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The role of Polycomb group proteins throughout development : f(l)avoring repression van der Stoop, P.M.

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Citation for published version (APA): van der Stoop, P. M. (2009). The role of Polycomb group proteins throughout development : f(l)avoring repression. Amsterdam: Nederlands Kanker Instituut - Antoni Van Leeuwenhoekziekenhuis.

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Download date: 27 Oct 2019 Chapter 4

Ubiquitin E3 Ligase Ring1b/Rnf2 of Polycomb Repressive Complex 1 Contributes to Stable Maintenance of Mouse Embryonic Stem Cells

Petra van der Stoop*, Erwin A. Boutsma*, Danielle Hulsman, Sonja Noback, Mike Heimerikx, Ron M. Kerkhoven, J. Willem Voncken, Lodewyk F.A. Wessels, Maarten van Lohuizen

* These authors contributed equally to this work

Adapted from: PLoS ONE (2008) 3(5): e2235

Ring1b regulates ES cell fate

Ubiquitin E3 Ligase Ring1b/Rnf2 of Polycomb Repressive Complex 1 Contributes to Stable Maintenance of Mouse Embryonic Stem Cells

Petra van der Stoop1*, Erwin A. Boutsma.1*, Danielle Hulsman1, Sonja Noback1, Mike Heimerikx2, Ron M. Kerkhoven2, J. Willem Voncken4, Lodewyk F.A. Wessels3, Maarten van Lohuizen1,5#

1Division of Molecular Genetics, and 2Center for Biomedical Genetics, and 3Bioinformatics and Statistics, Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, the Netherlands, 4Department of Molecular Genetics, Maastricht University Medical Centre, Maastricht, the Netherlands, 5Department of Neurogenetics, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands.

* These authors contributed equally to this work

Background Polycomb repressive complex 1 (PRC1) core member Ring1b/Rnf2, with ubiquitin E3 ligase activity towards histone H2A at lysine 119, is essential for early embryogenesis. To obtain more insight into the role of Ring1b in early development, we studied its function in mouse embryonic stem (ES) cells.

Methodology/Principle Findings We investigated the effects of Ring1b ablation on transcriptional regulation using Ring1b conditional knockout ES cells and large-scale gene expression analysis. The absence of Ring1b results in aberrant expression of key developmental genes and deregulation of specific differentiation-related pathways, including TGFbeta signaling, cell cycle regulation and cellular communication. Moreover, ES cell markers, including Zfp42/Rex-1 and Sox2, are downregulated. Importantly, retained expression of ES cell regulators Oct4, Nanog and alkaline phosphatase indicates that Ring1b-deficient ES cells retain important ES cell specific characteristics. Comparative analysis of our expression profiling data with previously published global binding studies shows that the genes that are bound by Ring1b in ES cells have bivalent histone marks, i.e. both active H3K4me3 and repressive H3K27me3, or the active H3K4me3 histone mark alone and are associated with CpG-‘rich’ promoters. However, deletion of Ring1b results in deregulation, mainly derepression, of only a subset of these genes, suggesting that additional silencing mechanisms are involved in repression of the other Ring1b bound genes in ES cells.

Conclusions Ring1b is essential to stably maintain an undifferentiated state of mouse ES cells by repressing genes with important roles during differentiation and development. These genes are characterized by high CpG content promoters and bivalent histone marks or the active H3K4me3 histone mark alone.

69 Chapter 4

Introduction 2000; Takihara et al, 1997; van der Lugt et al, 1994; Voncken et al, 2003). PcG proteins PcG proteins have initially been identified were shown to be important for self-renewal in Drosophila as transcriptional repressors and maintaining pluripotency of ES cells required for correct expression of homeotic (Boyer et al, 2006; Pasini et al, 2007; Valk- (Hox) genes. By means of chromatin Lingbeek et al, 2004). ES cells are derived remodeling, PcG proteins maintain stable from the inner cell mass of pre-implantation gene repression to ensure proper embryonic blastocysts (Evans and Kaufman, 1981). ES development. In mammals, two biochemically can self-renew and maintain a pluripotent, and functionally distinct PcG core complexes undifferentiated state under the right have been identified (Lund and van conditions in vitro, while retaining the Lohuizen, 2004). Mammalian polycomb capacity to differentiate into every cell type repressive complex 1 (PRC1) consists of required during development in vitro and close homologs of the Drosophila PRC1 in vivo when reintroduced back into a host core members Ph, Pc, Psc and dRing (Lund blastocyst (Beddington and Robertson, and van Lohuizen, 2004; Ringrose and Paro, 1989). Genome-wide and candidate-based 2004). Ring1b/Rnf2, the mouse homolog of studies revealed that PcG proteins maintain dRing, acts as a ubiquitin E3 ligase towards this undifferentiated state of ES cells through histone H2A at lysine 119 resulting in mono- direct repression of developmental genes ubiquitinated H2A (uH2A) (Buchwald et (Boyer et al, 2006; Bracken et al, 2006; Lee al, 2006; Wang et al, 2004). Ezh2/Kmt6, a et al, 2006; Squazzo et al, 2006; Tolhuis et methyltransferase that trimethylates histone al, 2006). Developmental genes are largely H3 at lysine 27 (H3K27me3), acts in complex associated with bivalent (i.e. both repressive with Suz12 and Eed, constituting Polycomb H3K27me3 and active H3K4me3) histone repressive complex 2 (PRC2) (Cao et al, marks and are silent or expressed at very 2002; Kirmizis et al, 2004; Kuzmichev et al, low levels in ES cells (Azuara et al, 2006; 2002). PRC1 and PCR2 do not physically Bernstein et al, 2006; Mikkelsen et al, 2007). interact, but the Ezh2 catalyzed histone mark Bivalent domains tend to resolve during H3K27me3 is recognized by PRC1 member development (Azuara et al, 2006; Bernstein Pc, providing a mechanism of communication et al, 2006; Mikkelsen et al, 2007). Therefore, between the two complexes (Fischle et it is suggested that this bivalent chromatin al, 2003). Furthermore, PRC1 binding to state poises genes for transcriptional chromatin requires PRC2, although PRC2- activation (loss of H3K27me3) or prolonged independent recruitment of PRC1 is also repression (loss of H3K4me3 or both marks) reported (Hernandez-Munoz et al, 2005b; during later developmental stages. Only Schoeftner et al, 2006). The counteracting recently, it was shown that Ring1b-mediated trithorax group (TrxG) proteins mediate an uH2A deposition at repressed bivalent active transcriptional state by methylation of genes restrains a poised RNA polymerase histone H3 at lysine 4 (H3K4me3) (Ringrose II (RNAPII) configuration (Stock et al, and Paro, 2004). 2007). Conditional deletion of Ring1b and Proper expression of PRC2 proteins subsequent loss of uH2A from the promoter is essential during early embryonic and coding region resulted in release of development, since mice lacking one of poised RNAPII and gene derepression. More these proteins die due to gastrulation evidence for a direct role of uH2A in negative defects (Faust et al, 1995; O’Carroll et al, transcriptional regulation was found by 2001; Pasini et al, 2004). In contrast, PRC1 another group, which investigated the role of members appear to be more important during histone H2A E3 ligase 2A-HUB in repressing later stages of development, with the clear chemokine genes in human monocytes exception of Ring1b, which evokes an early (Zhou et al, 2008). They showed that the embryonic lethal phenotype similar to PRC2 presence of uH2A in the promoter-proximal null mice (Akasaka et al, 1996; del Mar et al, region physically blocks the recruitment of

