Hedgehog controls neural stem cells through p53-independent regulation of Nanog
2010; Springer Nature; Volume: 29; Issue: 15 Linguagem: Inglês
10.1038/emboj.2010.131
ISSN1460-2075
AutoresAgnese Pò, Elisabetta Ferretti, Evelina Miele, Enrico De Smaele, Arianna Paganelli, Gianluca Canettieri, Sonia Coni, Lucia Di Marcotullio, Mauro Biffoni, Luca Massimi, Concezio Di Rocco, Isabella Screpanti, Alberto Gulino,
Tópico(s)Renal and related cancers
ResumoArticle25 June 2010free access Hedgehog controls neural stem cells through p53-independent regulation of Nanog Agnese Po Agnese Po Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Elisabetta Ferretti Elisabetta Ferretti Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Evelina Miele Evelina Miele Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Enrico De Smaele Enrico De Smaele Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Arianna Paganelli Arianna Paganelli Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Gianluca Canettieri Gianluca Canettieri Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Sonia Coni Sonia Coni Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Lucia Di Marcotullio Lucia Di Marcotullio Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Mauro Biffoni Mauro Biffoni Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanitá, Rome, Italy Search for more papers by this author Luca Massimi Luca Massimi Institute of Neurosurgery, Division of Pediatric Neurosurgery, Catholic University of Rome, Rome, Italy Search for more papers by this author Concezio Di Rocco Concezio Di Rocco Institute of Neurosurgery, Division of Pediatric Neurosurgery, Catholic University of Rome, Rome, Italy Search for more papers by this author Isabella Screpanti Isabella Screpanti Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Alberto Gulino Corresponding Author Alberto Gulino Department of Experimental Medicine, Sapienza University, Rome, Italy Department of Neuroscience, Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Italy Search for more papers by this author Agnese Po Agnese Po Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Elisabetta Ferretti Elisabetta Ferretti Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Evelina Miele Evelina Miele Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Enrico De Smaele Enrico De Smaele Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Arianna Paganelli Arianna Paganelli Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Gianluca Canettieri Gianluca Canettieri Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Sonia Coni Sonia Coni Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Lucia Di Marcotullio Lucia Di Marcotullio Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Mauro Biffoni Mauro Biffoni Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanitá, Rome, Italy Search for more papers by this author Luca Massimi Luca Massimi Institute of Neurosurgery, Division of Pediatric Neurosurgery, Catholic University of Rome, Rome, Italy Search for more papers by this author Concezio Di Rocco Concezio Di Rocco Institute of Neurosurgery, Division of Pediatric Neurosurgery, Catholic University of Rome, Rome, Italy Search for more papers by this author Isabella Screpanti Isabella Screpanti Department of Experimental Medicine, Sapienza University, Rome, Italy Search for more papers by this author Alberto Gulino Corresponding Author Alberto Gulino Department of Experimental Medicine, Sapienza University, Rome, Italy Department of Neuroscience, Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Italy Search for more papers by this author Author Information Agnese Po1,‡, Elisabetta Ferretti1,‡, Evelina Miele1, Enrico De Smaele1, Arianna Paganelli1, Gianluca Canettieri1, Sonia Coni1, Lucia Di Marcotullio1, Mauro Biffoni2, Luca Massimi3, Concezio Di Rocco3, Isabella Screpanti1 and Alberto Gulino 1,4 1Department of Experimental Medicine, Sapienza University, Rome, Italy 2Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanitá, Rome, Italy 3Institute of Neurosurgery, Division of Pediatric Neurosurgery, Catholic University of Rome, Rome, Italy 4Department of Neuroscience, Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Italy ‡These authors contributed equally to this work *Corresponding author. Department of Experimental Medicine, Sapienza University, 324 Viale Regina Elena, Rome 00161, Italy. Tel.: +39 6 446 4021; Fax: +39 6 446 1974; E-mail: [email protected] The EMBO Journal (2010)29:2646-2658https://doi.org/10.1038/emboj.2010.131 There is a Have you seen ...? (August 2010) associated with this Article. 