Title: OCT4/SOX2-independent<i>Nanog</i>autorepression modulates heterogeneous<i>Nanog</i>gene expression in mouse ES cells
Abstract: Article23 November 2012free access OCT4/SOX2-independent Nanog autorepression modulates heterogeneous Nanog gene expression in mouse ES cells Pablo Navarro Corresponding Author Pablo Navarro MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Nicola Festuccia Nicola Festuccia MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Douglas Colby Douglas Colby MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Alessia Gagliardi Alessia Gagliardi MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Nicholas P Mullin Nicholas P Mullin MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Wensheng Zhang Wensheng Zhang MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, ScotlandPresent address: Wellcome Trust Sanger Institute, Hinxton CB10 1HH, England Search for more papers by this author Violetta Karwacki-Neisius Violetta Karwacki-Neisius MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Rodrigo Osorno Rodrigo Osorno MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author David Kelly David Kelly Centre Optical Instrumentation Laboratory, Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Morag Robertson Morag Robertson MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, ScotlandPresent address: MRC Human Genetics Unit, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, Scotland Search for more papers by this author Ian Chambers Corresponding Author Ian Chambers MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Pablo Navarro Corresponding Author Pablo Navarro MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Nicola Festuccia Nicola Festuccia MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Douglas Colby Douglas Colby MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Alessia Gagliardi Alessia Gagliardi MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Nicholas P Mullin Nicholas P Mullin MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Wensheng Zhang Wensheng Zhang MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, ScotlandPresent address: Wellcome Trust Sanger Institute, Hinxton CB10 1HH, England Search for more papers by this author Violetta Karwacki-Neisius Violetta Karwacki-Neisius MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Rodrigo Osorno Rodrigo Osorno MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author David Kelly David Kelly Centre Optical Instrumentation Laboratory, Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Morag Robertson Morag Robertson MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, ScotlandPresent address: MRC Human Genetics Unit, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, Scotland Search for more papers by this author Ian Chambers Corresponding Author Ian Chambers MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland Search for more papers by this author Author Information Pablo Navarro 1, Nicola Festuccia1, Douglas Colby1, Alessia Gagliardi1, Nicholas P Mullin1, Wensheng Zhang1, Violetta Karwacki-Neisius1, Rodrigo Osorno1, David Kelly2, Morag Robertson1 and Ian Chambers 1 1MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland 2Centre Optical Instrumentation Laboratory, Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, Scotland *Corresponding authors. MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, School of Biological Sciences, 5 Little France Drive, Edinburgh EH16 4UU, Scotland. Tel:+44 131 651 9500; Fax:+44 131 651 9501; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2012)31:4547-4562https://doi.org/10.1038/emboj.2012.321 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info NANOG, OCT4 and SOX2 form the core network of transcription factors supporting embryonic stem (ES) cell self-renewal. While OCT4 and SOX2 expression is relatively uniform, ES cells fluctuate between states of high NANOG expression possessing high self-renewal efficiency, and low NANOG expression exhibiting increased differentiation propensity. NANOG, OCT4 and SOX2 are currently considered to activate transcription of each of the three genes, an architecture that cannot readily account for NANOG heterogeneity. Here, we examine the architecture of the Nanog-centred network using inducible NANOG gain- and loss-of-function approaches. Rather than activating itself, Nanog activity is autorepressive and OCT4/SOX2-independent. Moreover, the