70 Ring1b regulates ES cell fate

FACT, thereby causing RNAPII-dependent locus of Ring1b-/Lox ES cells, hereafter transcription elongation to pause. named Ring1b-/Lox;CreERT2 ES cells. The To explore the role of Ring1b in regulation of presence of loxP sequences in the genomic gene expression during early development, locus of Ring1b did not alter the expression we analyzed genome-wide changes in of Ring1b in ES cells. Accordingly, Ring1b-/ gene transcription following deletion of Lox and Ring1b-/Lox;CreERT2 mice developed Ring1b in ES cells. Hereto, we employed an indistinguishable from wild type mice (E.B, inducible knockout system and performed M.v.L, unpublished results). a genome-wide screen using microarrays Addition of 4-OHT to the culture medium to identify genes governed by Ring1b. We resulted in recombination of the Ring1b identify several processes and pathways conditional allele in Ring1b-/Lox;CreERT2 ES differentially regulated in Ring1b-deficient ES cells and subsequent depletion of Ring1b cells, which are implicated in differentiation protein as was shown by Southern and and embryogenesis. Importantly, we find that Western blot analysis (Figure 1B, 1C). We Ring1b-deficient ES cells retain expression observed cellular detachment and apoptosis of key stem cell regulators Oct4 and Nanog. concurrent with an increase in cells with Finally, by comparing our expression data with a differentiated phenotype after deleting previously published global binding studies, Ring1b in Ring1b-/Lox;CreERT2 ES cells, but we find that Ring1b preferentially represses not in similarly treated Ring1b+/+;CreERT2 genes with high CpG-content promoters and control ES cells. Based on Annexin V staining bivalent or H3K4me3 histone marks. This followed by flow cytometric analysis, about suggests that Ring1b has an important role 83% of all Ring1b-/Lox;CreERT2 ES cells were in maintaining an undifferentiated state of ES undergoing apoptosis four days following cells. Furthermore, this study provides more 4-OHT treatment (data not shown). Of the insight in the characteristics of the genes adherent population only 15% was Annexin that are under direct transcriptional control V positive, compared to 5% of the untreated of Ring1b in ES cells. population (data not shown). We therefore focused our studies on the immediate early effects of the first days following Cre- Results mediated deletion of Ring1b in the viable, adherent ES cells. Generating Ring1b conditional knockout mouse embryonic stem cells Ring1b is required for mono-ubiquitination To gain insight in the role of Ring1b in ES of H2A in ES cells cells, we generated Ring1b conditional Since Ring1b is a ubiquitin E3 ligase for knockout mouse ES cells. Previously, we histone H2A at lysine 119, we studied the have shown that deletion of the RING finger effect of Ring1b deletion on the levels domain of Ring1b, encoded by exon 3 and 4, of uH2A. Western blot analysis showed generates a functional null allele (Voncken that uH2A levels decreased to almost et al, 2003). Ring1b conditional knockout undetectable levels after deletion of Ring1b (Ring1b-/Lox) ES cells were then generated from Ring1b-/Lox;CreERT2 ES cells (Figure by a second targeting round in Ring1b+/- ES 1C). In addition, immunofluorescence cells (Voncken et al, 2003) using a targeting stainings confirmed that uH2A was lost in vector, which introduced loxP sequences ES cells that had lost Ring1b expression flanking these exons (Figure 1A; details (Figure 2A). The same held true for mouse in Materials and Methods). To generate embryonic fibroblasts (MEFs) derived from inducible Ring1b-/Lox ES cells, we targeted Ring1b-/Lox;CreERT2 mouse embryos, which a 4-hydroxytamoxifen (4-OHT) inducible showed no uH2A immunofluorescence R26CreERT2 construct (Cre-recombinase staining in Ring1b-negative cells (Figure fused to a mutated ligand binding domain of S1). Immunofluorescence stainings for the human estrogen receptor) to the ROSA26 H3K27me3, the polycomb repressive mark

71 Chapter 4 set by PRC2 member Ezh2, showed that Ring1b deficiency affects expression of this mark was present in all cells (Figure other PRC1 core members 2B). These results confirm that Ring1b is Studies in Drosophila, mouse and human the main E3 ligase for mono-ubiquitination cells have shown that PcG genes themselves of H2A in both mouse ES cells and MEFs are PcG protein binding targets, suggesting and suggest that deletion of Ring1b does not auto-regulation within the PcG family (Boyer affect H3K27 trimethylation. et al, 2006; DeCamillis et al, 1992; Lee et al, 2006). We therefore examined the

Figure 1. Generating Ring1b conditional knockout ES cells. (A) Schematic overview of the Ring1b locus targeting strategy in mouse Ring1b+/- 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+/- 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 5’end probe used for Southern blot analysis. (B) Southern blot analysis of HindIII digested genomic DNA of Ring1b-/Lox;CreERT2 ES cells showing recombination of the Ring1bLox allele following treatment with 4-OHT for the indicated time in days. The 5’end 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 Ring1b-/Lox;CreERT2 ES cells following treatment with 4-OHT for the indicated time in days. Tubulin serves as a loading control.

72 Ring1b regulates ES cell fate effect of Ring1b deletion on other PRC1 other PRC1 core members may explain and PRC2 members in ES cells. Microarray the severity of the Ring1b mouse knockout analysis (described in more detail in the phenotype compared to knockout models of following section) showed that the transcript other PRC1 members (Akasaka et al, 1996; levels of Ring1b and Phc1/Mph1 were del Mar et al, 2000; Takihara et al, 1997; van significantly downregulated over time, and der Lugt et al, 1994; Voncken et al, 2003). correspondingly decreased in protein levels Other polycomb group genes of which the (Figure 3A, Table S1, which is available as transcription had increased significantly supporting information at the PLoS ONE were Epc1, Phf1, and Phc2 (Table S1, which website). Interestingly, PRC1 member Bmi1 is available as supporting information at was derepressed transcriptionally in the the PLoS ONE website). No effects were absence of Ring1b, but its protein levels were observed on the protein or transcript levels down-regulated, suggesting that Bmi1 is post- of PRC2 members Ezh2 and Eed, or the transcriptionally regulated by Ring1b (Figure levels of H3K27me3, which is in agreement 3B). This is consistent with observations by with the immunofluorescence analysis others (Leeb and Wutz, 2007), and may be (Figure 3A, 3B, Table S1). The effect on explained by the fact that interaction between PcG protein and transcript levels after loss Bmi1 and Ring1b protects both proteins from ubiquitin-mediated degradation (Ben Saadon et al, 2006). The effect of loss of Ring1b on A 0 1 2 3 4

Ring1b A Ring1b uH2A DAPI Mph1

Eed untreated

Oct4

Tubulin 4-OHT B 0 1 2 3 4

Ring1b B Ring1b H3K27me3 DAPI Bmi1

Ezh2

untreated H3K27me3

LaminB

Figure 3. Downregulation of protein levels of 4-OHT PRC1, but not PRC2, following Ring1b deletion in ES cells. Western blot analysis of total cell protein Figure 2. Deletion of Ring1b results in loss extracts (A) or nuclear extracts (B) of Ring1b-/ of uH2A but not H3K27me3. Examples of Lox;CreERT2 ES cells shows that deletion of Ring1b immunofluorescence stainings in Ring1b-/ results in downregulation of Phc1/Mph1 and Bmi1 Lox;CreERT2 ES cells showing loss of uH2A (A), but protein levels, but not of Ezh2, Eed or H3K27me3 not H3K27me3 (B) in cells that have lost Ring1b levels following treatment with 4-OHT for the four days following 4-OHT treatment. indicated number of days.

73 Chapter 4 of Ring1b is in agreement with an earlier physiological processes’, ‘glyconeogenesis’, study performed in established Ring1b- and ‘cell cycle’ (Figure 4E; for a graphical deficient ES cells, with the exception of the overview of the overrepresented GO transcriptional downregulation of Phc1 (Leeb categories, see Figure S2; for the BiNGO and Wutz, 2007). This might therefore reflect analysis, see Table S2). These data confirm an indirect effect of acute loss of Ring1b in that Ring1b is involved in regulating cell ES cells. These results suggest that deletion cycle, developmental and chromatin-related of Ring1b affects the transcript and protein processes and indicate that deletion of Ring1b levels of members of PRC1, but not of PRC2, affects glyconeogenesis and the regulation of and underscores the importance of Ring1b cellular physiological processes. Given that for PRC1 stability. loss of Ring1b affects the undifferentiated state of ES cells, the regulated processes are Regulation of developmental processes likely to include genes that are deregulated through derepression of Ring1b bound due to indirect effects. genes By using a genome-wide ChIP-on-chip To obtain insight in the genome-wide effects analysis Boyer and colleagues identified on gene transcription after deletion of Ring1b genes that are bound by Ring1b in mouse in ES cells, we performed microarray analysis. ES cells (Boyer et al, 2006). By combining We analyzed the temporal changes in gene our microarray expression data with the data transcription in Ring1b-/Lox;CreERT2 ES cells from this Ring1b binding study, we were one to four days after deletion of Ring1b. As able to determine which genes were direct a reference, we used Ring1b+/+;CreERT2 ES transcriptional targets of Ring1b. Of the 1219 cells similarly treated with 4-OHT to correct Ring1b bound genes, identified by Boyer for any effects induced by 4-OHT or CreERT2. and colleagues, 1078 were represented on We identified 2365 differentially regulated our microarray. Remarkably, we found that genes, i.e. genes that were significantly only a subset of 141 Ring1b bound genes (p<0.05 according to the Rosetta-error had significantly changed expression (13% model, for details see Materials and Methods) (141/1078). These genes were predominantly up- or downregulated (7%; 2365/31769; upregulated, which is in agreement with Figure 4A, 4B, Table S1, which is available Ring1b being a transcriptional repressor as supporting information at the PLoS ONE (Figure 4C). The partial derepression implies website). The reliability of the microarray that other regulatory mechanisms are active data was confirmed by quantitative real-time at the other Ring1b target genes in addition PCR analysis (QPCR) on a selection of 18 to Ring1b, and reflects that a subset of genes genes (Figure 4D). is deregulated due to indirect effects of loss To gain insight into the processes that are of Ring1b in ES cells. deregulated in absence of Ring1b in mouse Next, we assessed which of the deregulated ES cells, we analyzed the deregulated genes within an overrepresented GO genes based on their ontology classification. category (most down-stream in the GO- To this end, we employed the gene ontology tree, see also Figure S2) were bound by (GO) tool BiNGO to determine which GO Ring1b. We found that on average 30% of categories are statistically overrepresented. the deregulated genes within an enriched By mapping the predominant functional GO ontology was bound by Ring1b, in particular categories in a hierarchical graph BiNGO in the ontologies organ development and facilitates insight in the hierarchical structure morphogenesis, nucleosome assembly, and and relations between the ontologies (Maere regulation of cellular physiological process et al, 2005). We found that the outliers were (Figure 4F). This was substantially higher involved in various processes, of which the than what would be expected considering most downstream in the GO-tree were: that only 6% (141/2365) of all deregulated ‘organ development’, ‘organ morphogenesis’, genes was bound by Ring1b. This indicates ‘nucleosome assembly’, ‘regulation of cellular that Ring1b is involved in direct transcriptional

74 Ring1b regulates ES cell fate regulation of a substantial large fraction of specifically important for dynamic repression the deregulated genes in these ontologies. of developmental, chromatin-related, cell Together, these data suggest that Ring1b is cycle and metabolic genes in ES cells.

