gata4 blocks somatic cell reprogramming by directly repressing nanog

Author contributions: F.S.: Collection and/or assembly of data, data analysis and interpretation, manuscript writing; C.F.C.: Collection and/or assembly of data, data analysis and interpretation; M.B.: Collection and/or assembly of data; J.T.: Conception and design, manuscript writing; J.V.C.: Financial support; R.B.: Conception and design, financial support, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript.

Correspondence: Roque Bort PhD, Unidad de Hepatología Experimental, CIBERehd, Instituto de Investigación Sanitaria La Fe, Escuela de Enfermeria, Avda Campanar 21, 46009, Spain. Telephone +34-96-1973485; Fax: +34-96-1973018; [email protected]; Gata4 represses Nanog Page; Received May 02, 2012; accepted for publication October 04, 2012; 1066-5099/2012/$30.00/0 as doi: 10.1002/stem.1272

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi:10.1002/stem.1272

STEM CELLS®

EMBRYONIC STEM CELLS/INDUCED PLURIPOTENT STEM CELLS

Gata4 Blocks Somatic Cell Reprogramming by Directly Repressing Nanog

Felipe Serrano1, Carles F. Calatayud1,Marina Blazquez1, Josema Torres2, Jose V. Castell1, and Roque Bort1

1Unidad de Hepatología Experimental, CIBERehd, Instituto de Investigación Sanitaria La Fe, Valencia 46009, Spain; 2Departamento de Biología Celular, Universidad de Valencia, E-46100 Spain.

Key words. Induced Pluripotent Stem Cells iPS cells Nuclear Reprogramming Pluripotency Nanog

ABSTRACTSomatic cells can be reprogrammed to induced-pluripotent stem (iPS) cells by ectopic expression of the four factors Oct4, Klf4, Sox2 and Myc. Here we investigated the role of Gata4 in the reprogramming process and present evidence for a negative role of this family of transcription factors in the induction of pluripotency. Coexpression of Gata4 with Oct4, Klf4, Sox2 with or without Myc in mouse embryonic fibroblasts greatly impaired reprogramming and endogenous Nanog expression The lack of Nanogupregulation was associated with a blockade in the transition from the initiation phase of reprogramming to the full pluripotent state characteristic of iPS cells. Addition of Nanog to the reprogramming cocktail blocked the deleterious effects observed with Gata4 expression. Downregulation of endogenous Gata4 by short

hairpin RNAs during reprogramming both accelerated and increased the efficiency of the process, and augmented the mRNA levels of endogenous Nanog. Using comparative genomics we identified a consensus binding site for Gata factors in an evolutionary conserved region located 9 kilobases upstream of the Nanog gene. Using chromatin immunoprecipitation, gel retardation and luciferase assays we found that Gata4 bound to this region and inhibited Nanog transcription in mouse embryonic stem (ES) cells. Overall, our results describe for first time the negative effect of Gata4 in the reprogramming of somatic cells and highlight the role of Gata factors in the transcriptional networks that control cell lineage choices in the early embryo.

INTRODUCTION

Reprogramming mouse embryonic fibroblasts (MEF) into induced-pluripotent stem (iPS) cells by ectopic expression of the four reprogramming factors (Oct4, Klf4, Sox2 and Myc) is a stochastic and inefficient process characterized by an organized sequence of events that begins with a mesenchymal to epithelial transition (MET) associated with the downregulation of fibroblast markers [1, 2].

Simultaneously, cells activate p53-mediated stress response that leads to senescence, which constitutes the primary barrier to reprogramming [3]. Consistent with the notion that loss of replicative potential provides a barrier for reprogramming, inhibition of senescence effectors such as p16Ink4a or p19Arf

or genetic ablation of p53 significantly increases the yield of iPS cell colonies [3, 4]. The cells that successfully overcome this barrier begin to express the transcriptional

Gata4 represses Nanog

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program characteristic of reprogrammed cells, followed by upregulation of essential pluripotent genes such as Nanog or Lin28during the maturation and stabilization steps of the process. It is noteworthy that Nanog-deficient cells undergo the initiation phase of reprogramming but fail to acquire full reprogramming to pluripotency [5]. In fact, Nanog is essential to achieve full reprogramming to ground state pluripotency by coordinating the pluripotency networks that allow self-sustained configuration of iPS cells. Reprogramming to a full pluripotent state causes the resetting of epigenetic marks in the somatic cells to a state similar to that of embryonic stem (ES) cells [6].

