Ring1b Regulates ES Cell Fate Figure 2. Deletion of Ring1b results in loss of uH2A but not H3K27me3. Examples of immunofluorescence stainings in Ring1b2/Lox;CreERT2 ES cells showing loss of uH2A (A), but not H3K27me3 (B) in cells that have lost Ring1b four days following 4- OHT treatment. doi:10.1371/journal.pone.0002235.g002 Figure S2; for the BiNGO analysis, see Table S2). These data confirm that Ring1b is involved in regulating cell cycle, developmental and chromatin-related processes and indicate that deletion of Ring1b affects glyconeogenesis and the regulation of cellular physiological processes. Given that loss of Ring1b affects the undifferentiated state of ES cells, the regulated processes are likely to include genes that are deregulated due to indirect effects. By using a genome-wide ChIP-on-chip analysis Boyer and colleagues identified genes that are bound by Ring1b in mouse ES cells [21]. By combining our microarray expression data with the data from this Ring1b binding study, we were able to determine which genes were direct transcriptional targets of Ring1b. Of the 1219 Ring1b bound genes, identified by Boyer and colleagues, 1078 were represented on our microarray. Remarkably, we found that only a subset of 141 Ring1b bound genes had significantly changed expression (13% (141/1078). These genes were predominantly upregulated, which is in agreement with Ring1b being a transcrip- tional repressor (Figure 4C). The partial derepression implies that other regulatory mechanisms are active at the other Ring1b target genes in addition to Ring1b, and reflects that a subset of genes is deregulated due to indirect effects of loss of Ring1b in ES cells. Figure 3. Downregulation of protein levels of PRC1, but not PRC2, following Ring1b deletion in ES cells. Western blot analysis of total cell protein extracts (A) or nuclear extracts (B) of Ring1b2/Lox;CreERT2 ES cells shows that deletion of Ring1b results in downregulation of Phc1/Mph1 and Bmi1 protein levels, but not of Ezh2, Eed or H3K27me3 levels following treatment with 4-OHT for the indicated number of days. doi:10.1371/journal.pone.0002235.g003 Next, we assessed which of the deregulated genes within an overrepresented GO category (most down-stream in the GO-tree, see also Figure S2) were bound by Ring1b. We found that on average 30% of the deregulated genes within an enriched ontology was bound by Ring1b, in particular in the ontologies organ development and morphogenesis, nucleosome assembly, and regulation of cellular physiological process (Figure 4F). This was substantially higher than what would be expected considering that only 6% (141/2365) of all deregulated genes was bound by Ring1b. This indicates that Ring1b is involved in direct transcriptional regulation of a substantial large fraction of the deregulated genes in these ontologies. Together, these data suggest that Ring1b is specifically important for dynamic repression of developmental, chromatin-related, cell cycle and metabolic genes in ES cells. Regulation of specific differentiation-related pathways in Ring1b-deficient ES cells The analysis based on the ontology classification shows that absence of Ring1b results in aberrant expression of genes involved in developmental and various cellular processes. We set out to identify specific pathways that are affected in Ring1b-deficient ES cells. To remain unbiased in the analysis of our set of outliers, we used the bioinformatics tool DAVID, which uses information of well-defined pathways available in the KEGG pathway database (http://david.abcc.ncifcrf.gov). Interestingly, we found that the PLoS ONE | www.plosone.org 4 May 2008 | Volume 3 | Issue 5 | e2235