D stem cell genes Ring1b bound genes other genes A 1 2 3 4 22 untreated 12 day 2 10 day 3 8 6 4

relative mRNA levels relative 2 0 Id1 Id2 Kit1 Yy1 Bin1 Fgf4 Pitx2 Oct4 Sox2 Fgf15 Sfmbt Gata3 Nanog Ccnd2 Dppa3 Lmx1a Notch1 Krt1-19

alcohol biosynthesis E monosaccharide biosynthesis 3.5 hexose biosynthesis gluconeogenesis pyruvate metabolism 3 carboxylic acid metabolism organic acid metabolism cellular physiological process 2.5 cell cycle organ development metabolism cellular metabolism 2 nucleobase, ..., nucleic acid metabolism nucleosome assembly B 1400 chromatin assembly 1.5 1200 regulation of physiological process 1000 primary metabolism regulation of cellular physiological process 800 chromatin assembly or disassembly 1 600 organ morphogenesis 400 regulation of cellular process regulation of biological process 200 0.5 morphogenesis Numberof regulated genes 0 DNA metabolism 1 2 3 4 development 0 1 2 3 4 (-log10)p-value

120 C F # R1b # R1b 100 % Up bound % Down bound

80 gluconeogenesis 40 (2/5) 1 60 (3/5) 0 cell cycle 62 (24/39) 4 38 (15/39) 0 60 organ development 63 (36/57) 14 37 (21/57) 1 40 nucleosome assembly 93 (13/14) 8 7 (1/14) 0 20 Numberof regulated Ring1b bound genes organ morphogenesis 80 (37/46) 16 20 (9/46) 0 0 reg of cellular phys 100 (109/109) 32 0 (0/109) 0 1 2 3 4

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 (Boyer et al, 2006). (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).

75 Chapter 4

of S phase (derepressed Ccnd2/Cyclin Regulation of specific differentiation- D2 and Cul1), G2/M arrest (derepressed related pathways in Ring1b-deficient ES Gadd45g and Sfn/14-3-3-sigma) or bypass cells of G2/M arrest (downregulated Gadd45a) The analysis based on the ontology and mitotic exit (derepressed Cdc20 and classification shows that absence of Ring1b Cdc27). This could imply that Ring1b has a results in aberrant expression of genes dual role in cell cycle regulation in ES cells, involved in developmental and various which is supported by the observation that cellular processes. We set out to identify Ring1b is involved in direct repression of cell specific pathways that are affected in Ring1b- cycle promoting gene Ccnd2 and cell cycle deficient ES cells. To remain unbiased inhibitory genes Cdkn1c and Gadd45g. in the analysis of our set of outliers, we Various components of cellular used the bioinformatics tool DAVID, which communication pathways were deregulated uses information of well-defined pathways in the absence of Ring1b. These comprise available in the KEGG pathway database ECM genes (derepressed Col4a2 and Thbs1) (http://david.abcc.ncifcrf.gov). Interestingly, involved in focal adhesion, gap junction we found that the identified pathways have genes (derepressed Gja1 and Csnk1d and roles in development and differentiation of downregulated Itpr1), and adherens junction ES cells. A selection is listed in Table 1. A pathways, which are linked to the actin complete overview of the pathway analysis cytoskeleton organization (downregulated can be found in Table S3. Vcl and Actn3, derepressed Iqgap1). Most revealing was the identification of the Several of these genes were reported to TGFbeta signaling pathway of which Bmp/ change expression during differentiation and Id signaling (derepressed Bmp7, Bmpr1a, development (Schenke-Layland et al, 2007; Id1, Id2, and Id3) and Nodal/Pitx2 signaling Wong et al, 2004; Yamauchi et al, 2007). (derepressed Nodal, Pitx2) were suggested to Summarizing, this suggests that Ring1b be transcriptionally upregulated upon loss of deficiency induces changes in cellular Ring1b. Genome-wide binding studies have pathways, such as Bmp/TGFbeta identified that PcG proteins are associated signaling, cell cycle regulation, and cellular with members of the TGFbeta signaling communication, which strongly suggests a pathway and show PcG dissociation from link to differentiation programs in ES cells. the genes that were derepressed following differentiation (Boyer et al, 2006; Lee et al, Retained expression of stem cell 2006). These data support a role for Ring1b/ regulators in Ring1b-deficient ES cells PcG in direct repression of TGFbeta signaling ES cells, derived from the epiblast of early in ES cells. blastocysts, can self-renew and maintain In agreement with the GO classification a pluripotent, undifferentiated state in vitro analysis, we found altered expression by extrinsic factors, such as LIF and Bmp, of several components of the cell cycle and intrinsic factors, including stem cell pathway. Changes in cell cycle regulation regulators Oct4 and Nanog (Boyer et al, have been observed following differentiation 2005; Chambers et al, 2003; Niwa et al, 2000; of ES cells, characterized by amongst Ying et al, 2003). Upon induction of lineage- others activation of the pRb-E2f controlled specific genes, these stem cell regulators G1 checkpoint, which is inoperative in ES are downregulated through a negative- cells (Burdon et al, 2002; White et al, 2005). feedback-loop by not yet understood In line with this, key cell cycle regulators mechanisms (Fujikura et al, 2002; Niwa et al, involved in activation of the G1/S checkpoint 2000; Ying et al, 2003). In line with this, the (derepressed Cdkn1c; downregulated E2f1, transcript levels of stem cell specific genes Cdc7 and Mcm6) were transcriptionally Sox2, Dppa3/Stella and Zfp42/Rex-1 were deregulated. However, deregulation of other strongly downregulated in Ring1b-deficient key cell cycle genes suggest promotion ES cells (Boyer et al, 2005; Nakamura et al,

76 Ring1b regulates ES cell fate

2007) (Figure 4D, Table S1). However, QPCR outlier on the microarray and Western blot analysis showed that Nanog transcript levels analysis showed no significant changes in had not altered and that Oct4 transcript levels Oct4 protein levels (Figure 3A, Table S1). had reduced only to about 55% (Figure 4D). To examine Oct4 expression in more detail, Importantly, Oct4 was not identified as an we also quantified the immunofluorescence

Table 1. A selection of the deregulated genes and corresponding pathways affected in Ring1b-deficient ES cells.

Symbol EntrezID Regulation bound Symbol EntrezID Regulation Bound Focal Adhesion TGFbeta signaling Actg2 11464 up 0 Amhr2 110542 down ND Actn3 11474 down 0 Bmp7 12162 up 1 Capn2 12334 up 0 Bmpr1a 12166 up 0 Capn5 12337 down 0 Cul1 26965 up 0 Ccnd1 12443 up 0 Id1 15901 up 0 Ccnd2 12444 up 1 Id2 15902 up 0 Col4a2 12827 up 1 Id3 15903 up 1 Grb2 14784 down 0 Lefty1 13590 up 0 Ilk 16202 up 0 Mapk3 26417 down ND Nras 18176 down 0 Nodal 18119 up 0 Pdgfc 54635 up ND Pitx2 18741 up 0 Pten 19211 up 0 Thbs1 21825 up 0 Rras2 20130 down 0 Shc2 216148 down ND Cell cycle Thbs1 21825 up 0 Anapc10 68999 up 0 Vegfa 22339 down 0 Ccnd2 12444 up 1 Zyx 22793 up 0 Cdc20 107995 up 0 Cdc27 217232 up 0 Gap junction Cdc7 12545 down 0 Itpr1 16438 down 0 Cdkn1c 12577 up 1 Adcy2 210044 up 1 Chek1 12649 down 0 Csnk1d 104318 up 0 Cul1 26965 up 0 Edg2 14745 down 0 Gadd45a 13197 down 0 Gja1 14609 up 1 Gadd45g 23882 up 1 Grb2 14784 down 0 Mcm6 17219 down 0 Nras 18176 down 0 Sfn 55948 up ND Pdgfc 54635 up ND Smc1l2 140557 down 0 Rras2 20130 down 0

Adherens junctions Actn3 11474 down 0 Baiap2 108100 up 0 Iqgap1 29875 up 0 Lmo7 380928 down ND Mapk3 26417 down ND Pvrl4 71740 down 0 Vcl 22330 down 0 Wasl 73178 up 0

DAVID Pathway analysis revealed the pathways deregulated in Ring1b-deficient ES cells. Shown are the deregulated genes per pathway, their EntrezIDs, the deregulated expression state, and binding of Ring1b to the promoter (1=bound, 0=not bound, ND=no data). For a complete overview of all identified pathways see Table S3.