Reprogramming to pluripotency can be accelerated by facilitating the overcoming of roadblocks to pluripotency. In this regard,

BMP agonists and TGF inhibitors increase reprogramming efficiency by favoring MET [2, 7]. Reprogramming can also be accelerated by the coexpression of transcription factors that favor the acquisition of pluripotency like Glis1 or Nanog [8, 9]. Also, combined inhibition of GSK3-beta and MAPK signaling pathways not only accelerates reprogramming but also induces partially reprogrammed cells into full pluripotency [10]. On another hand, chromatin modifying molecules such as HDAC inhibitors or DNA methyltransferases enhance reprogramming, suggesting that the complete erasure of epigenetic marks could also be considered as barrier to pluripotency [11]. In conclusion, cell reprogramming efficiency can be manipulated by different strategies that target the essential roadblocks of the process.

Hhex, Foxa2 and Gata4/6 are essential for proper definitive endoderm formation [12-14]. Hhex is required for normal development of ventrally derived endoderm-related organs such as thyroid and liver. Foxa2 deficient embryos rescued for the embryonic-extraembryonic constriction, lack foregut and midgut endoderm. Gata4/6 are highly conserved, functionally redundant and capable of binding identical nucleotide sequences in genomic DNA to regulate gene expression [15-17]. Gata4/6 are key players for early cell fate decisions in the mouse Inner Cell Mass (ICM)

when cells segregate into epiblast and primitive endoderm. In fact, formation of these embryonic layers is governed by the activity of the transcription factors Nanog and Gata4/6 [18]. Nanog protein is initially found throughout the early embryo [19] and then becomes restricted to a subset of cells in the ICM, the epiblast progenitors. It is proposed that induction of Gata4/6 by Fgf signaling through Grb2 may promote primitive endoderm in a “salt and pepper” distribution of Nanog-positive and Gata-positive cells in the ICM. Nanog and Gata4/6 are not coexpressed in any cell throughout the E3.5 blastocyst [19], suggestive of reciprocal inhibition. The mechanisms exerted by Gata4/6 to downregulate Nanog expression and override epiblast commitment are still unclear.

Here we investigated the role of Gata factors in the reprogramming of somatic cells and found that these factors constitute a negative control of the process. We show for first time that Gata4 binds to a distal enhancer 9 kb upstream of the Nanog gene and inhibits its transcription. Our studies support the hypothesis that Gata4 expression in ICM constitutes a mechanism to inhibit Nanog gene expression, allowing the segregation of primitive endoderm in the early embryo.

MATERIALS AND METHODS

Plasmids and sequence analysis. The retroviral construct pMIGR1-Hhex was generated by subcloning the Hhex cDNA into the XhoI-NotI restriction sites of pMIGR1 (a kind gift from Dr.Pellicer, NYU Medical Center, USA). pPYCAG-IP-HAGata4 was obtained by PCR using the primers described in Table S2 and pBABE-Gata4 (a kind gift from Dr. Ken Zaret, University of Pennsylvania, USA) as template. pBABE-Foxa2 and pPYCAG-IP were kind gifts from Dr. Ken Zaret and Dr. Austin Smith (Wellcome Trust Centre for Stem Cell Research, U.K.) respectively. To generate pBabe-Gata6, mouse Gata6 was amplified by PCR from mouse lung cDNA and subcloned into the BamHI-SalI sites of pBabe-puro. To obtain the pGL3-Nanog-enh/prom reporter plasmid, the enhancer region of mouse Nanog


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was amplified by PCR from mouse genomic DNA (Figure S10) and subcloned into the KpnI-SacI sites of the pGL3-Nanog vector (a kind gift from Dr. Paul Robson [20]). All constructs were sequence verified. The plasmids encoding the reprograming factors pMXs-Oct4, pMXs-Sox2, pMXs-Klf4, pMXs-Myc, pMXs-Nanog and pMXs-RFP were from Addgene (www.addgene.com).

Evolutionary conserved regions within the Nanog gene were extracted by multiple alignments between mouse (mm9), human (hg19), monkey (rheMac2), opossum (monDom4), pufferfish (fr2), junglefowl (galGal3) and frog (xenTro2) genomes available using the ECR Browser web-based tool (http://ecrbrowser.dcode.org [21]). We searched the evolutionary conserved regions identified above for transcription factor binding sites that showed more than 85% sequence similarity using rVISTA 2.0 [22].