influence of Nanog on Oct4 and Sox2 expression is minimal. Using Nanog:GFP reporters, we show that Nanog autorepression is a major regulator of Nanog transcription switching. We conclude that the architecture of the pluripotency gene regulatory network encodes the capacity to generate reversible states of Nanog transcription via a Nanog-centred autorepressive loop. Therefore, cellular variability in self-renewal efficiency is an emergent property of the pluripotency gene regulatory network. Introduction For stem cell populations to remain effective, they must balance manifestation of their two defining properties: self-renewal and differentiation (Silva and Smith, 2008). This is achieved by non-genetic heterogeneity, a prominent topic at the forefront of stem cell research (Huang, 2009). Indeed, heterogeneous gene expression is a recurrent property of stem cells that underpins their developmental potency and plasticity (Graf and Stadtfeld, 2008; Martinez-Arias and Brickman, 2009). This has made stem cells a useful model system to study how heterogeneity in gene expression is generated and used by individual cells to undertake decision-making processes (Balazsi et al, 2011). A paradigmatic example is provided by embryonic stem (ES) cell populations, where a subset of the cells do not express NANOG, the master regulator of the efficiency of self-renewal (Chambers et al, 2003, 2007; Mitsui et al, 2003; Singh et al, 2007; Kalmar et al, 2009). Consequently, NANOG-negative ES cells possess an increased differentiation propensity compared with the highly self-renewing NANOG-positive subpopulation, in which high NANOG levels shield cells from commitment signals (Chambers et al, 2003, 2007). Moreover, NANOG expression is mosaic in the inner cell mass of the blastocyst from which ES cells are derived (Chazaud et al, 2006; Dietrich and Hiiragi, 2007; Plusa et al, 2008; Nichols and Smith, 2011). While NANOG-positive cells are the founders of the epiblast from which the embryo proper originates, NANOG-negative cells give rise to the primitive endoderm, which contributes to extra-embryonic tissues. Therefore, heterogeneous NANOG expression enables important fate decisions and its relevance is illustrated by the observation that elimination of NANOG heterogeneity is associated with a failure to undergo normal embryogenesis and with a considerable resistance of ES cells to differentiate (Chambers et al, 2003, 2007; Nichols et al, 2009). The mechanisms associated with heterogeneous NANOG expression are largely unknown and ill-defined. However, it is known that NANOG heterogeneity is governed by transcriptional switching of Nanog (Chambers et al, 2007; Kalmar et al, 2009). Thus, instead of acting as a static regulatory platform continuously preserving the undifferentiated state, the gene regulatory network supporting self-renewal is dynamic, intermittently silencing Nanog to provide temporal opportunities for differentiation. When this study was initiated, the view of the network proposed that Nanog and other regulators form a stable, self-sustaining circuitry consisting of positive autoregulatory and feed-forward loops (Jaenisch and Young, 2008). In particular, NANOG was believed to activate transcription of Oct4 and Sox2, two additional pluripotency factors, which in turn activate themselves, each other and Nanog (Figure 1A). Although this architecture appears intuitively advantageous for the efficient maintenance and exit from pluripotency, it predicts the emergence of coherent expression patterns of OCT4, SOX2 and NANOG. However, fluctuating Nanog transcription occur within cells expressing relatively uniform levels of OCT4/SOX2 (Chambers et al, 2007). Figure 1.Negative correlation between NANOG protein levels and transcription activity of the Nanog locus. (A) Architecture of the core pluripotency network inferred from genome-wide analyses (Jaenisch and Young, 2008). (B) Schematic diagram of WT E14Tg2a, NANOG overexpressing EF4 and Nanog−/− RCNβH-B(t) cells. Note the presence of Nanog intron 1 sequences at the endogenous Nanog locus of all lines: this is the region were the RT–(Q)PCR primers (black dots within the E14Tg2a diagram) and the RNA-FISH probe (black line within the E14Tg2a diagram) were designed and used to detect the activity of the Nanog locus. A full description of these cell lines can be found in previous publications (Chambers et al, 2003, 2007). Tg, transgene. (C) Analysis of the