77 Chapter 4 levels of Oct4 and Ring1b stainings in (Figure 5A, 5B). However, whereas four individual cells. We compared this to guided days following Ring1b deletion, 81% of all neural differentiation induced by retinoic acid cells were negative for Ring1b, only 5% had (RA) treatment, which was demonstrated lost Oct4 expression (Figure 5A, 5B, 5C). To to result in significant downregulation of assess the extent of neural differentiation, Oct4 (Minucci et al, 1996). As expected, we next examined the expression of we found that three days after RA induced neuronal-lineage marker Nestin (Cattaneo neural differentiation Oct4 was completely and McKay, 1990). We found that three downregulated, in about 65% of the cells days after RA treatment approximately

A Oct4 DAPI Merge B untreated C untreated 60 80 60 40 40 20 20 0 0

untreated 0 0

4-OHT 4-OHT 60 80 60 40 40 4-OHT 20 20 0 0 0 0 RA RA

RA 60 80 60 40 40 20 20 0 0 number of cells D Nestin DAPI Merge 0 number of cells 0 relative intensity Oct4 relative intensity Ring1b

E 100 ubiquitous 80 filamentous

untreated 60 40 % cells % 20 0 u 4OHT RA 4-OHT F untreated 4-OHT RA RA

Figure 5. Ring1b-deficient ES cells retain expression of stem cell specific regulators. (A and D) Examples of immunofluorescence stainings in Ring1b-/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 and 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 Ring1b-/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 Ring1b-/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 Ring1b-/Lox;CreERT2 ES cells either untreated, or treated for four days with 4-OHT or three days with RA.

78 Ring1b regulates ES cell fate

44% of the cells showed filamentous Nestin staining, compared to 32% of the cells after Ring1b represses bivalent or H3K4me3 deletion of Ring1b (Figure 5D, 5E). This marked genes with CpG-rich promoters is remarkable considering the number of Ring1b-mediated H2A mono-ubiquitination cells expressing Oct4. This suggests that was shown to restrain a poised RNAPII expression of lineage markers co-exists with configuration specifically at the promoters stem cell regulators in absence of Ring1b in of bivalent genes, which were derepressed ES cells. Finally, we examined the activity upon loss of Ring1b and uH2A (Stock et of alkaline phosphatase, which is highly al, 2007). Therefore, we investigated the expressed in undifferentiated, pluripotent chromatin state of the genes that were ES cells (Pease et al, 1990). We found that deregulated in absence of Ring1b in ES cells. after deleting Ring1b a substantial number Hereto, we used a data set from Mikkelsen of cells still showed high activity of alkaline and colleagues, who generated genome- phosphatase compared to RA treated ES wide chromatin-state maps for mouse ES cells, which is indicative for a pluripotent, cells, neural progenitor cells (NPC) and undifferentiated state of ES cells (Figure MEFs (Mikkelsen et al, 2007). Of 948 of the 5F). Combined, our findings suggest that 1078 Ring1b bound genes (Boyer et al, 2006) Ring1b-deficient ES cells show features of represented on our microarray chromatin- differentiated cells, while retaining important state maps were retrieved for the histone ES cell characteristics. modifications H3K4me3 and H3K27me3 (Mikkelsen et al, 2007). Notably, virtually all Ring1b represses genes that are co- (99%) of the Ring1b bound genes that were occupied by stem cell regulators Oct4 mapped as marked with both H3K27me3 and and Nanog in ES cells H3K4me3 or H3K27me3 alone, according The stem cell-specific transcription factors to Mikkelsen-data set (Mikkelsen et al, Oct4 and Nanog control ES cell pluripotency 2007), were associated with H3K27me3 by regulating expression of stem cell specific according to the H3K27me3 mapping data genes and repression of differentiation- by Boyer et. al. (Boyer et al, 2006), indicating specific genes, of which a subset is co- a strong overlap between the two data occupied by PcG proteins (Boyer et al, 2005; sets for this histone mark. The chromatin- Lee et al, 2006; Loh et al, 2006). Considering state maps also contain information about the retained expression of Oct4 and Nanog, the CpG content of promoters genome- we were interested to see the effect on gene wide. Mikkelsen and colleagues show that transcription of these co-occupied genes in genes with high-CpG content promoters absence of Ring1b. We therefore analyzed (HCP) and H3K4me3 marks generally the changes in expression of genes that were have ‘housekeeping’ functions, including bound by Ring1b (Boyer et al, 2006) and by replication and basic metabolism, while Oct4 and/or Nanog in mouse ES cells as genes with HCP and bivalent marks mainly was identified by Loh and co-workers (Loh include key developmental transcription et al, 2006). We found that the expression factors, morphogens, and cell surface of 25 of the 212 Nanog/Oct4/Ring1b bound markers. Genes with low-CpG content genes represented on our microarray was promoters (LCP) are associated with highly altered in Ring1b-deficient ES cells (Figure tissue-specific genes. A third class of genes 4D, Table S1). We found that 18 of these 25 with intermediate CpG promoter content genes were derepressed, including Fgf15, (ICP) was added to ensure discrimination Bmp7, Gata3, Bmi1, Msx2, Podxl, Col4a2, between the HCPs and LCPs. Gadd45g, Gja1 and Eif4g3. This suggests Comparative analysis showed that about that Ring1b is required to repress a specific two-third of the promoters genome-wide, subset of Oct4 and Nanog bound genes to i.e represented on our microarrays, or maintain a pluripotent, undifferentiated ES differentially regulated in Ring1b-deficient ES cell state. cells, had HCPs (Figure 6A, 6B). Remarkably,

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Ring1b almost exclusively associated with We next analyzed the chromatin state of the and regulated transcription of genes with Ring1b bound genes with HCPs. We found HCPs (93%; 882/948 and 94%; 119/126, that these genes were strongly associated respectively; Figure 6C, 6D). This suggests with bivalent marks (74%; 653/882). A that Ring1b primarily regulates transcription similar fraction of deregulated Ring1b bound of CpG-‘rich’ promoters. genes with HCPs was bivalently marked

genes deregulated genes A B E HCP F ICP genome-w ide genome-w ide 100 100 ICP; 204 ICP; 2402 LCP; 108 80 80 K4 K4+K27 LCP; 2146 60 60 K27 HCP; 8784 HCP; 1079 40 40 none

number of genes (%) 20 20 C Ring1b bound D deregulated Ring1b genes bound genes 0 0 ICP; 54 ICP; 7 LCP; 12 LCP; 0

eregulated

eregulated

deregulated Ring1bbound genome-wide Ring1bbound d

Genome-wide deregulated Ring1bbound genome-wide Ring1bbound d

Genome-wide

HCP; 882 HCP; 119 H R1b K4+K27 up (n=75) I R1b K4+K27 down (n=12) 2 1 5 1 G 0 0

(log2) -1 4 -1 -2

3 expression ratio 1 2 3 4 1 2 3 4 J 2 R1b K4 up (n=21) K R1b K4 down (n=11)

1 1 1

expression level (log10) 0 0 0 (log2) K4 K4+K27 K4 K4+K27 K4 K4+K27 -1 -1

genome-wide R1b bound dereg. R1b bound expression ratio 1 2 3 4 1 2 3 4

Figure 6. Deregulation of genes bivalent or H3K4me3 marked genes with HCPs in Ring1b-deficient ES cells. (A-D) Circle graphs show that Ring1b primarily occupies (C) and regulates (D) transcription of genes with high CpG content promoters (HCPs) rather than intermediate (ICP) or ‘low’ (LCP) CpG content promoters, compared to the distribution of HCPs in all genes (A) or only the deregulated genes (B) at the microarray (‘genome-wide’). (E) Bar graph showing the distribution (percentages) of the chromatin state of HCPs for all genes on the whole microarray (‘genome-wide’); only the genes deregulated in absence of Ring1b; Ring1b bound genes; only deregulated Ring1b bound genes in Ring1b-deficient ES cells. Graph shows that Ring1b binds and regulates transcription of bivalent, and to a lesser extend H3K4me3 marked genes (final two columns), in contrast to the genome-wide distribution of these marks on promoters (first two columns). (F) as (E), but for genes with ICPs. Graph shows that bivalent Ring1b bound genes with ICPs are not deregulated in Ring1b-deficient ES cells. G( ) Blotplot representing Affimetrix array data of RNA expression levels in wild type ES cells (Mikkelsen et al, 2007), for the genes represented in (E). The blotplot shows that the median expression levels of H3K4me3 marked genes on the whole microarray (‘genome-wide’ ) are higher compared to the median RNA expression levels of H3K4me3+H3K27me3 marked genes (first two columns). This is similar for genes that are bound by Ring1b (middle two columns), and for Ring1b bound genes that are deregulated in Ring1b-deficient ES cells (last two columns). Black diamond represents median RNA expression level. (H-K) Line graphs showing changes (log2 ratio) in gene expression of bivalently (H-I) or H3K4me3 (J-K) marked genes bound by Ring1b following deletion of Ring1b in Ring1b-/Lox;CreERT2 ES cells. Time is indicated in number of days of 4-OHT treatment.