Cell Culture and Cell Imaging. CCE1.19mouse ES cells were a kind gift of Dr. M. Gassman [23]. Cells were grown in ES cell medium as described [24]: Glasgow Minimum Essential Medium (GMEM) supplemented with 10% fetal bovine serum, 0.1mM -mercaptoethanol, 1x non-essential amino acids, 1x sodium pyruvate, 1x penicillin/streptomycin (Life-Technologies), 2mM glutamine in the presence of LIF. MEF were isolated from E13 mouse embryos from pregnant CD1 and Tg(Nanog-GFP,Puro) mice [25] by trypsin digestion. MEF were maintained in Dulbecco's Modified Eagle Medium (DMEM) with Glutamax, supplemented with 10 % FBS and 1x penicillin/streptomycin (Life-Technologies). When indicated, colonies were hand-picked and maintained in 2i+LIF medium, i.e. ES cell medium containing 3 Mof the GSK3 inhibitor CHIR99021and 1 M of the MAPK inhibitor PD0325901 [26]. All cells were maintained at 37 °C with 5% CO2 and were regularly examined with an Olympus CKX41 microscope. Images were taken with a Leica DFC350 FX digital camera connected to a Leica DMI 4000 B fluorescence microscope using Leica Application Suite software.

Generation of virus, viral infections and reprogramming. Ecotropic retroviruses were generated in 293T cells by cotransfection of 10 µg of shuttle vector with 10 µg of pCL-eco (replication-incompetent helper vector pCL-eco) by the calcium phosphate method. Supernatants containing the retroviral particles were collected after 48 hours, filtered through a 0.45µm nitrocellulose filter, supplemented with 8µg/ml polybrene and immediately used. Reprogramming of somatic cells was done as described [27].

For the knockdown of the endogenous Gata4, a collection of target sequences of short hairpin RNAs (shRNA) in vector pLKO.1 was purchased from Open Biosystems (catalogue number RMM4534). A shRNA vector against luciferase (pLKO.1-shLuc) was used as a negative control. For lentivirus production, 293T cells were cotransfected with 10 g of pLKO.1 shuttle vector, 7.5 g of psPAX2 and 5 g of pMD2.G. Supernatants containing the viruses were collected 48 h post transfection, filtered through a 0.45 m-nitrocellulose filter, supplemented with 8 µg/ml polybrene and immediately used.

Reverse Transcription and real-time quantitative RT-PCR. Total RNA was extracted using the RNeasy mini kit (Qiagen) and reverse-transcribed using MMLV Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. PCR amplification was performed using Expand High Fidelity PCR system (Roche) following the manufacturer´s instructions. Quantitative RT-PCR (qRT-PCR) was run on a Light Cycler 480 II Real-Time PCR System (Roche) using the Light Cycler 480 SYBR Green I Master (Roche).The PCR reaction consisted of 7.5 µl of SYBR Green Master I, 0.75 µl of 6 µM of forward primer, 0.75 µl of 6 µM reverse primer, 3 µl water and 3 µl template cDNA (1/20) in a total volume of 15 µl. Cycling was performed 10 minutes at 95°C, followed by 40 rounds of 10 seconds at 95°C, 10 seconds at 57-64°C and 72°C for 20 seconds, and extension at 72°C for 5 minutes. The relative expression of each gene was normalized against mouse Beta-actin (Actb). The specificity of the amplified PCR products was


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confirmed by analysis of the melting curve and agarose gel electrophoresis. Primers used for the qRT-PCR are shown in Table S2.

Immunoblotting. Protein extracts (total, nuclear or cytoplasmic) were obtained by M-PER or NE-PER protein extraction reagent following the manufacturer's instructions. Samples were quantified with Coomassie Plus (Bradford) Protein Assay (Pierce). Equal quantities of protein were loaded for each sample and subjected to polyacrylamide gel electrophoresis. Gels were subsequently transferred to PVDF membranes using an iBlot gel transfer system (Invitrogen). PVDF membranes were blocked in TBS-T containing 5% nonfatmilk powder and incubated with primary antibody overnight at 4o C. Next day, membranes were washed followed by incubation with the secondary antibody for 45 minutes at RT. Immunoreactive proteins were visualized using the ECL detection reagent system. Commercial antibodies used were purchased from Santa Cruz Biotechnology: anti-Gata4 (sc#9053), anti-HA(sc#57592) and anti-Tubulin (sc#8035).