level of NANOG protein, Nanog mRNA and Nanog-derived pre-mRNA (E14Tg2a RNA levels set to 1) in EF4, E14Tg2a and RCNβH-B(t) ES cells (n=2; error bars represent s.e.m.). (D) Co-transfection of either a luciferase reporter driven by a 6-kb-long Nanog promoter (left), or by a Rex1 promoter (right panel) in supertransfectable E14/T ES cells (Chambers et al, 2003) with either an empty vector (EV, set to 1), a NANOG-expressing vector (NANOG) or a vector expressing a mutant form of NANOG unable to bind DNA (NANOG:N51-A). n=2; error bars represent s.e.m. Download figure Download PowerPoint As autoregulation is widely associated with the dynamic behaviour of regulatory networks (Balazsi et al, 2011), we aimed to examine the details of Nanog autoregulation. To do so, we used a genetic approach consisting of inducible systems of gain- and loss-of-function combined with Nanog:GFP reporters. In agreement with a recent report (Fidalgo et al, 2012), we establish that the current architecture of the core pluripotency network must be overturned: Nanog activity is autorepressive. Moreover, we report that the NANOG-mediated control of Oct4/Sox2 expression is minimal. We further show that the autorepressive mechanism does not involve OCT4/SOX2 and, importantly, that Nanog autorepression controls switching of Nanog transcription to modulate Nanog gene expression heterogeneity. Results NANOG negatively influences Nanog transcription In several regulatory networks associated with fluctuating gene expression, one or more of the components are negatively autoregulated, either directly or indirectly (Balazsi et al, 2011). However, in the case of the pluripotency gene regulatory network, NANOG is considered to act as a transcriptional activator of Nanog gene expression (Figure 1A; Jaenisch and Young, 2008). To experimentally test the validity of this idea, we used quantitative RT–PCR (RT–(Q)PCR) to determine the level of pre-messenger RNA produced by the Nanog locus in cell lines expressing differing levels of NANOG (Figure 1B and C). We used five primer pairs located within a region of Nanog intron 1 that remains intact in Nanog-null ES cells to assess the transcriptional activity of the Nanog locus in wild-type (WT) ES cells (E14Tg2a), Nanog-null ES cells (RCNβH-B(t)) and in cells overexpressing NANOG from a randomly integrated cDNA transgene (EF4). In contrast to the accepted model, we found a negative correlation between the level of Nanog mRNA and protein (derived from the endogenous alleles in E14Tg2a and from both the endogenous alleles and the transgene in EF4) and the level of transcription of the endogenous Nanog locus (Figure 1C). This may suggest that NANOG negatively affects transcription of the Nanog gene. In agreement, we found that a luciferase gene driven by a 6-kb-long Nanog promoter region is repressed by co-transfecting a vector expressing WT NANOG but not a variant in which the DNA-binding homeodomain carries a point mutation known to abolish binding of homeodomain proteins to DNA (Pomerantz and Sharp, 1994; NANOG:N51-A, Figure 1D). Conversely, a Rex1 promoter-driven luciferase gene was shown to be trans-activated by NANOG (Figure 1D), confirming that NANOG can both activate or repress transcription from distinct pluripotency-associated promoters. The inducible loss of NANOG leads to increased Nanog transcription To address whether the upregulation of Nanog transcription is a primary response to the loss of NANOG, we first analysed the dynamics of pre-messenger transcription from the endogenous Nanog locus in inducible Nanog-null cells by using RCNβH cells, the parental line from which RCNβH-B(t) cells were derived (Chambers et al, 2007). RCNβH cells are Nanog-null cells that express Nanog mRNA from a constitutive transgene from which the Nanog ORF can be deleted by Tamoxifen treatment. Upon deletion of the Nanog transgene, GFP is brought under the control of the constitutive CAG promoter (Figure 2A). Figure 2.Endogenous Nanog transcription is rapidly upregulated upon loss of exogenous NANOG expression. (A) Schematic diagram of Tamoxifen-inducible Nanog-null ES cells. In addition to the different features shown, RCNβH ES cells carry a Cre-ERT2 transgene knocked-in to Rosa26. (B) FACS profiles monitoring the deletion of the Nanog cDNA transgene after 12, 24 and 48 h of Tamoxifen treatment. (C) Analysis of NANOG protein (top), Nanog mRNA (middle), and Nanog pre-mRNA expression (bottom) in