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 Hedgehog (Hh) pathway has a pivotal function in development and tumorigenesis, processes sustained by stem cells (SCs). The transcription factor Nanog controls stemness acting as a key determinant of both embryonic SC self-renewal and differentiated somatic cells reprogramming to pluripotency, in concert with the loss of the oncosuppressor p53. How Nanog is regulated by microenvironmental signals in postnatal SC niches has been poorly investigated. Here, we show that Nanog is highly expressed in SCs from postnatal cerebellum and medulloblastoma, and acts as a critical mediator of Hh-driven self-renewal. Indeed, the downstream effectors of Hh activity, Gli1 and Gli2, bind to Nanog-specific cis-regulatory sequences both in mouse and human SCs. Loss of p53, a key event promoting cell stemness, activates Hh signalling, thereby contributing to Nanog upregulation. Conversely, Hh downregulates p53 but does not require p53 to control Nanog. Our data reveal a mechanism for the function of Hh in the control of stemness that represents a crucial component of an integrated circuitry determining cell fate decision and involved in the maintenance of cancer SCs. Introduction Nanog is a core intrinsic determinant of self-renewal and pluripotent cellular state of embryonic stem cells (ESCs) of the blastocyst inner cell mass, and its expression is downregulated on differentiation (Chambers et al, 2003; Mitsui et al, 2003; Boiani and Scholer, 2005; Pan and Thomson, 2007). Nanog is also required for early embryo development, as Nanog-deficient mice fail to develop beyond the blastocyst stage (Mitsui et al, 2003). Reprogramming experiments of differentiated somatic cells by expression of Oct4, Sox2, c-Myc and Klf4, reactivate the production of Nanog+ pluripotent stem cells (SCs) (Takahashi and Yamanaka, 2006; Brambrink et al, 2008), indicating that Nanog has an important function in determining stemness features. Consistently, overexpression of Nanog gives rise to pluripotent SCs (Yu et al, 2007; Hanna et al, 2009). Recently, Nanog has been described to be reactivated in cancer cells (e.g. glioma, pancreas, colorectal, endometrial) possibly as a reminiscence of stemness properties of cancer SCs (Clement et al, 2007; Hubbard et al, 2009; Jeter et al, 2009; Ji et al, 2009; Meng et al, 2010; Wen et al, 2010). These observations have emphasized the notion that cancer cells and SCs might be governed by common mechanisms. Such conclusions are also supported by recent works demonstrating that loss of the tumor suppressor p53 is another key determinant of stemness and of transcriptional activation of Nanog (Lin et al, 2005; Hong et al, 2009; Kawamura et al, 2009). Thus, the elucidation of the poorly understood events that regulate Nanog in postnatal SCs represents a fundamental aspect to elucidate both SC and tumor biology. Hedgehog (Hh) pathway has a pivotal function in cell development and tumorigenesis in a wide variety of tissues, both processes sustained by SCs. More specifically, Hh signalling sustains embryonic and postnatal NSCs of forebrain subventricular zone and hippocampus (Ahn and Joyner, 2005; Lai et al, 2003; Machold et al, 2003; Palma et al, 2005; Palma and Ruiz i Altaba, 2004), as well as cerebellar NSCs and glioma SCs overexpressing a stemness gene signature (e.g. Nanog, Oct4, Sox2, CD133) (Clement et al, 2007; Stecca and Ruiz i Altaba, 2009). A paradigmatic Hh-target organ is cerebellum, where Hh is critically required to keep transit-amplifying granule cell progenitors (GCPs) undifferentiated, to promote