Luciferase Reporter Assay. Cells were transfected using Lipofectamine 2000 (Invitrogen) following manufacturer’s protocol. Renilla luciferase plasmid (pRL-TK from Promega) was cotransfected to correct variations in transfection efficiency. The luciferase activity in the lysate was measured using the Dual-Luciferase reporter assay system (Promega) in a 96-well luminometer reader (Berthold detection systems).

Low cell number chromatin immunoprecipitation (ChIP). ChIP using low cell numbers was done as described [28]. Primers are described in Table S2. Specificity of the Gata4 and HA antibody was validated by immunoblotting as shown in Figure S11.

Bromodeoxyuridine incorporation assay.Cell proliferation was assessed using the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche) following manufacturer’s instructions. Alkaline Phosphatase staining. Alkaline phosphatase activity was assessed using the Alkaline Phosphatase Detection Kit from

Millipore (SCR004) following manufacturer's instructions.

Electrophoretic Mobility Shift Assays (EMSA).Nuclear extracts from CCE1.19 mouse ES cells were obtained using NE-PER protein extraction reagent following the manufacturer's instructions. Probe labeling, gel shift reaction, transfer and detection were done using the DIG Gel Shift kit, 2nd Generation (Roche), with minor modifications. DNA binding reactions consisted in15 g of nuclear extract, 157 fmol labeled probe, 1 g/ l Poly L-lysine and 1 g/ l poly(dIdC) in a final volume of 20 l buffer (12mM HEPES pH7.9,4 mM Tris-HCl pH 8.0, 12 mM glycerol, 5mM MgCl2, 60mM KCl, 0.6 mM EDTA pH 7.9,0.28 mg/ml BSA and 0.6 mM DTT). When indicated, 1 M unlabeled double-stranded competitor was included prior to the addition of nuclear extracts. Binding reactions were incubated for 20 minutes at room temperature. Where indicated, 2 l of anti-Gata4 antibody (Santa Cruz Biotechnology) were added 10 minutes after the addition of the nuclear extracts and left to proceed for 10 minutes more, after this time reactions were stopped and further processed as indicated below. Binding reactions were resolved on a prerun 6% non-denaturing TBE-polyacrylamide gel (16.5 × 20 cm) in 0.5× Tris-borate-EDTA (TBE) at 20mA (constant current). Gel was electroblotted in 0.4% TBE buffer using Hybond N+ transfer membrane during 2 hours at 400 mA with a semi-dry blotter. DNA was crosslinked by UV-light (306nm) in a Gel-Logic 200 transilluminator for 12 minutes.

Microarray hybridization and analysis. Total RNA was extracted from mouse ES cells, MEF and isolated clones of MEF infected with distinct retroviral combinations using RNeasy extraction kit (Qiagen). RNA was then quantified and analized using the Agilent 2100 bioanalyzer. RNA Integrity number (RIN) values range from 10 (intact) to 1 (totally degraded), and our samples had a RIN >9. Gene expression analysis was performed according to the Affymetrix-recommended protocol using Affymetrix Mouse Gene 1.0ST array. GeneChips containing probes for more than 28000 genes. Total RNA (300 ng per


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sample) was labeled using the Affymetrix Gene Chip WT cDNA Synthesis and Amplification kit protocol, and hybridized to the arrays as described by the manufacturer (Affymetrix, Santa Clara, CA, USA). The complementary RNA hybridization cocktail was incubated overnight at 45ºC while rotating in a hybridization oven. After 16 hours of hybridization, the cocktail was removed and the arrays were washed and stained in an Affymetrix Gene Chip fluidics station 450, according to the Affymetrix-recommended protocol. Arrays were scanned on an Affymetrix Gene Chip Scanner 3000 7G.Three different samples per group were examined. Data (.CEL files) were normalized using the robust multiarray algorithm (RMA) [29]. Next, we employed a conservative probe-filtering step excluding probes not reaching a log2

expression value of 5 in at least one sample, which resulted in the selection of a total of 19621 probes from the original set of 28853. To identify genes differentially expressed between the different microarray study groups, we employed Linear Models for Microarray Data R-package [30].