RCNβH cells treated with Tamoxifen for 0 (set to 1 for the RT–(Q)PCR), 12, 24 and 48 h (n=2; error bars represent s.e.m.). (D) Western blot analysis of OCT4 and SOX2 at the same time points of Tamoxifen treatment. (E) Relative expression of Oct4, Sox2, Esrrb and Klf4 transcripts after 48 h of Tamoxifen treatment (untreated cells set to 1; n=7; error bars represent s.e.m.). Download figure Download PowerPoint After 12 h of Tamoxifen treatment, around 75% of the cells have undergone the deletion of the Nanog transgene as evaluated by FACS analysis (Figure 2B). However, exogenous Nanog mRNA and protein is only reduced by half and this is accompanied by a modest upregulation of endogenous Nanog locus transcription (Figure 2C). After 48 h of treatment, when 98% of the cells are GFP-positive (Figure 2B) and exogenous NANOG protein and mRNA become essentially undetectable (Figure 2C), the production of pre-mRNA from the endogenous Nanog locus has increased three-fold (Figure 2C). Importantly, OCT4 and SOX2 protein (Figure 2D) and mRNA (Figure 2E) remained expressed following loss of exogenous NANOG expression, suggesting efficient maintenance of the undifferentiated state. However, other pluripotency genes such as Klf4 and Esrrb were downregulated after 48 h of Tamoxifen treatment (Figure 2E). The inducible restoration of NANOG leads to reduced Nanog transcription In a complementary approach, we introduced a transgene encoding a NANOG-ERT2 fusion protein to an independent Nanog-null ES cell line (TβC44Cre6; Chambers et al, 2007) in order to restore nuclear NANOG expression upon Tamoxifen treatment (44NERT; Figure 3A). Three independent clones were generated, two expressing Nanog transcripts at similar levels to WT ES cells (44NERTc1&2) and one in which Nanog transcripts are increased (44NERTc3; Figure 3B). However, immunoblot analyses indicated that in the three clones, and in particular in 44NERTc3, NANOG-ERT2 is overexpressed as compared with the level of WT NANOG detected in E14Tg2a cells (Figure 3C). The nuclear translocation of NANOG-ERT2 triggered by Tamoxifen (Figure 3D) leads to an accompanying reduction of endogeneous Nanog pre-mRNA expression to ∼50% of starting levels by 6 h (Figure 3E), in cells that display unchanged levels of OCT4 and SOX2 (Figure 3F). After 24 h of Tamoxifen treatment, endogenous Nanog downregulation is maintained while OCT4 and SOX2 mRNA and protein levels remain unaffected (Figure 3G and H). In contrast, increased levels of Esrrb and Klf4 mRNA (Figure 3G) and pre-mRNA (Figure 3I) were detected upon Tamoxifen treatment, mirroring the results observed in RCNβH cells. Figure 3.Endogenous Nanog transcription is rapidly downregulated upon restoration of nuclear NANOG expression. (A) Schematic diagram of Tamoxifen-inducible 44NERT cells. (B) Relative expression of Nanog mRNA in WT E14Tg2a cells, Nanog-null TβC44Cre6 (a schematic picture of this line is shown in Figure 7C), and three independent 44NERT clones (n=3; error bars represent s.e.m.). (C) Immunoblot analysis of NANOG expression in 44NERT and WT E14Tg2a ES cells (n.s. designates a non-specific band). (D) Immunofluorescence detection of NANOG (red) and the RNAPII (green) in 44NERTc3 cells before (left) and after 30 min of Tamoxifen treatment (right), on DAPI-stained nuclei (blue). (E) Relative quantification of endogenous Nanog-driven pre-mRNA after 0 (set to 1 for each clone), 3 and 6 h of Tamoxifen treatment in three independent 44NERT clones (n=2; error bars represent s.e.m.). (F) Immunoblot analysis of OCT4 and SOX2 after 3 and 6 h of Tamoxifen treatment of 44NERTc3 cells. (G) Relative expression of Nanog pre-mRNA, and of four pluripotency transcripts (Oct4, Sox2, Esrrb and Klf4) after 24 h of Tamoxifen treatment (n=6; error bars represent s.e.m.). (H) Immunoblot analysis of OCT4 and SOX2 in untreated and 24 h-treated 44NERT clones. (I) Relative quantification of Esrrb and Klf4 pre-mRNA production after 3 h of Tamoxifen treatment in the three 44NERT clones. (J) Relative quantification of Nanog locus pre-mRNA in the three 44NERT clones treated for 2.5 h with either Cycloheximide (Chx, set to 1 for each clone and shown as a single bar; Control), or Cycloheximide plus Tamoxifen (n=2; error bars represent s.e.m.). (K) Schematic representation of the three luciferase reporter constructs used in this study. (L) Relative luciferase activity of 44NERTc3 cells transfected with the Long Pr. construct and treated