80 Ring1b regulates ES cell fate

(73%; 87/119; Figure 6E). These deregulated we cannot exclude that differentiation- bivalently marked Ring1b bound genes associated changes affect transcription of included mostly developmental genes, the Ring1b bound genes, these data suggest such as Bin1, Bmi1, Bmp7, Col4a2, Ccnd2, that Ring1b predominantly represses bivalent En1, Gata3, Lmx1a and Wnt6, and were genes, and is also involved in modulating, predominantly derepressed in absence rather than silencing, the transcriptional of Ring1b (86%; 75/87; Figure 6H, 6I). activity of genes with the active H3K4me3 Interestingly, we found that Ring1b was also histone mark in ES cells. associated with a considerable number of Strikingly, a specific subset of Ring1b bound HCPs with the H3K4me3 mark alone (25%; genes was not affected following Ring1b 218/882; Figure 6E). The lack of repressive deletion. This subset contains LCPs, which H3K27me3 marks at these genes, catalyzed were all without H3K27me3 or H3K4me3 by PRC2, implies that Ring1b might be marks, and bivalently marked genes with recruited independent from PRC2 for these ICPs (Figure 6D, 6F). This implies that other genes in ES cells. Of the deregulated mechanisms are involved in regulation of H3K4me3 marked Ring1b bound genes these genes in ES cells. with HCPs 66% (21/32) was derepressed, Summarizing, these data suggest that including Eif4g3, Gja1 and Sgce, and Ring1b is involved in direct regulation of various histone encoding genes, such as RNAPII controlled promoters with ‘high’ CpG Hist1h2bn, Hist1h2bf, Hist1h3i, Hist1h4h, and content and epigenetically distinct histone Hist1h4f (Figure 6J, 6K). Not all promoters marks, namely bivalent marks or the active associated with the active H3K4me3 mark H3K4me3 mark, in mouse ES cells. are actively transcribed (Mikkelsen et al, 2007). Considering the fact that Ring1b is a transcriptional repressor, we predicted that Discussion the H3K4me3 marked genes with relative low expression levels were bound by Ring1b. To investigate the role of Ring1b in mouse We therefore examined the RNA expression ES cells, we employed a conditional levels of these genes in wild type ES cells knockout system. By performing microarray by using the Affimetrix RNA expression data analysis, we studied the genome-wide that was available in the chromatin-state changes in gene transcription that occur mapping study by Mikkelsen et. al. (Mikkelsen following Ring1b deletion in ES cells. We et al, 2007). Unexpectedly, Ring1b binding found that in Ring1b-deficient ES cells key did not correlate with low expression levels developmental genes were derepressed, of H3K4me3 marked genes, but instead including a subset of genes bound by stem seemed to associate with these actively cell regulators Oct4 and Nanog, suggesting marked genes indifferent of their level of that Ring1b deficiency results in activation expression (Figure 6G). Notably, in more of the differentiation program. Accordingly, committed NPCs and MEFs (89% and 83%, specific differentiation-related pathways respectively) most of the Ring1b bound were differentially deregulated, including H3K4me3 marked genes retain this histone TGFbeta signaling, cellular communication mark and corresponding transcriptional and cell cycle pathways. Importantly, state, according to the chromatin-state maps retained expression of stem cell regulators generated for these cell types (Mikkelsen Oct4 and Nanog indicates that Ring1b- et al, 2007) (Figure S3). In contrast, of the deficient ES cells still have important ES bivalently marked genes only 11% and 51% cell specific characteristics. Based on re- retain both histone marks in NPCs and analysis of previously published global MEFs, respectively (Mikkelsen et al, 2007) binding studies performed in mouse ES (Figure S3), indicating that Ring1b does not cells, we found that Ring1b predominantly act to silence the H3K4me3 marked genes binds and represses genes with CpG-‘rich’ in ES cells or during development. Although promoters. Furthermore, these genes are

81 Chapter 4 associated with bivalent domains or the 1999), resulting in the delay of embryonic H3K4me3 histone mark alone. We suggest death of about 4 days (Voncken et al, 2003). that Ring1b contributes to stable maintenance Since relief of the Ink4a/Arf-mediated of pluripotency of ES cells by repressing antiproliferative block only bypasses the bivalently or actively marked genes. proliferative defects induced by loss of Ring1b, this underscores the importance for We found that Ring1b is required to repress Ring1b in regulating developmental genes the differentiation program in ES cells, during early embryogenesis. which is consistent with the observation The observation that only a subset of Ring1b that Ring1b-deficient ES cells are prone bound genes is derepressed following for differentiation (Leeb and Wutz, 2007). deletion of Ring1b in ES cells, suggests that Comparative analysis with genome-wide additional mechanisms mediate silencing of Ring1b binding data of mouse ES cells the other Ring1b target genes. This would (Boyer et al, 2006), indicated that Ring1b is be in correspondence with observations involved in direct transcriptional regulation by others, who show that only part of the of key developmental genes, such as Bmp7, PcG bound genes are deregulated after Gata3 and Msx. This is in agreement with RNAi-mediated depletion of PcGs in other genome-wide and candidate-based human embryonic fibroblasts and in ES PcG binding studies performed in mouse cells deficient for Eed or Suz12 (Boyer et and human ES cells (Azuara et al, 2006; al, 2006; Bracken et al, 2006; Pasini et al, Boyer et al, 2006; Bracken et al, 2006; Lee 2007). Partial derepression could be also et al, 2006; Leeb and Wutz, 2007; Squazzo be explained by functional redundancy by et al, 2006; Tolhuis et al, 2006). Notably, related Ring1a, which possess ubiquitin E3 by directly repressing these extracellular ligase activity towards histone H2A, alike and down-stream effector components of Ring1b, although with less efficiency (Cao et differentiation-related pathways, such as al, 2005; de Napoles et al, 2004; Wei et al, Bmp7 and Id3 of the Bmp/TGFbeta signaling 2006). In addition, the composition of PRCs pathway, and Col4a2, Gja1 and Ccnd2 of the can vary per tissue and developmental cellular communication pathways, Ring1b stage (de Napoles et al, 2004; Kuzmichev may have an important role in influencing et al, 2005; Pasini et al, 2007; Squazzo et lineage choice and other developmental al, 2006). Therefore, Ring1b might not be events. For example, expression of ECM essential in every promoter-bound PcG protein Col4a2 was shown to induce repressive complex. mesodermal differentiation of ES cells in vitro, We found that Ring1b represses genes that while repression of Bmp7 at the dorsal site of are co-occupied by Oct4 and Nanog. This is zebrafish gastrula was shown to allow cellular consistent with earlier reports, which show migration as is required for limb development. a role for PcG proteins in repressing a set (Nishikawa et al, 1998; von der Hardt et al, of developmental genes that are bound by 2007). In Ring1b knockout embryos, several Oct4 and Nanog in ES cells (Lee et al, 2006; developmental markers are misexpressed Loh et al, 2006). A possible link between at early gastrulation stages, extraembryonal these proteins is provided by Wang and mesoderm is over-represented, epiblast colleagues, who co-purified Oct4, Yy1 has expanded improperly, and embryonic and Ring1b with stem cell specific protein growth is delayed (Voncken et al, 2003). Rex-1 from ES cells (Wang et al, 2006). In This is in line with the aberrant expression Drosophila, PcG-specific sequences (named of developmental markers and induction Polycomb response elements or PREs) of apoptosis observed in Ring1b knockout mediate PcG repression (Mulholland et al, ES cells. The early embryonic lethality is 2003). However, in mammalians, PREs have partially rescued in a Cdkn2a-knockout not been identified. Therefore, one might background, one of the PcG transcriptional speculate that recruitment of PcG-silencing targets (Bracken et al, 2007; Jacobs et al, complexes to specific genes involves

82 Ring1b regulates ES cell fate interactions with site-specific DNA-binding proteins are involved in direct repression of factors. This could be the recruitment of the stem cell regulators in differentiated cells PcG silencing complex to key developmental (Azuara et al, 2006). genes through interaction with stem cell Comparative analysis of our expression specific factors Rex-1 and/or Oct4 in order profiling data with recently published to maintain an undifferentiated state of ES global chromatin-state maps generated of cells. This suggestion is supported by the mouse ES cells showed that Ring1b almost observation that Suz12 dissociates from exclusively binds to genes with CpG-rich PcG/Oct4 co-occupied promoters upon promoters. This is in consistent with a report downregulation of Oct4 (Pasini et al, 2007). showing that a strong correlation exists Interestingly, others have found that histone between promoters with highly conserved H2A E3 ligase 2A-HUB, but not RING1B, large CpG islands and Suz12 binding is selectively required for H2A mono- domains in undifferentiated human ES cells ubiquitination and subsequent repression of (Tanay et al, 2007). Furthermore, our data chemokine genes following recruitment by confirm that PcG proteins are involved in co-repressor N-CoR in human monocytes repressing bivalently marked developmental (Zhou et al, 2008). Together, this suggests genes in ES cells (Azuara et al, 2006; that different site-specific factors are involved Bernstein et al, 2006; Mikkelsen et al, 2007; in the recruitment of distinct H2A E3 ligases Stock et al, 2007). This comparative analysis to mediate repression of a specific set of also revealed that a considerable number genes. of actively transcribed H3K4me3 marked Ring1b-deficient ES cells retain expression Ring1b bound genes are regulated (mainly of important stem cell regulators Oct4, repressed) in ES cells. The absence of Nanog and alkaline phosphatase, which H3K27me3 at these promoters suggests was also observed in other PcG-deficient that Ring1b is recruited independently from ES cells (Leeb and Wutz, 2007; Pasini et PRC2. This is in agreement with other al, 2007; Stock et al, 2007). Derepression of observations indicating that Xist RNA, but developmental genes and downregulation not PRC2, is required for PRC1 recruitment of other stem cell markers, such as Rex-1, during X inactivation in differentiating ES cells Sox2 and Dppa3, suggests activation of (Schoeftner et al, 2006). The association the differentiation program. During ES of Ring1b to actively transcribed genes is cell differentiation, repression of Oct4 consistent with global and candidate-based and Nanog is essential, considering that binding studies that show overlap between ectopic expression of Oct4 can block PcG and RNAPII occupancy or association of differentiation, while expression of Nanog is PcG proteins with expressed genes in mouse enough to maintain ES cell self-renewal and and human ES cells, neural progenitor cells pluripotency, even in the absence of extrinsic and embryonic fibroblasts (Bracken et al, factor LIF or Oct4 expression (Chambers et 2006; Breiling et al, 2004; Lee et al, 2006; al, 2003; Hochedlinger et al, 2005; Niwa et Pasini et al, 2007; Squazzo et al, 2006). al, 2000). Retained expression of Oct4 and This suggests that Ring1b does not function Nanog could indicate that PcG proteins as a silencer of these genes in ES cells. are either directly or indirectly involved in Moreover, a large subset of actively marked regulation of Oct4 and Nanog. A direct role genes retain this mark and corresponding for PcG proteins in regulating the expression transcriptional state in more committed of Oct4 and Nanog has been suggested NPCs and MEFs (Mikkelsen et al, 2007). by Pasini and co-workers, who show that Thus, rather than poising for activation or PRC2 proteins bind to the Oct4 and Nanog prolonged inactivation, as was proposed for promoters in ES cells (Pasini et al, 2007). the bivalent genes, Ring1b might be required Moreover, the H3K27me3 histone mark to modulate the transcriptional activity of has been found at the Oct4 promoter in this subset of actively marked genes in ES more committed cells, suggesting that PcG cells and during later developmental stages.

