Ring1b Regulates ES Cell Fate Figure 5. Ring1b-deficient ES cells retain expression of stem cell specific regulators. (A,D) Examples of immunofluorescence stainings in Ring1b2/Lox;CreERT2 ES cells of (A) Oct4 (red) and DAPI (blue), and (D) Nestin (green) and DAPI (blue), either untreated (upper panels) or treated for four days with 4-OHT (middle panels) or three days with RA (lower panels). (B,C) Histograms represent the distribution of the mean immunofluorescence levels per cell retrieved after quantification of immunofluorescence stainings of Oct4 (B) or Ring1b (C) in Ring1b2/Lox;CreERT2 ES cells either untreated, or treated for four days with 4-OHT or three days with RA. (E) Graph representing the number of Ring1b2/Lox;CreERT2 ES cells either untreated, or treated for four days with 4-OHT or three days with RA that show ubiquitous (black bars) or filamentous (grey bars) Nestin staining. (F) Alkaline phosphatase activity stainings in Ring1b2/Lox;CreERT2 ES cells either untreated, or treated for four days with 4-OHT or three days with RA. doi:10.1371/journal.pone.0002235.g005 lack of repressive H3K27me3 marks at these genes, catalyzed by PRC2, implies that Ring1b might be recruited independent from PRC2 for these genes in ES cells. Of the deregulated H3K4me3 marked Ring1b bound genes with HCPs 66% (21/32) was derepressed, including Eif4g3, Gja1 and Sgce, and various histone encoding genes, such as Hist1h2bn, Hist1h2bf, Hist1h3i, Hist1h4h, and Hist1h4f (Figure 6J, 6K). Not all promoters associated with the active H3K4me3 mark are actively transcribed [30]. Considering the fact that Ring1b is a transcriptional repressor, we predicted that the H3K4me3 marked genes with relative low expression levels were bound by Ring1b. We therefore examined the RNA expression levels of these genes in wild type ES cells by using the Affimetrix RNA expression data that was available in the chromatin-state mapping study by Mikkelsen et al. [30]. Unexpectedly, Ring1b binding did not correlate with low expression levels of H3K4me3 marked genes, but instead seemed to associate with these actively marked genes indifferent of their level of expression (Figure 6G). Notably, in more committed NPCs and MEFs (89% and 83%, respectively) most of the Ring1b bound H3K4me3 marked genes retain this histone mark and corre- PLoS ONE | www.plosone.org 8 May 2008 | Volume 3 | Issue 5 | e2235