for 24 h with Tamoxifen (untreated cells set to 1; n=2; error bars represent s.e.m.). (M) Relative luciferase activity of 44NERTc3 cells transfected with either the WT-Enh & Min Pr. or the Mut-Enh & Min Pr. constructs and treated for 24 h with Tamoxifen (untreated cells transfected with the WT construct were set to 1; n=2; error bars represent s.e.m.). Download figure Download PowerPoint Our results show that the Nanog gene responds rapidly to the inducible depletion and restoration of NANOG. Whether this effect is a direct consequence of NANOG activity was investigated by treating 44NERT cells with Tamoxifen and Cycloheximide, a potent inhibitor of protein synthesis. Compared with cells treated with Cycloheximide alone, cells treated with Tamoxifen and Cycloheximide for 2.5 h displayed a 25% (clone#1), 18% (clone#2) and 35% (clone#3) downregulation of endogenous Nanog pre-mRNA (Figure 3J). Thus, the effect of NANOG is independent of any additional putative repressor of Nanog, whose expression may be activated by NANOG. NANOG represses Nanog through unknown binding sites Our results contrast markedly with the generally accepted model of Nanog autoregulation, and in particular with a previous report showing that a putative NANOG-binding site located 5-kb upstream of the Nanog transcription start site conferred high transcriptional activity to a luciferase gene driven by the minimal Oct4 promoter (Wu et al, 2006). To investigate this discrepancy, we established a luciferase strategy in 44NERTc3 cells, in which transfection of the 6-kb-long Nanog promoter/luciferase construct (Long Pr. construct, Figure 3K) recapitulates the NANOG-mediated repression of Nanog-driven transcription upon Tamoxifen treatment (Figure 3L). We then generated a chimaeric reporter in which the −5 kb region was positioned adjacent to the minimal Nanog promoter instead of the Oct4 promoter used by Wu et al (WT-Enh & Min Pr. Figure 3K). We found that Tamoxifen treatment of 44NERTc3 cells transfected with this DNA recapitulated the ∼50% reduction in luciferase activity observed with the Long Pr. construct (Figure 3M). Next, we introduced the same deletion of the putative NANOG-binding site previously assessed (Mut-Enh & Min Pr., Figure 3K; Wu et al, 2006). When this reporter was transfected into 44NERTc3 cells, reduced luciferase activity was observed compared with the WT-Enh & Min. Pr reporter (Figure 3M), as previously shown (Wu et al, 2006). However, this decrease was observed in cells that were not treated with Tamoxifen (i.e., in the absence of nuclear NANOG), indicating that the reduced activity conferred by this mutation is not due to a lack of NANOG binding as speculated previously. Moreover, addition of Tamoxifen reduced the activity of the mutant construct by around 50%, similar to the reduction observed with the WT-Enh & Min Pr. (Figure 3M). We conclude from this that the previously identified putative NANOG-binding site is not responsible for NANOG-mediated regulation of Nanog. While it is also clear that an as yet unknown transcriptional activator binds to the deleted region, our results indicate strongly that Nanog is subject to direct autorepression through NANOG binding to unknown sites within the −5 kb region. Dose response of NANOG-mediated repression of Nanog After 3 and 6 h of Tamoxifen treatment, Nanog pre-mRNA is slightly more downregulated in 44NERTc3 cells than in the other two clones (Figure 3E). Similarly, FACS analyses of GFP expression, which in 44NERT cells is expressed from one of the endogenous Nanog alleles (Figure 3A), confirmed both that endogenous Nanog gene activity is reduced upon Tamoxifen treatment and that this reduction is more prominent in 44NERTc3 than in 44NERTc1 and c2 cells (Figure 4A). This was particularly obvious when the relative Nanog:GFP population median was calculated (Figure 4B). Given that 44NERTc3 expresses the highest levels of NANOG-ERT2 among the three 44NERT clones (Figure 3C), this suggests that the NANOG-mediated repression of Nanog might be dose responsive. Figure 4.Dose responsiveness of NANOG-mediated repression of Nanog. (A) FACS profiles of the three 44NERT clones before (blue) and after 48 h of Tamoxifen treatment (red). (B) Relative Nanog:GFP population median in untreated (blue, set to 1 for each clone and shown as a single bar; Control) and Tamoxifen-treated 44NERT clones (n=2; error bars represent s.e.m.). (C) Schematic diagram of