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Only recently, association of Ring1b and DNA and a 5’end external probe in intron 1 to uH2A was shown to mediate a silenced discriminate between the Ring1b wild type (11 state of RNAPII present at promoters and kb), conventional knockout (5 kb) (Voncken et al, coding regions of bivalent genes (Stock et 2003), Lox (7 kb), and Del (4 kb) allele. The Del allele follows from deletion of exons 3 and 4 after al, 2007). Dissociation of Ring1b and uH2A 4-OHT-mediated activation of CreERT2 in Ring1b-/ from these promoters results in activation of Lox;CreERT2 ES cells. the RNAPII complex without major changes in the levels of PRC2 proteins or association Cell culture of H3K27me3. In addition, others showed ES cells were cultured on dishes coated with 0.1% that the presence of uH2A in the promoter- gelatin (Sigma) without feeders in 60% conditional proximal region blocks recruitment of FACT Buffalo Rat Liver (BRL) medium, which consisted resulting in pausing of RNAPII and inhibition of 60% BRL medium and 40% standard ES of transcriptional elongation (Zhou et al, medium. Standard ES cell medium consisted of 2008). One could speculate that Ring1b- GMEM (Gibco), 10% foetal calf serum (Sigma), supplemented with 1% non-essential amino mediated mono-ubiquitination of histone acids, 1 mM sodium pyruvate, L-glutamate, beta- H2A also interferes with the RNAPII complex mercaptoethanol and 103 units LIF (all Gibco). at active H3K4me3 marked genes, but how BRL medium was derived by filtering (0.2 µM) remains to be investigated. the supernatant of BRL cells cultured for one Altogether, our data suggest that Ring1b is week in standard ES cell medium without beta- important to maintain an undifferentiated mercaptoethanol or LIF. state of ES cells through repression of key Ring1b conditional ES cells or Ring1b+/+;CreERT2 developmental genes. The finding that this control ES cells were treated with 200 nM 4-OHT involves direct transcriptional repression of at and MEFs with 1 µM 4-OHT for the indicated time. least two epigenetically distinct sets of genes MEFs were isolated from 14.5 day old embryo’s and cultured as described before (Jacobs et al, helps to understand and further explore the 1999). For RA treatment ES cells were cultured role of Ring1b during development. in standard ES medium without LIF and in the presence of 500 nM all-trans-RA (ATRA) (Sigma). Materials and Methods Protein isolation and Western blot analysis Generating Ring1b conditional knockout ES Proteins were extracted from whole cells (RIPA cells lysates, protocol as described previously) The Ring1b wild type allele in Ring1b+/- ES cells (Hernandez-Munoz et al, 2005a) or from the (Voncken et al, 2003) was targeted with a construct nucleus (nuclear extraction). For nuclear extracts containing loxP sequences at the EcoRI site in cells were washed with PBS, incubated on ice intron 2 and at the XhoI site in intron 4, followed for 5 min in Triton lysis buffer (0.1 M NaCl, 0.3 M by a third LoxP site enclosing a hygromycin sucrose, 3 mM MgCl2, 50 mM HEPES pH 6.8, 1 cassette to allow for selection of targeted ES mM EGTA, 0.2% Triton-X100) supplemented with cells. Correct targeting of hygromycin resistant Complete™ protease inhibitor cocktail (Roche) Ring1b-/LoxHyg ES cell clones was confirmed by and 10 mM N-ethylmaleimide (NEM) followed by Southern blot analysis. Next, to obtain Ring1b-/Lox spinning for 5 min at 3000 rpm at 4°C to pellet ES clones the hygromycin cassette was removed the nuclei. Nucleic proteins were recovered by by transiently expressing adenoviral Cre and incubating the nuclei in SDS-lysis buffer (0.1% subsequently screening for hygromycin sensitivity. SDS, 50 mM TrisHCl pH 7.5, 0.15 M NaCl) for 10 Correct removal of the hygromycin cassette in the min on ice, followed by sonification (10 pulses at selected Ring1b-/Lox ES clones was confirmed 50%, level 4) and spinning for 1 min at 14.000 rpm by Southern blot analysis. Finally, to generate to remove insoluble material. Protein samples inducible conditional Ring1b-/Lox;CreERT2 ES cells were assayed with SDS-PAGE using pre-cast a puromycin-selectable R26CreERT2 construct gradient gels (Invitrogen) and conventional was targeted to the ROSA26 locus. CreERT2 Western blotting techniques. could be activated by adding 200 nM 4-OHT (Sigma, dissolved in absolute ethanol) to the ES Immunostaining, alkaline phosphatase, and cell medium. Annexin V staining Southern blot analysis of HindIII digested genomic ES cells were cultured for four days with 200

84 Ring1b regulates ES cell fate

nM 4-OHT or three days with 500 nM RA on were analyzed with Image-Pro 5.1 software (Media coverslips coated with 0.1% gelatin. Cells were Cybernetics). In brief, mean fluorescence intensity fixed in 4% paraformaldehyde for 10 min at RT levels of the immunofluorescence-labeled protein and permeabilization in 0.25% Triton-X100/ per cell were measured for the nucleic area as was PBS for 5 min. Cells were then incubated for 30 determined by the DAPI staining for on average minutes at RT in blocking solution (5% foetal calf 250 cells per quantification and three independent serum (Gibco), 5% normal goat serum (Vector stainings. The distribution of the mean intensity laboratories), 0.02% Triton-X100/PBS). Next, cells per cell is displayed in histogram figures with were incubated with the 1st antibody for 1 hour at same x-axis scale. RT in blocking solution and afterwards washed five times 5 minutes with 0.02% Triton-X100/PBS. RNA isolation, real-time QPCR and microarray Hereafter, cells were incubated for 1 hour at RT analysis with fluorescence labeled 2nd antibody in blocking Ring1b-/Lox;CreERT2 and Ring1b+/+;CreERT2 solution, followed by five washes of 5 min each control ES cells (used as a reference for QPCR with 0.02% Triton-X100/PBS. Cover slips were and microarray) were cultured under the same mounted on object slides using Vectashield conditions and RNA was isolated at the indicated with DAPI (Vector Laboratories). For the uH2A time points. Total RNA was isolated using TRIzol immunostaining cells were permeabilized prior to reagent (Invitrogen) according to the manufactures fixation by in cytoskeletal buffer (100 mM NaCl, protocol followed by DNase treatment and aRNA 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES pH amplification (Invitrogen). Real-time QPCR 6.8) for 30 sec, followed by 30 sec in cytoskeletal conditions and primer sequences are as described buffer containing 0.5% Triton-X100, and 30 sec in elsewhere (Boyer et al, 2006). Changes in RNA cytoskeletal buffer without Triton-X100, all on ice. levels following 4-OHT treatment of Ring1b-/ For the alkaline phosphatase staining cells were Lox;CreERT2 relative to Ring1b+/+;CreERT2 ES cells cultured on dishes, followed by fixation and were determined based on the results of three stained according to the manufactures protocol QPCRs. (Sigma). The oligonucleotide arays were printed at the CMF To determine the level of apoptosis, cells were core facility of the NKI/AvL with mouse Operon collected by trypsinization, or first washed twice microarray version 3 (http://microarrays.nki.nl/ with PBS to obtain only the adherent cells, followed download/geneid.html). Detailed information by staining with FITC-conjugated Annexin V-FITC about the labeling and hybridization protocols (Roche) according to the manufactures protocol and array analysis can be found on the NKI/AvL and analyzed by flow cytometry. microarray website (http://microarrays.nki.nl/ download/protocols.html). In brief, amplified RNA Antibodies was labeled with ULS Cy5 or Cy3 (Kreatech EA- H3K27me3 rabbit polyclonal was a kind gift from T. 006, Amsterdam) was pooled and co-hybridized to Jenuwein. Ezh2 mouse monoclonal antibody was the microarray (each experiment was performed a kind gift from K. Helin. Ring1b mouse monoclonal in dye swap fashion and controlled with self-self and rabbit polyclonal antibodies were obtained hybridization experiments to correct for dye-bias). from H. Koseki. Oct4 mouse monoclonal antibody After 16 hours of hybridization the microarrays C-10 and Lamin-B goat polyclonal antibody M20 were washed and scanned using an Agilent DNA from Santa Cruz, Nestin mouse monoclonal Microarray scanner (Agilent Technologies, Cat# antibody from BD, Eed rabbit polyclonal and G2505B). ImaGene v6.0 software (BioDiscovery Bmi1 and uH2A mouse monoclonal antibodies Inc) was used to quantify the RNA expression from Upstate. Secondary immunofluorescence levels using the TIFF images produced by the antibodies were Alexa 488- or 568-conjucated scanner. The background-corrected intensities anti-mouse, 568- or 488-conjucated anti-rabbit from the Cy5- and Cy3 channel were used to from Molecular Probes. calculate log2 transformed ratios. These ratios were normalized using a lowest fit per subarray Image analysis and quantification (Yang et al, 2002). A weighted average ratio and To quantify the fluorescence levels of confidence level (P-value) was calculated per immunofluorescence-labeled proteins in individual gene by a NKI platform adjusted error model cells, we took sequential images of DAPI and (Hughes et al, 2000), which was fine-tuned by the fluorescence labeled protein using a Leica self-self hybridizations. Significantly differentially confocal research microscope with a 63x lens and expressed genes (outliers) between sample and fixed laser settings per protein. Files were saved in reference were selected based on their P-value TIFF format with a 512x512 resolution. The images (a gene with a P-value <0.05 was considered an

