Ring1b Regulates ES Cell Fate Figure 1. Generating Ring1b conditional knockout ES cells. (A) Schematic overview of the Ring1b locus targeting strategy in mouse Ring1b+/2 ES cells. The wild type Ring1b allele is shown with main restrictions sites, introns and exons, in the targeted genomic region. The Ring1bLoxHyg allele was generated by targeting a floxed exon3+4/Hygromycin replacement vector to the wild type Ring1b locus of the Ring1b+/2 ES cells. The hygromycin cassette allowed for selection after the initial targeting and was removed by transiently expressing adenoviral Cre to generate the conditional Ring1bLox allele. Acute loss of Ring1b from Ring1bLox ES cells was induced by adding 4-OHT to the medium to activate CreERT2 (Cre- recombinase fused to a mutated ligand binding domain of the human estrogen receptor (ER), targeted to the ROSA locus, not shown) resulting in deletion of exons 3 and 4, represented as the Ring1bDel allele. Exons are indicated by white boxes with the corresponding exon number, loxP sequences by black triangles, and HYG indicates the hygromycin cassette. Main restriction sites are indicated (H = HindIII, E = EcoRI, X = XhoI) as well as the location of the 59end probe used for Southern blot analysis. (B) Southern blot analysis of HindIII digested genomic DNA of Ring1b2/Lox;CreERT2 ES cells showing recombination of the Ring1bLox allele following treatment with 4-OHT for the indicated time in days. The 59end probe visualizes the conventional knockout allele (5 kb), the conditional Lox allele (7 kb) and the recombined Del allele (4 kb). (C) Western blot analysis showing loss of Ring1b protein and uH2A in Ring1b2/Lox;CreERT2 ES cells following treatment with 4-OHT for the indicated time in days. Tubulin serves as a loading control. doi:10.1371/journal.pone.0002235.g001 exception of the transcriptional downregulation of Phc1 [34]. This might therefore reflect an indirect effect of acute loss of Ring1b in ES cells. These results suggest that deletion of Ring1b affects the transcript and protein levels of members of PRC1, but not of PRC2, and underscores the importance of Ring1b for PRC1 stability. Regulation of developmental processes through derepression of Ring1b bound genes To obtain insight in the genome-wide effects on gene transcription after deletion of Ring1b in ES cells, we performed microarray analysis. We analyzed the temporal changes in gene transcription in Ring1b2/Lox;CreERT2 ES cells one to four days after deletion of Ring1b. As a reference, we used Ring1b+/+;CreERT2 ES cells similarly treated with 4-OHT to correct for any effects induced by 4-OHT or CreERT2. We identified 2365 differentially regulated genes, i.e. genes that were significantly (p,0.05 according to the Rosetta-error model, for details see Materials and Methods) up- or downregulated (7%; 2365/31769; Figure 4A, 4B, Table S1). The reliability of the microarray data was confirmed by quantitative real-time PCR analysis (QPCR) on a selection of 18 genes (Figure 4D). To gain insight into the processes that are deregulated in absence of Ring1b in mouse ES cells, we analyzed the deregulated genes based on their ontology classification. To this end, we employed the gene ontology (GO) tool BiNGO to determine which GO categories are statistically overrepresented. By mapping the predominant functional GO categories in a hierarchical graph BiNGO facilitates insight in the hierarchical structure and relations between the ontologies [36]. We found that the outliers were involved in various processes, of which the most downstream in the GO-tree were: ‘organ development’, ‘organ morphogenesis’, ‘nucleosome assembly’, ‘regulation of cellular physiological processes’, ‘glyconeogenesis’, and ‘cell cycle’ (Figure 4E; for a graphical overview of the overrepresented GO categories, see PLoS ONE | www.plosone.org 3 May 2008 | Volume 3 | Issue 5 | e2235