their proliferation (Dahmane and Ruiz i Altaba, 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999) and to cause medulloblastoma (Mb) (Ruiz i Altaba, 2006). Aberrant Hh signalling activation occurs in Mb as a consequence of genetic or epigenetic changes affecting several components of the pathway (recently reviewed in Teglund and Toftgard, 2010). For instance, loss-of-function mutation of Patched1 (Ptc1) (the receptor of the Hh ligands) mimics the ligand-induced relief of the inhibition on the transducer Smoothened (Smo), leading to constitutive pathway activation. Downstream effectors of Smo activity are Gli transcription factors, which act on a set of target genes promoting cell proliferation and reducing cell differentiation. These target genes include Gli1 itself, thus autoreinforcing the signalling strength and representing a sensitive read out of the pathway. The identity of Hh/Gli-target genes involved in the control of stemness in NSCs and cancer SCs is poorly understood. Here, we show that both Nanog and Gli1 are highly expressed in postnatal cerebellar NSCs and in Hh-dependent mouse and human Mb SCs. Nanog is required as a critical mediator of Hh-driven self-renewal of NSCs, as Hh acts through transcriptional activation of both mouse and human Nanog. Our data also suggest Nanog involvement in the Hh-dependent stemness properties of brain tumors. Results Cerebellar neurospheres coexpress Nanog and Gli1 Earlier studies have shown the existence of a subpopulation of NSCs in mouse postnatal cerebellum (Klein et al, 2005; Lee et al, 2005). Thus, to investigate a functional connection between Hh and intrinsic stemness determinants, we sorted Prominin1+ cells, (Supplementary Figure S1A) from cerebella of 4-day-old mice. We found that these cells displayed enhanced expression of stemness markers (Nanog, Oct4, Sox2, Bmi1, Musashi1, Prominin1, Nestin) together with Hh pathway target genes (Ptc1, Hip1, Cyclin D1–2 and Gli1), compared with Prominin1-negative cells (Figure 1A). Conversely, differentiation markers (e.g. the granule cell lineage markers Math1 and Zic1) were significantly reduced (Figure 1A; Supplementary Figure S1B), suggesting that Hh activity preferentially associates to stemness features. Figure 1.Cerebellar neurospheres coexpress Nanog and Gli1. (A, B) Heat map of RT–qPCR gene expression (stemness, Hh pathway and differentiation (Diff) markers) of FACS-sorted Prominin1+ versus Prominin1− cells from eight cerebella of 4-day-old mice (three representative experiments) (A) or 24 h cultured cerebellar cells (T0) versus derived neurospheres (B). A red–green colour scale (−10 to +30) depicts markers expression normalized to three housekeeping genes. (C) Western blot analysis of endogenous Gli1 and stemness markers levels in cerebellar cells (T0) and derived neurospheres (NS). (D) Immunofluorescence or nuclear Hoechst (blue) staining with Nanog (green), Gli1 (red) or both antibodies in disgregated neurospheres from murine cerebellum. The relative percentage of either Nanog or Gli1 or double-positive (marked) cells is indicated on the right. (E) mRNA levels evaluated by RT–qPCR of Hh (Gli1, Gli2) or stemness (Nanog, Sox2) markers in mouse embryonic fibroblasts (MEFs) Ptc1−/− respect to wild-type MEFs (wt). *P<0.05 versus wild-type MEFs. (F) Western blot analysis of Gli1 and Nanog levels in Ptc1 wt versus Ptc1−/− MEFs. (G) Western blot analysis of endogenous Gli1 and Nanog in Mb cells (T0) and derived neurospheres (NS). (H) Immunofluorescence or nuclear Hoechst (blue) staining with Nanog (green) and Gli1 (red) antibodies in disgregated neurospheres from mouse Ptc1+/− Mb. Download figure Download PowerPoint Cerebellar SCs formed primary neurospheres and were further propagated as secondary neurospheres from single-cell suspensions indicating that they were able to self-renew. To characterize SCs, we analysed mRNA and protein expression of stemness markers, differentiation markers and Hh activity (Gli1 expression) in neurospheres with respect to pre-neurosphere cells. Although we found loss of differentiation markers