RESULTS

Foxa2 and Gata4 block Nanog upregulation during reprogramming. We sought to investigate the role of endodermal transcription factors in the reprogramming of somatic cells. We selected three transcription factors essential for proper differentiation of the definitive endoderm, Foxa2, Gata4 and Hhex [12-14]. MEF were subjected to a reprogramming protocol by introducing these endoderm-specific transcription factors together with the reprogramming factors Oct4, Klf4 and Sox2 in the presence or absence of Myc (OKSM or OKS, respectively) by retroviral transduction (Table S1). The specific time point in which colonies were readily detected in the cultures was different for each condition. In particular, the addition of Foxa2 and Gata4 to OKSM delayed the process significantly. Infection of MEF with the OKS or OKSM combination of retroviruses rendered colonies with a distinct edge, containing cells of typical mouse ES cell morphology with a round shape, large nucleoli

and scant cytoplasm. However, the addition of Foxa2 and Gata4 to the OKS or OKSM mixtures of retroviruses resulted in granular colonies with irregular and non-distinctive borders, composed of small cells resembling fibroblasts rather than ES cells (Figure S1A). When colonies acquired a critical size they were hand-picked and expanded independently for molecular analysis (six colonies per group).

Nanog expression is a hallmark of pluripotency [5, 31]. To assess which clones had achieved full reprogramming, we determined the levels of Nanog mRNA by quantitative RT-PCR (qRT-PCR) after 4 consecutive passages. Clones obtained with the OKSM combination (OKSM-clones) showed the highest levels of Nanog mRNA (Figure 1A). Addition of Hhex alone (H) or in combination with Foxa2 (H+F) to the reprogramming retroviral cocktails rendered clones with either similar or slightly diminished Nanog mRNA expression compared to their OKS- or OKSM-clone counterparts, suggesting that both factors do not severely limit fibroblast reprogramming. Remarkably, addition of Foxa2 and Gata4 (F+G) to the OKS or OKSM reprogramming cocktails rendered colonies with no expression of Nanog mRNA (Fig 1A), suggesting that these two factors impair the reprogramming of MEF. Nonetheless, the absence of NanogmRNA could also be the consequence of the differentiation of early-stage Nanog-positive cells into Nanog-negative cells. To explore this possibility, we performed a time-course analysis of Nanog mRNA expression during the first four weeks after the initial transduction of the cells with the retroviral mixtures (Figure 1B). In the cultures infected with OKSM, Nanog mRNA levels became detectable by day 7 (inset in Figure 1B) and increased steadily until day 28. However, Nanog expression levels remained below the detection limit in MEF infected with OKSM+F+G at all time-points. These results suggest that the addition of Foxa2 in combination with Gata4 impairs reprogramming and the expression of Nanog in MEF subjected to OKS/M transduction.


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Foxa2 and Gata4 do not reprogram MEF into endoderm-like lineages. ES cell differentiation is accompanied by Nanog downregulation [24]. Given the pivotal role of Foxa2 and Gata4 in embryonic endoderm formation [12, 13, 32-34], we speculated that isolated clones from OKS/M+F+G were in fact endodermal progenitor cells or cells committed towards an embryonic endodermal lineage, i.e. definitive, visceral, parietal, extraembryonic or primitive. To test this hypothesis, we performed Affymetrix gene expression profiling of the isolated clones obtained after MEF reprogramming in presence of Foxa2 and Gata4, using MEF and mouse ES cells as controls. We compiled the relative expression levels of specific markers for each embryonic layer into a color-coded graph focusing in endoderm-related genes (Figure 1C). The expression levels of Lamb1, Sparc, Sox7, Afpand Ttr were validated by qRT-PCR (Figure S1B). Sox7, Afp and Ttr, which are highly expressed in visceral endoderm, were not detected. Likewise, expression levels of markers for all other endodermal cell types indicates that the cell clones obtained with the OKS+F+G combination are not committed to an endodermal fate.

Next, the pluripotent state of the OKS+F+G-clones was investigated using global gene profiling. To do so, we extended our gene profile analysis to clones isolated from OKSM infection that showed very low or no expression of Nanog by qRT-PCR. We used hierarchical cluster analysis to sort the different mRNA samples based on expression of 19621probes expressed above background level (Figure S2). OKS+F+G-clones clustered together with Nanog-negative OKSM-clones, indicating that their expression patterns are more similar than those of non-reprogrammed cell types (MEF and mouse ES cells).

