overlap between the two data sets for this histone mark. The chromatin-state maps also contain information about the CpG content of promoters genome-wide. Mikkelsen and colleagues show that genes with high-CpG content promoters (HCP) and H3K4me3 marks generally have ‘house- keeping’ functions, including replication and basic metabolism, while genes with HCP and bivalent marks mainly include key developmental transcription factors, morphogens, and cell surface markers. Genes with low-CpG content promoters (LCP) are associated with highly tissue-specific genes. A third class of genes with intermediate CpG promoter content (ICP) was added to ensure discrimination between the HCPs and LCPs. Comparative analysis showed that about two-third of the promoters genome-wide, i.e represented on our microarrays, or differentially regulated in Ring1b-deficient ES cells, had HCPs (Figure 6A, 6B). Remarkably, Ring1b almost exclusively associated with and regulated transcription of genes with HCPs (93%; 882/ 948 and 94%; 119/126, respectively; Figure 6C, 6D). This suggests that Ring1b primarily regulates transcription of CpG- ‘rich’ promoters. We next analyzed the chromatin state of the Ring1b bound genes with HCPs. We found that these genes were strongly associated with bivalent marks (74%; 653/882). A similar fraction of deregulated Ring1b bound genes with HCPs was bivalently marked (73%; 87/119; Figure 6E). These deregulated bivalently marked Ring1b bound genes included mostly developmental genes, such as Bin1, Bmi1, Bmp7, Col4a2, Ccnd2, En1, Gata3, Lmx1a and Wnt6, and were predominantly derepressed in absence of Ring1b (86%; 75/87; Figure 6H, 6I). Interestingly, we found that Ring1b was also associated with a considerable number of HCPs with the H3K4me3 mark alone (25%; 218/882; Figure 6E). The PLoS ONE | www.plosone.org 7 May 2008 | Volume 3 | Issue 5 | e2235