Math1 and Zic1 (Figure 1B), we observed enhanced expression of stemness markers together with Hh pathway activation in neurospheres (Figure 1B and C). Notably, we also found, by immunofluorescence, coexpression of Gli1 and Nanog in about 40% of cells within neurospheres (Figure 1D), whereas Nanog did not stain rare cells positive for neuronal differentiation marker MEF2D (Supplementary Figure S1C, upper panels). These results suggest us a possible functional connection between Hh signalling and Nanog. To analyse this relationship, we studied Nanog expression ex vivo in conditions of Hh pathway constitutive activation using the Ptc1−/− mouse embryo fibroblasts (MEFs) model, in which the activation of Gli is a consequence of Ptc1 deletion (Goodrich et al, 1997). In this context, we observed increased Nanog mRNA and protein levels (Figure 1E and F). Accordingly, in neurospheres derived from murine Ptc1+/− Mbs, both Nanog and Gli1 levels were higher than in pre-neurosphere cells and mostly displayed cellular coexpression (Figure 1G and H). Collectively, these observations further support the notion that Nanog and Hh signalling are functionally connected. Function of Hh–Gli and Nanog in cerebellar neurospheres Cerebellar neurosphere multipotency is indicated by the ability to differentiate into distinct lineages (Figure 2A) when cultured under appropriate conditions (Lee et al, 2005), whereas the reduction of Gli1, Gli2, Nanog and Sox2 expression suggests that Hh activity and stemness features were coregulated during differentiation (Figure 2B). To understand the function of Hh in the control of cerebellar NSCs, we tested the effects of the activation of the pathway by the Smo-agonist SAG (Chen et al, 2002; Ruiz i Altaba, 2006). SAG enhanced Gli1, Nanog and Prominin1 mRNA and protein levels (Figure 2C and D; Supplementary Figure S2A) as well as the percentage of neurosphere-forming cells (Figure 2E). Conversely, treatment with the Hh inhibitor KAAD cyclopamine had a suppressive effect on NSCs self-renewal and on Gli1 and Nanog expression (Figure 2E; Supplementary Figure S2B). Importantly, SAG-induced Gli1 is coexpressed with Nanog (Supplementary Figure S2C), indicating that both are targets of Hh activity. Consistently, inactivation of Hh signalling by siRNA-mediated depletion of Smo significantly reduced the levels of Smo, Gli1 and Nanog (Figure 2F) as well as the self-renewal ability, both after SAG treatment and under basal conditions (Figure 2G). Figure 2.Function of Hh–Gli and Nanog in cerebellar neurospheres. (A, B) Percentage of positive cells detected with immunofluorescence staining (A) or western blot (B) with the indicated antibodies in neurospheres cultured in D-poly-ornithine-coated chamber slides with N2 medium plus PDGF (10 ng/ml) for 7 days (A, Gran, Granule cells; Purk, Purkinje cells; Astr, astrocytes; Olig, oligodendrocytes) (B, differentiated (Diff)). (C, D) mRNA (C) and protein levels (D) of Gli1 and Nanog evaluated by RT–qPCR or western blot relative to housekeeping controls, in neurospheres before (Ctrl) and after 48-h SAG treatment. (E) Neurosphere-forming assay of single cells derived from secondary neurospheres cultured in basal stem medium (−) or in the presence of SAG or KAAD cyclopamine for 10 days. (F, G) Western blot (F) or neurosphere-forming assay (G) after transfection with control (siCtrl) or Smo siRNA (siSmo) in the absence or in the presence of SAG for 10 days. *P<0.05 versus untreated cells. Download figure Download PowerPoint As Hh enhances the proliferation of GCPs (Dahmane and Ruiz i Altaba, 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999), we hypothesized that it could favour NSCs towards the expansion of the granule cell lineage. Treatment of neurospheres with SAG for 4 or 10 days followed by shift to differentiation medium did not influence the multipotency of cerebellar NSCs, as no significant change in the generation of the various cerebellar cell lineages was observed compared with untreated neurospheres (Supplementary Figure S2D). Overall, these findings show that Hh signalling