Ring1b Regulates ES Cell Fate Figure 4. Gene expression profiling in Ring1b-deficient ES cells. (A) Expression profiling data clustered by the software program Genesis shows regulation of gene expression (p,0.05) after deletion of Ring1b in mouse ES cells over time. (B) Graph representing the number of all upregulated (red) and downregulated (green) genes (p,0.05). (C) Graph representing the number of up- (red) or downregulated (green) Ring1b bound genes specifically [21]. (D) QPCR validation of microarray data for a selection of genes. Time indicates number of days of 4-OHT treatment. (E) Heatmap of the statistically overrepresented GO categories, as identified by BiNGO analysis. The color scale bar represents the relation between the color intensity and the level of significance (–log10(p-value)). (F) Table showing the significantly enriched GO categories and the percentage (numbers between brackets) of up- or downregulated genes and the number of Ring1b bound genes per GO category (the ones most-downstream in the GO-tree, see also Figure S2). doi:10.1371/journal.pone.0002235.g004 identified pathways have roles in development and differentiation of ES cells. A selection is listed in Table 1. A complete overview of the pathway analysis can be found in Table S3. Most revealing was the identification of the TGFbeta signaling pathway of which Bmp/Id signaling (derepressed Bmp7, Bmpr1a, Id1, Id2, and Id3) and Nodal/Pitx2 signaling (derepressed Nodal, Pitx2) were suggested to be transcriptionally upregulated upon loss of Ring1b. Genome-wide binding studies have identified that PcG proteins are associated with members of the TGFbeta signaling pathway and show PcG dissociation from the genes that were PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2235