Doxycycline-inducible 44iN cells. (D) Relative Nanog mRNA expression measured in 44iN cells treated for 48 h with the indicated concentrations of Doxycycline (3000, ng/ml set to 1; n=3; error bars represent s.e.m.). (E) Immunoblot analysis of NANOG expression in WT E14Tg2a and in 44iN cells treated for 48 h with the indicated concentrations of Doxycycline. (F) FACS profiles of untreated and Doxycycline-treated 44iN cells (48 h at the indicated doses). (G) Relative Nanog:GFP population median in untreated (blue, set to 1) and Doxycline-treated 44iN cells (48 h at the indicated doses; untreated cells set to 1; n=3; error bars represent s.e.m.). (H) Luciferase activity driven by a 6-kb-long Nanog promoter transfected in 44iN cells and treated with different doses of Doxycycline for 24 h (untreated cells set to 1; n=2; error bars represent s.e.m.). Download figure Download PowerPoint To more rigorously assess the dose-responsiveness of the NANOG-mediated repression of Nanog, in particular at low cellular doses, we used an independent genetic system of NANOG restoration (44iN cells; Festuccia et al, 2012). Like 44NERT cells, 44iN were derived from TβC44Cre6 cells and therefore express the GFP from one Nanog allele. Moreover, a WT version of NANOG can be expressed upon addition of Doxycycline to 44iN cultures (Figure 4C). Treatment of 44iN cells with increasing concentrations of Doxycycline leads to a progressive increase of exogenous Nanog mRNA expression (Figure 4D). Immunoblot analyses (Figure 4E) showed that among the tested conditions, two were associated with levels of NANOG clearly below WT levels (15 and 30 ng/ml of Doxycycline), whereas three other conditions produced elevated levels of NANOG expression (100, 300 and 3000, ng/ml of Doxycycline). Analysis of the FACS profiles showed that at all concentrations of Doxycycline, Nanog:GFP is downregulated as compared with untreated 44iN cells (Figure 4F). Interestingly, when the Nanog:GFP population median was plotted, a gradual and progressive downregulation of Nanog:GFP expression was observed (Figure 4G), starting from the lowest concentration of Doxycycline which is associated with very low levels of exogeneous NANOG (Figure 4D and E). Moreover, when untreated 44iN cells were transfected with the 6-kb-long Nanog promoter/luciferase construct and cultivated for 24 h in the same range of Doxycycline concentrations, a gradual reduction in luciferase activity was observed with increasing doses of Doxycycline (Figure 4H). We conclude that even at low concentrations, NANOG represses endogeneous Nanog transcription in a clear dose-response manner. Transcriptional foundation of Nanog autorepression The results presented above, in which pre-mRNA production by the Nanog locus responds rapidly to the loss and restoration of NANOG function (Figures 2, 3, 4), suggest that transcriptional mechanisms underlie NANOG-mediated repression of Nanog. In line with the idea of direct and transcriptional Nanog autorepression, chromatin immunoprecipitation (ChIP) analyses have shown that NANOG binds at the Nanog locus. Indeed, it is clear from genome-wide studies that two hotspots of transcription factor binding control Nanog transcription in ES cells: the promoter and a 5-kb upstream region where NANOG binding occurs (Loh et al, 2006; Chen et al, 2008; Marson et al, 2008; Kim et al, 2008). Using ChIP with 22 primer pairs covering 9 kb of the Nanog locus from −6 kb to +3 kb relative to the transcription start site (Figure 5A), we observed maximal binding of NANOG at the −5 kb region (Figure 5B). Importantly, NANOG binding is appropriately abolished (Figure 5C) and restored (Figure 5D) upon Tamoxifen treatment of RCNβH and 44NERT ES cells, respectively. Figure 5.Transcriptional foundation of Nanog autorepression. (A) (Top) Schematic representation of the 5′ end of the Nanog locus analysed by ChIP. Each vertical bar represents a primer pair. The five primer pairs coloured in red are located within Nanog intron 1 and were used to detect pre-mRNA transcription from the endogenous locus (also represented in Figure 1B). The arrow represents the transcription start site (TSS) of Nanog, and the grey box Nanog exon 1. (Bottom) ChIP profile obtained in E14Tg2a using an irrelevant IgG as a negative control (n=2). (B–D) ChIP analysis of NANOG across the Nanog 5′ region in the indicated lines and conditions (B, E14Tg2a, n=4; C, RCNβH, n=6; D, 44NERT, n=6). (E) Tamoxifen-indu