genes in ES cells. Moreover, a large subset of actively marked genes retain this mark and corresponding transcriptional state in more committed NPCs and MEFs [30]. Thus, rather than poising for activation or prolonged inactivation, as was proposed for the bivalent genes, Ring1b might be required to modulate the transcriptional activity of this subset of actively marked genes in ES cells and during later developmental stages. Only recently, association of Ring1b and uH2A was shown to mediate a silenced state of RNAPII present at promoters and coding regions of bivalent genes [31]. Dissociation of Ring1b and uH2A from these promoters results in activation of the RNAPII complex without major changes in the levels of PRC2 proteins or association of H3K27me3. In addition, others showed that the presence of uH2A in the promoter-proximal region blocks recruitment of FACT resulting in pausing of RNAPII and inhibition of transcriptional elongation [32]. One could speculate that Ring1b-mediated mono-ubiquitination of histone H2A also interferes with the RNAPII complex at active H3K4me3 marked genes, but how remains to be investigated. Altogether, our data suggest that Ring1b is important to maintain an undifferentiated state of ES cells through repression of key developmental genes. The finding that this involves direct transcriptional repression of at least two epigenetically distinct sets of genes helps to understand and further explore the role of Ring1b during development. Materials and Methods Generating Ring1b conditional knockout ES cells The Ring1b wild type allele in Ring1b+/2 ES cells [17] was targeted with a construct containing loxP sequences at the EcoRI site in intron 2 and at the XhoI site in intron 4, followed by a third LoxP site enclosing a hygromycin cassette to allow for selection of targeted ES cells. Correct targeting of hygromycin resistant Ring1b2/LoxHyg ES cell clones was confirmed by Southern blot analysis. Next, to obtain Ring1b2/Lox ES clones the hygromycin cassette was removed by transiently expressing adenoviral Cre and subsequently screening for hygromycin sensitivity. Correct remov- al of the hygromycin cassette in the selected Ring1b2/Lox ES clones was confirmed by Southern blot analysis. Finally, to generate inducible conditional Ring1b2/Lox;CreERT2 ES cells a puromycin- selectable R26CreERT2 construct was targeted to the ROSA26 locus. CreERT2 could be activated by adding 200 nM 4-OHT (Sigma, dissolved in absolute ethanol) to the ES cell medium. Southern blot analysis of HindIII digested genomic DNA and a 59end external probe in intron 1 to discriminate between the Ring1b wild type (11 kb), conventional knockout (5 kb)[17], Lox (7 kb), and Del (4 kb) allele. The Del allele follows from deletion of exons 3 and 4 after 4-OHT-mediated activation of CreERT2 in Ring1b2/Lox;CreERT2 ES cells. Cell culture ES cells were cultured on dishes coated with 0.1% gelatin (Sigma) without feeders in 60% conditional Buffalo Rat Liver (BRL) medium, which consisted of 60% BRL medium and 40% standard ES medium. Standard ES cell medium consisted of GMEM (Gibco), 10% foetal calf serum (Sigma), supplemented with 1% non-essential amino acids, 1 mM sodium pyruvate, L- glutamate, 0.1 M beta-mercaptoethanol and 103 units LIF (all Gibco). BRL medium was derived by filtering (0.2 uM) the supernatant of BRL cells cultured for one week in standard ES cell medium without beta-mercaptoethanol or LIF. Ring1b conditional ES cells or Ring1b+/+;CreERT2 control ES cells were treated with 200 nM 4-OHT and MEFs with 1 mM 4- Ring1b Regulates ES Cell Fate OHT for the indicated time. MEFs were isolated from 14.5 day old embryo’s and cultured as described before [55]. For RA treatment ES cells were cultured in standard ES medium without LIF and in the presence of 500 nM all-trans-RA (ATRA) (Sigma). Protein isolation and Western blot analysis Proteins were extracted from whole cells (RIPA lysates, protocol as described previously) [65] or from the nucleus (nuclear extraction). For nuclear extracts cells were washed with PBS, incubated on ice for 5 min in Triton lysis buffer (0.1 M NaCl, 0.3 M sucrose, 3 mM MgCl2, 50 mM HEPES pH 6.8, 1 mM EGTA, 0.2% Triton-X100) supplemented with Complete protease inhibitor cocktail and 10 mM N-ethylmaleimide (NEM) followed by spinning at for 5 min 3000 rpm at 4uC to pellet the nuclei. Nucleic proteins were recovered by incubating the nuclei in SDS-lysis buffer (0.1% SDS, 50 mM TrisHCl pH 7.5, 0.15 M NaCl) for 10 min on ice, followed by sonification (10 pulses at 50%, level 4) and spinning for 1 min at 14.000 rpm to remove insoluble material. Protein samples were assayed with SDS-PAGE using pre-cast gradient gels (Invitrogen) and conventional Western blotting techniques. Immunostaining, alkaline phosphatase, and Annexin V staining ES cells were cultured four days with 200 nM 4-OHT or three days with 500 nM RA on coverslips coated with 0.1% gelatin. Cells were fixed in 4% paraformaldehyde for 10 min at RT and permeabilization in 0.25% Triton-X100/PBS for 5 min. Cells were then incubated for 30 minutes at RT in blocking solution (5% foetal calf serum (Gibco), 5% normal goat serum (Vector laboratories), 0.02% Triton-X100/PBS). Next, cells were incubat- ed with the 1st antibody for 1 hour at RT in blocking solution and afterwards washed five times 5 minutes with 0.02% Triton-X100/ PBS. Hereafter, cells were incubated for 1 hour at RT with fluorescence labeled 2nd antibody in blocking solution, followed by five washes of 5 min each with 0.02% Triton-X100/PBS. Cover slips were mounted on object slides using Vectashield with DAPI (Vector Laboratories). For the uH2A immunostaining cells were permeabilized prior to fixation by in cytoskeletal buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES pH6.8) for 30 sec, followed by 30 sec in cytoskeletal buffer containing 0.5% Triton-X100, and 30 sec in cytoskeletal buffer without Triton- X100, all on ice. For the alkaline phosphatase staining cells were cultured on dishes, followed by fixation and stained according to the manufactures protocol (Sigma). To determine the level of apoptosis, cells were collected by trypsinization, or first washed twice with PBS to obtain only the adherent cells, followed by staining with FITC-conjugated Annexin V-FITC (Roche) according to the manufactures protocol and analyzed by flow cytometry. Antibodies H3K27me3 rabbit polyclonal was a kind gift from T. Jenuwein. Ezh2 mouse monoclonal antibody was a kind gift from K. Helin. Ring1b mouse monoclonal and rabbit polyclonal antibodies were obtained from H. Koseki. Oct4 mouse monoclonal antibody C-10 and Lamin-B goat polyclonal antibody M20 from Santa Cruz, Nestin mouse monoclonal antibody from BD, Eed rabbit polyclonal and Bmi1 and uH2A mouse monoclonal antibodies from Upstate. Secondary immunofluorescence antibodies were Alexa 488- or 568-conjucated anti-mouse, 568- or 488-conjucated anti-rabbit from Molecular Probes. PLoS ONE | www.plosone.org 11 May 2008 | Volume 3 | Issue 5 | e2235