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outlier). Ring1B generates self atypical mixed ubiquitin The microarray details and results are submitted to chains required for its in vitro histone H2A the ArrayExpress database (http://www.ebi.ac.uk/ ligase activity. Mol. Cell 24: 701-711. arrayexpress/), reference number E-NCMF-14. Bernstein B.E., Mikkelsen T.S., Xie X., Kamal Clustering of the microarray data was performed M., Huebert D.J., Cuff J., Fry B., Meissner A., using the bioinformatics software program Wernig M., Plath K., Jaenisch R., Wagschal A., Genesis (Sturn et al, 2002). GO classification Feil R., Schreiber S.L., and Lander E.S. (2006) analysis was performed using bioinformatics A bivalent chromatin structure marks key program BiNGO (Maere et al, 2005). Pathway developmental genes in embryonic stem cells. analysis was performed using the bioinformatics Cell 125: 315-326. software program DAVID based on the KEGG Boyer L.A., Lee T.I., Cole M.F., Johnstone S.E., pathway database available online (http://david. Levine S.S., Zucker J.P., Guenther M.G., abcc.ncifcrf.gov). Kumar R.M., Murray H.L., Jenner R.G., Gifford D.K., Melton D.A., Jaenisch R., and Young R.A. (2005) Core transcriptional regulatory circuitry Acknowledgements in human embryonic stem cells. Cell 122: 947- We are indebted to all members of the van 956. Boyer L.A., Plath K., Zeitlinger J., Brambrink T., Lohuizen lab for technical assistance and Medeiros L.A., Lee T.I., Levine S.S., Wernig M., suggestions, and thank Sophia Bruggeman, Tajonar A., Ray M.K., Bell G.W., Otte A.P., Vidal Anke Sparmann, Alexandra Pietersen for M., Gifford D.K., Young R.A., and Jaenisch critical comments and Anders Lund and R. (2006) Polycomb complexes repress Inmaculada Hernández-Munõs for helpful developmental regulators in murine embryonic discussions. We greatly appreciate Kathrin stem cells. Nature 441: 349-353. Plath for technical assistance and critical Bracken A.P., Dietrich N., Pasini D., Hansen K.H., comments. We thank Ate Loonsta and and Helin K. (2006) Genome-wide mapping of Anton Berns for the R26CreERT2 targeting Polycomb target genes unravels their roles in construct and sharing unpublished results. cell fate transitions. Genes Dev. 20: 1123-1136. Bracken A.P., Kleine-Kohlbrecher D., Dietrich We thank Coenraad Kuijl, Lauran Oomen and N., Pasini D., Gargiulo G., Beekman C., Lenny Brocks for assistance with confocal Theilgaard-Monch K., Minucci S., Porse B.T., microscopy and immunofluorescence Marine J.C., Hansen K.H., and Helin K. (2007) analysis, and members of the NKI microarray The Polycomb group proteins bind throughout core facility for assistance with the microarray the INK4A-ARF locus and are disassociated in analysis. senescent cells. Genes Dev. 21: 525-530. Breiling A., O’Neill L.P., D’Eliseo D., Turner B.M., and Orlando V. (2004) Epigenome changes in References active and inactive polycomb-group-controlled regions. EMBO Rep. 5: 976-982. Akasaka T., Kanno M., Balling R., Mieza M.A., Buchwald G., van der Stoop P., Weichenrieder O., Taniguchi M., and Koseki H. (1996) A role for Perrakis A., van Lohuizen M., and Sixma T.K. mel-18, a Polycomb group-related vertebrate (2006) Structure and E3-ligase activity of the gene, during theanteroposterior specification Ring-Ring complex of polycomb proteins Bmi1 and Ring1b. EMBO J. : 2465-2474. of the axial skeleton. Development 122: 1513- 25 1522. Burdon T., Smith A., and Savatier P. (2002) Azuara V., Perry P., Sauer S., Spivakov M., Signalling, cell cycle and pluripotency in Jorgensen H.F., John R.M., Gouti M., Casanova embryonic stem cells. Trends Cell Biol. 12: M., Warnes G., Merkenschlager M., and Fisher 432-438. A.G. (2006) Chromatin signatures of pluripotent Cao R., Tsukada Y., and Zhang Y. (2005) Role of Bmi-1 and Ring1A in H2A ubiquitylation and cell lines. Nat. Cell Biol. 8: 532-538. Beddington R.S. and Robertson E.J. (1989) An Hox gene silencing. Mol. Cell 20: 845-854. assessment of the developmental potential Cao R., Wang L., Wang H., Xia L., Erdjument- of embryonic stem cells in the midgestation Bromage H., Tempst P., Jones R.S., and Zhang Y. (2002) Role of histone H3 lysine 27 mouse embryo. Development 105: 733-737. Ben Saadon R., Zaaroor D., Ziv T., and methylation in Polycomb-group silencing. Ciechanover A. (2006) The polycomb protein Science 298: 1039-1043. Cattaneo E. and McKay R. (1990) Proliferation and

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Supplementary figures

untreated 6 days 4-OHT Figure S1. Loss of uH2A after deletion of Ring1b in MEFs. Example of immunofluorescence stainings -/Lox T2 Ring1b of Ring1b ;CreER MEFs 6 days following 4-OHT treatment showing loss of uH2A in MEFs that have lost Ring1b.

uH2A

DAPI

Merge

NPC MEF Figure S3. Ring1b bound H3K4me3 100% marked genes with CpG-rich promoters retain this mark in NPC and MEFs. Bar graph showing the 80% distribution of the chromatin-state K4 of the Ring1b bound genes with 60% K4/K27 high-CpG content promoters (HCP) K27 in neural progenitor cells (NPC) or 40% none MEF that were marked with either H3K4me3 or H3K4me3+H3K27me3 20% in ES cells. Graph represents the chromatin state of all Ring1b 0% bound genes that are represented all dereg all dereg all dereg all dereg on the microarray (‘all’), and only

K4/K27 K4 K4/K27 K4 the Ring1b bound genes that are deregulated in Ring1b deficient ES chromatin state in ES cells cells (‘dereg’). These data show that most H3K4me3 marked genes, retain this mark in NPC and MEFs.

90 Ring1b regulates ES cell fate

Gene Ontology

biological process

cellular physiological process process

cellular physiological metabolism process

cellular primary macromolecule biosynthesis metabolism metabolism metabolism

carbohydrate cellular metabolism biosynthesis

alcohol cellular macromolecule metabolism carbohydrate biosynthesis metabolism

generation of organic monosaccharidealcohol carbohydrate precursor metabolitesacid metabolism biosynthesis biosynthesis and energy metabolism

energy derivationcarboxylic acid hexose monosaccharide by oxidation of metabolism metabolism biosynthesis organic compounds

pyruvate main pathways ofglucose hexose metabolism carbohydrate metabolism biosynthesis metabolism

gluconeogenesis

Figure S2a. Graphical representation of GO categories that are significantly enriched one day after 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells (see also Table S2a). Graphical map shows the hierarchical relations between the significantly enriched GO categories (yellow circles) identified by the BiNGO bioinformatics tool.