is required to maintain both self-renewal and Nanog expression in cerebellar NSCs. Nanog is required for Hh-induced NSCs self-renewal To understand the function of Nanog in postnatal NSCs and its connection with Hh signalling, we studied the self-renewal potential of Nanog-positive cells. To this purpose, we infected NSCs with a Nanog-GFP lentiviral vector expressing GFP protein under the control of Nanog promoter (Nanog-GFP). We observed a significant expression of GFP in NSCs, whereas we did not detect any expression in GCPs obtained from primary cerebellar cell cultures (as described in Di Marcotullio et al, 2006) (Figure 3A). GCPs are ‘already committed’ precursor cells, which do not express stem markers but are characterized by neuronal markers (MEF2D) as shown in Supplementary Figure S1C. Figure 3.Nanog is required for Hh-induced NSC self-renewal. (A) Histograms showing the percentage of GFP+ cells in postnatal cerebellar secondary neurospheres (NS) and GCPs 48 h after infection with Nanog-GFP or Zeo-GFP control lentiviral vector. *P<0.05 versus Zeo-GFP-positive cells. (B) mRNA and protein levels of Nanog and Gli1 in neurospheres (NS) or GCPs before (T0) or after 2 days culture (DIV2). (C) Western blots of Gli1 and Nanog in Nanog-GFP versus Zeo-GFP-sorted neurosphere-derived cells. (D) Secondary neurosphere-forming assay of GFP-sorted cells as in panel (A). (E) mRNA (upper panel) and protein levels (bottom panel) of Nanog and Prominin1 in neurospheres after siRNA-mediated silencing of Nanog (siNanog +) versus control siRNA (−). (F) Secondary neurosphere-forming assay after control siRNA (siCtrl) or Nanog siRNA (siNanog), treated (+) or not (−) with SAG. *P<0.05 versus untreated or Zeo-GFP cells. (G) Representative bright field images of cerebellar neurosphere after transfection with control siRNA (siCtrl) or Nanog siRNA (siNanog). Download figure Download PowerPoint As control, a CMV-driven-GFP lentivector (Zeo-GFP) was similarly expressed in both cell types. Of relevance, Nanog-GFP expression paralleled the one of endogenous Nanog in NSCs, whereas both were not expressed in differentiated GCPs (Figure 3B and C). To show the function of Nanog in NSC self-renewal, we sorted NanogGFP+ cells and we found that they were able to form secondary neurospheres to a significantly higher extent compared with Zeo-GFP-positive cells (Figure 3D). Accordingly, silencing of Nanog reduced the expression of Prominin1 (Figure 3E) and significantly impaired the ability to form neurospheres compared with control (Figure 3F and G), elucidating its function in self-renewal. Moreover, Nanog depletion abrogated the enhancing activity of SAG on neurosphere-forming assay (Figure 3F), indicating that Nanog is required for Hh effects on NSCs. Hh regulates SC self-renewal through a p53-independent pathway As p53 suppresses Nanog transcription and self-renewal ability in ESCs (Lin et al, 2005) and can be repressed by Hh signalling through Mdm2 (Abe et al, 2008; Stecca and Ruiz i Altaba, 2009), we addressed the function of p53 in Hh-induced regulation of Nanog and NSC self-renewal. To this end, cerebellar NSCs have been isolated from p53-deficient mice (Jacks et al, 1994). The derived neurospheres displayed a significantly enhanced expression of stemness markers including Nanog (Figure 4A and B) and, consistently, a higher clonogenic activity with respect to wild-type cells (Figure 4C). Interestingly, p53-deficient neurospheres also displayed higher levels of Gli1 (Figure 4A and B), suggesting that p53 downregulates Hh signalling and confirming recent observations (Stecca and Ruiz i Altaba, 2009). Importantly, abrogation of Hh signalling in p53-deficient neurospheres by means of siRNA-mediated depletion of Smo, still reduced Nanog levels and neurosphere-forming ability (Figure 4D–F), suggesting that p53 is at least in part dispensable in this context. Therefore, in addition to p53-mediated control, Hh regulates Nanog and NSCs self-renewal also in a p53-independent manner. Figure 4.Hh regulates stem cell self-renewal through a p53-independent pathway. (A, B) RT–qPCR (A) and western blot (B) analysis of stemness markers and Gli1 in neurospheres from p53-deficient mouse cerebella (p53−/−). (A) Results are expressed as mean values of five different neurosphere cultures from p53−/− mice with respect to p53+/+ wild type (dashed line). (C) Secondary neurosphere-forming assay of p53−/− cells with respect to cells from p53 wild-type mice. (D) Western blot analysis of Smo, Gli1 and Nanog in p53−/− neurospheres after transfection with siRNA against Smo (siSmo) compared with control siRNA (Ctrl). (E, F) Secondary neurosphere-forming assay (E) and representative bright field images (F) of cerebellar p53−/− cells after transfection with siRNA against Smo (siSmo) or control siRNA (siCtrl). *P<0.05 versus untreated p53+/+ cells (A, C) or siCtrl (E). Download figure Download PowerPoint Hh/Gli activate Nanog transcription To address how Hh signalling affects Nanog expression, we performed a time course of SAG treatment. SAG increased Nanog mRNA (as early as 3–6 h) and protein levels (9 h) mimicking the induction of a direct Hh target, Gli1 (Figure 5A and B). Of note, other stemness markers, such as Sox2 (Figure 5A and B), were not affected, suggesting that Hh-mediated enhancement of Nanog could depend not only on expansion of NSC population, but also on specific transcriptional regulation. Indeed, a number of potential Gli-binding sequences were observed in a region spanning from −1060 to −200 bp of Nanog (Figure 5C; Supplementary Figure S3A). As Gli2 is an early transducer of SAG-dependent Smo activation leading to its accumulation into the nucleus (Supplementary Figure S3B) (Park et al, 2000; Ikram et al, 2004; Lipinski et al, 2006), we performed chromatin immunoprecipitation (ChIP) experiments to determine the in vivo occupancy of endogenous Gli2 onto the Nanog promoter in mouse NSCs. Gli2 was recruited to the S1 (−200 bp) and S2 to S5 (−775, −935, −1000 and −1060 bp) sites (but not to −2100 bp site devoid of Gli consensus sequence) following 2.5 and 5 h SAG treatment (Figure 5D and F). Gli2 recruitment was accompanied by an increase of acetylated histone H3, indicating that this promoter was transcriptionally activated by SAG (Figure 5D and F). As SAG-activated Gli2 enhances Gli1 transcription, representing the second wave of Hh activity, we determined Gli1 recruitment to Nanog promoter after SAG treatment. At later time, Gli1 substituted Gli2 on the S1 and S2–S5 sites, together with hyperacetylation of histone H3 (Figure 5E and F). According to p53-mediated regulation of Hh, a similar enhanced recruitment of Gli1 was observed in p53-deficient NSCs in the absence of SAG treatment (Figure 5E). A basal recruitment of Gli2 and Gli1 onto Nanog promoter was also detected, although lower than that found after SAG treatment with respect to an unrelated control gene (actin) or to non-specific −2100 bp Nanog site devoid of Gli consensus sequence. Consistently, the activation of Nanog and Gli1 expression observed in neurospheres derived from Ptc1+/− Mbs was associated with an enhanced Gli1 occupancy of Nanog promoter (Figure 5G). Figure 5.Hh/Gli activate Nanog transcription. (A, B) RT–qPCR (A) and western blot (B) analysis of Nanog, Sox2 and Gli1 levels in neurosphere cultures after SAG treatment up to 24 h (means±s.d. from four different experiments). *P<0.05 versus untreated cells. (C) Representation of Nanog promoter showing putative Gli-responsive elements (GliRE). (D–F) ChIP (D, E) and real-time qPCR-ChIP (F) assays from untreated (nt), 2.5 h (F), 5 h (D, F) and 24 h (E, F) SAG-treated neurospheres or p53−/− neurospheres (E), using anti-Gli2 (D, F) or anti-Gli1 (two different antibodies, see Materials and methods and panels (E, F)) and anti-acetyl-H3 antibodies. Eluted DNA was PCR amplified with primers shown in Supplementary Figure S4B. Real-time qPCR-ChIP results are expressed as fold induction versus endogenousβ-actin-amplified ChIP controls. Bars represent the mean of three independent experiments±s.d. (*P<0.05 versus nt). (G) RT–qPCR ChiP assay from untreated murine cerebellar neurospheres (mNSC) and murine medulloblastoma-derived neurospheres (mMbSC) using anti-Gli1 antibody. Eluted DNA was PCR amplified with primers shown in Supplementary Figure S4B. Bars represent the mean of three independent experiments±s.d. (*P<0.05 versus mNSC). A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint To verify whether these Gli-binding sites were responsible for the transcriptional activation of Nanog, we determined the effect of Gli transcription factors overexpression on mouse Nanog promoter-luciferase reporter. The luciferase activity was strongly induced by Gli1 and Gli2, whereas this induction was significantly reduced by deletion of the (−1060 to −935) Gli-binding sites or mutations of critical nucleotides in S1 to S5 sites (Figure 6A; Supplementary Figure S3A). Figure 6.Transcriptional activity of Gli-responsive sites of Nanog promoter. (A) Relative luciferase activity driven by either mouse Nanog (−2500/+1 bp) or (−877/+1 bp) region or (−2500/+1 bp) reporter constructs carrying either mutagenized S1 to S5 Gli consensus sequences (indicated in Supplementary Figure S3A) or Ptc1 promoter/reporter or PgL4 empty vector, transfected into mouse cerebellar neurospheres together with mock (continuous line), Gli1, Gli2 or CREB (as a negative control). Data are indicated as mean ratios with respect to pRL-CMV-Renilla Luciferase control (ctrl). *P<0.05 versus Ctrl. **P<0.05 versus wild-type constructs (dashed lines). (B) Relative luciferase activity driven by Nanog promoter in p53−/− compared with p53 wt neurospheres (continuous line). The day before Gli1 and Gli2 overexpression, cells were transfected with siRNA against Smo (siSmo) or control. *P<0.05 versus Mock-transfected cells; **P<0.05 versus siCtrl. Download figure Download PowerPoint Moreover, to further strengthen the p53-independent and direct regulation of Nanog by Gli factors, we investigated the effect of the modulation of Hh signalling on Nanog promoter-luciferase reporter in a p53−/− neurospheres. We first observed that in this context, Nanog promoter has a basal higher activity when compared to wild type. The modulation of Hh signalling by Gli1 or Gli2 overexpression or by Smo siRNA increased or reduced, respectively, the luciferase activity, indicating that p53 is dispensable for Hh regulation (Figure 6B). Overall, these findings indicate the presence of discrete Gli-binding sites in the cis-regulatory sequences of Nanog that are responsible for its Hh-mediated transcriptional activation. Conservation of human Nanog regulation by Hh To investigate whether Hh-induced transcriptional regulation is also active on human Nanog, we treated human neurospheres (human neuronal precursor cells, HNPCs) with SAG. This resulted in enhanced transcription of Gli1 as well as of Nanog mRNA and protein (Figure 7A and B), suggesting that Hh signalling may enhance also human Nanog transcription. Interestingly, the −256 to −118 bp region of Nanog, including the S1 Gli site, is conserved between mouse and human (Figure 7C; Pan and Thomson, 2007). Accordingly, the activity of a human Nanog reporter (−2586/+1 bp), spanning from −2586 bp to the transcription start site, was upregulated by Gli1 and Gli2 overexpression in human cells (Figure 7D; Supplementary Figure S3C). Figure 7.Conservation of human Nanog regulation by Hh. (A, B) RT–qPCR (A) of Nanog, Sox2 and Gli1 levels and western blot analysis (B) of Nanog and Gli1 levels in HNPC after SAG treatment up to 24 h (means±s.d. from four different experiments). *P<0.05 versus untreated cells. (C) Alignment of mouse and human Nanog upstream cis-regulatory sequences, showing a good homology in the −256 to −118 bp region, encompassing a conserved Gli consensus site at −200 bp. (D) Relative luciferase activity driven by human Nanog (−2586/+1 bp) reporter transfected into human HNPC together with Mock, Gli1 and Gli2. Data are indicated as mean ratios with respect to pRL-CMV-Renilla Luciferase control (ctrl). *P<0.05 versus ctrl. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint Hh-depen
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