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Gene Ontology Gene Ontology

biological process biological process

cellular physiological process process development

organ cellular development physiological process

cell cycle

Figure S2b. Graphical representation of GO Figure S2c. Graphical representation of GO categories that are significantly enriched two days categories that are significantly enriched three after 4-OHT treatment of Ring1b-/Lox;CreERT2 ES days after 4-OHT treatment of Ring1b-/Lox;CreERT2 cells (see also Table S2b). Graphical map shows ES cells (see also Table S2c). Graphical map shows the hierarchical relations between the significantly the hierarchical relations between the significantly enriched GO categories (yellow circles) identified enriched GO categories (yellow circles) identified by the BiNGO bioinformatics tool. by the BiNGO bioinformatics tool.

92 Ring1b regulates ES cell fate

Gene Ontology

biological process

cellular physiological process process

cellular physiological metabolism process

cell cellular macromolecule primary organization metabolism metabolism metabolism and biogenesis

organelle organization nucleobase, and biogenesis biopolymer nucleoside, metabolism nucleotide and nucleic acid metabolism

chromosome DNA metabolism organization and biogenesis chromosome organization and biogenesis (sensu Eukaryota)

DNA packaging protein metabolism cellular establishment and/or maintenance of chromatin architecture macromolecule metabolism

chromatin assembly cellular development regulation of or disassembly protein biological process metabolism

chromatin assembly organ morphogenesis regulation of regulation of development cellular process physiological process protein complex assembly

organ regulation of nucleosome morphogenesis cellular physiological assembly process

Figure S2d. Graphical representation of GO categories that are significantly enriched four days after 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells (see also Table S2d). Graphical map shows the hierarchical relations between the significantly enriched GO categories (yellow circles) identified by the BiNGO bioinformatics tool.

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File created with BiNGO (c) on 6-aug-2007 at 15:35:09

ontology: process curator: GO

Selected ontology file : D:\Cytoscape 22\cytoscape-v2.2\plugins\BiNGO\GO_Biological_Process Selected annotation file : D:\Cytoscape 22\cytoscape-v2.2\plugins\BiNGO\M_musculus_default Overrepresentation Selected statistical test : Hypergeometric test Selected correction : Bonferroni Family-Wise Error Rate (FWER) correction Selected significance level : 0.1

Testing option : Test cluster versus network

Number of genes selected : 285 Total number of genes in annotation : 10855

GO-ID p-value corr p-value# selected # total Description 46165 1,99E-06 1,66E-03 6 16 alcohol biosynthesis 46364 1,99E-06 1,66E-03 6 16 monosaccharide biosynthesis 19319 1,99E-06 1,66E-03 6 16 hexose biosynthesis 6094 1,99E-05 1,65E-02 5 14 gluconeogenesis 6090 5,75E-05 4,79E-02 5 17 pyruvate metabolism 19752 9,71E-05 8,09E-02 22 342 carboxylic acid metabolism 6082 9,71E-05 8,09E-02 22 342 organic acid metabolism

Table S2a. BiNGO ontology analysis of genes deregulated after one day of 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells, using EntrezIDs.

File created with BiNGO (c) on 6-aug-2007 at 15:31:04

ontology: process curator: GO

Selected ontology file : D:\Cytoscape 22\cytoscape-v2.2\plugins\BiNGO\GO_Biological_Process Selected annotation file : D:\Cytoscape 22\cytoscape-v2.2\plugins\BiNGO\M_musculus_default Overrepresentation Selected statistical test : Hypergeometric test Selected correction : Bonferroni Family-Wise Error Rate (FWER) correction Selected significance level : 0.1

Testing option : Test cluster versus network

Number of genes selected : 386 Total number of genes in annotation : 10855

GO-ID p-value corr p-value# selected # total Description 8151 1,23E-05 1,14E-02 296 7262 cellular physiological process 7049 2,65E-05 2,45E-02 36 489 cell cycle

Table S2b. BiNGO ontology analysis of genes deregulated after two days of 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells, using EntrezIDs.

94 Ring1b regulates ES cell fate

File created with BiNGO (c) on 6-aug-2007 at 15:10:50

ontology: process curator: GO

Selected ontology file : D:\Cytoscape 22\cytoscape-v2.2\plugins\BiNGO\GO_Biological_Process Selected annotation file : D:\Cytoscape 22\cytoscape-v2.2\plugins\BiNGO\M_musculus_default Overrepresentation Selected statistical test : Hypergeometric test Selected correction : Bonferroni Family-Wise Error Rate (FWER) correction Selected significance level : 0.1

Testing option : Test cluster versus network

Number of genes selected : 466 Total number of genes in annotation : 10855

GO-ID p-value corr p-value# selected # total Description 48513 4,50E-05 4,46E-02 54 725 organ development

Table S2c. BiNGO ontology analysis of genes deregulated after three days of 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells, using EntrezIDs.

File created with BiNGO (c) on 6-aug-2007 at 13:48:33

ontology: process curator: GO

Selected ontology file : D:\Cytoscape 22\cytoscape-v2.2\plugins\BiNGO\GO_Biological_Process Selected annotation file : D:\Cytoscape 22\cytoscape-v2.2\plugins\BiNGO\M_musculus_default Overrepresentation Selected statistical test : Hypergeometric test Selected correction : Bonferroni Family-Wise Error Rate (FWER) correction Selected significance level : 0.1

Testing option : Test cluster versus network

Number of genes selected : 639 Total number of genes in annotation : 10855

GO-ID p-value corr p-value# selected# total Description 8152 1,77E-07 2,10E-04 374 5286 metabolism 44237 3,15E-07 3,73E-04 354 4969 cellular metabolism 6139 9,90E-07 1,17E-03 180 2221 nucleobase, nucleoside, nucleotide and nucleic acid metabolw. 6334 1,14E-06 1,35E-03 14 52 nucleosome assembly 8151 1,32E-06 1,56E-03 481 7262 cellular physiological process 31497 1,68E-06 1,98E-03 15 61 chromatin assembly 50791 2,03E-06 2,41E-03 175 2169 regulation of physiological process 44238 3,29E-06 3,89E-03 335 4747 primary metabolism 51244 4,13E-06 4,89E-03 169 2102 regulation of cellular physiological process 6333 5,22E-06 6,18E-03 17 83 chromatin assembly or disassembly 9887 5,55E-06 6,57E-03 44 369 organ morphogenesis 48513 6,39E-06 7,56E-03 72 725 organ development 50794 6,57E-06 7,77E-03 175 2209 regulation of cellular process 50789 1,20E-05 1,42E-02 185 2384 regulation of biological process 9653 1,25E-05 1,48E-02 68 686 morphogenesis 6259 1,60E-05 1,89E-02 46 409 DNA metabolism 7275 2,20E-05 2,60E-02 129 1560 development

Table S2d. BiNGO ontology analysis of genes deregulated after four days of 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells, using EntrezIDs.

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Term (based on KEGG pathway database) # genes % PValue Day 1 MMU04110:Cell cycle 8 1,74% 0,03953 MMU00051:Fructose and mannose metabolism 6 1,30% 0,020727 MMU00710:Carbon fixation 4 0,87% 0,0305 MMU00030:Pentose phosphate pathway 4 0,87% 0,037932 MMU00010:Glycolysis/gluconeogenesis 6 1,30% 0,0257

Day 2 MMU00330:Arginine and proline metabolism 6 0,93% 0,059059 MMU00480:Glutathione metabolism 5 0,78% 0,093433 MMU00271:Methionine metabolism 4 0,62% 0,018164 MMU04520:Adherens junction 8 1,24% 0,02595 MMU00010:Glycolysis/gluconeogenesis 6 0,93% 0,062923 MMU00590:Arachindonic acid metabolism 7 1,09% 0,071111 MMU00970:Aminoacyl-tRNA synthetases 5 0,78% 0,021118 MMU00220:Urea cycle and metabolism of amino groups 4 0,62% 8,36E-02 MMU04 730:Long-term depression 7 1,09% 0,074783

Day 3 MMU04540:Gap junction 9 1,15% 0,058752 MMU00910:Nitrogen metabolism 5 0,64% 0,017028 MMU04510:Focal adhesion 17 2,17% 0,032601 MMU04350:TGF-beta signaling pathway 9 1,15% 0,04452

Day 4 MMU04110:Cell cycle 13 1,26% 0,048647 MMU00340:Histidine metabolism 7 0,68% 0,07664 MMU03030:DNA polymerase 6 0,58% 0,030831 MMU00280:Valine, leucine and isoleucine degradation 9 0,87% 0,020638 MMU00030:Pentose phosphase pathway 6 0,58% 0,022619 MMU00271:Methionine metabolism 4 0,39% 0,07396 MMU00260:Glycine, serine, and threonine metabolism 8 0,77% 0,046524 MMU00010:Glycolysis/gluconeogenesis 8 0,77% 0,077236 MMU00564:Glycerophospholipid metabolism 11 1,06% 0,028432 MMU04350:TGF-beta signaling pathway 12 1,16% 0,026717 MMU00970:Aminoacyl-tRNA syntetases 6 0,58% 0,035555 MMU04320:Dorso-ventral axis formation 6 0,58% 0,052289

Table S3. Pathway analysis of genes transcriptionally deregulated following Ring1b deletion. Excel file of DAVID pathway analysis (selecting the KEGG pathway database option) of all outliers, based on EntrezIDs, per day of 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells.

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