To get a clearer picture of the pluripotent status of our OKS+F+G-clones, we performed an additional analysis of the microarray data based on a selected battery of genes. In this regard, Mikkelsen et al. dissected the transition from MEF to iPS cells by an integrative genomic approach [35]. They found that stable

partially reprogrammed cell lines show the reactivation of a distinctive subset of stem-cell-related genes and an incomplete repression of lineage-specifying transcription factors, suggesting that some cells may become trapped in partially reprogrammed states due to incomplete repression or activation of specific transcription factors. In agreement with these findings, the analysis of the gene expression profile of the Nanog-negative OKSM-clones showed their identity as partially reprogrammed iPS (pr-iPS) cells (Figure 2A). Conversely, OKS+F+G-clones did not downregulate structural genes such as Col6a2 or growth arrest mediators such as Gas1. In addition, upregulation of genes directly linked to early pluripotency such as Zic3 and Fgf4, did not take place in the OKS+F+G-clones while the upregulation of several pluripotency markers such as Utf1, Gdf3 or Nodal, was very limited. Our results show that although the OKS+F+G-clones displayed the expression of several pluripotency markers at low levels, they are not pr-iPS cells. A new hierarchical analysis based on this subset of genes, clustered the five OKS+F+G-clones together and at a lower relative distance to MEF than to the Nanog-negative OKSM clones, indicating that their expression patterns are similar to non-reprogrammed MEF (Figure 2B).

Pluripotency of reprogrammed cells can be definitively assessed by culturing the iPS cell-like clones in a selective serum-free medium containing inhibitors of the MAPK and GSK3 kinases in the presence of LIF (2i+LIF [26]). In this medium, partially reprogrammed cells will be promoted to full pluripotency while null-pluripotent cells will eventually die [10]. Nanog-negative OKSM- and OKS-clones grew in the presence of 2i+LIF (Figure 2C), acquired a typical ES cell morphology, expressed pluripotency markers such as Lin28, Nanog,Oct4 or Prdm14, and formed uniform spheres when induced to differentiate as embryoid bodies, all characteristics of fully pluripotent cells. OKS/M+F+G-clones did not survive in this selective media. In conclusion, addition of Foxa2 in combination with Gata4 to the OKS/M reprogramming cocktail results in the formation of proliferative clones that show low expression of pluripotency genes and are


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different from the previously described pr-iPS cells.

Gata4 but not Foxa2 expression abolishes Nanog induction and clonal expansion during reprogramming. Reprogramming blockade by Foxa2 and Gata4 is specific, since the addition of Hhex alone or in combination with Foxa2 did not affect the expression of Nanog induced by infection of MEF with the OKS/M mixture of viral supernatants (Figure 1A). To identify the transcription factor responsible for the impairment of Nanog induction in the reprogramming assays, we infected MEF with OKSM alone or together with Foxa2 (OKSM+F) or Gata4 (OKSM+G) and assessed the induction of pluripotency by analyzing Nanog expression. Nanog mRNA was induced in cells transduced with OKSM or OKSM+F, while no expression of this pluripotency marker was observed with the addition of Gata4 (Figure 3A). In agreement with the observed Nanog expression profile, the addition of Foxa2 to the reprogramming cocktail induced the formation of cell aggregates that resembled ES cell colonies and displayed expression of pluripotency markers after extensive culture (n = 6) (data not shown). Cell colonies obtained upon transduction of MEF with the OKSM+G showed a fibroblast-like morphology and could not be isolated or further expanded in culture (n=20). Moreover, transduction of MEF derived from the Tg (Nanog-GFP:Puro) mice [25] with OKSM+F activated the Nanog-GFP transgene whereas reporter activation was not observed in the presence of Gata4 in the retroviral mixture (Figure 3B). To evaluate pluripotency, we cultured MEF infected with the OKSM+G or OKSM+F retroviral mixtures in 2i+LIF medium and assessed their morphology for 10 days after switching to the selective media. Cells infected with OKSM+G died within 7 days (Figure 3C). In sharp contrast, 2i+LIF medium favored the growth of multiple colonies in the cells infected with OKSM+F.

Our results suggest that Gata4 is a negative regulator of somatic cell reprogramming. In agreement with this hypothesis it has been

described that Gata4 expression is induced at the initial stages of reprogramming and maintained until the last stages of reprogramming. In addition, iPS cells have been shown to express Gata4 mRNA at lower but detectable levels [2]. Therefore we reasoned that downregulation of Gata4 shouldincrease the efficiency of cell reprogramming. To test this, we generated lentiviral vectors expressing short hairpin RNAs specific for mouse Gata4 and validated their ability to downregulate Gata4 expression in 3T3 cells (Figure S3). All four lentiviral constructs reduced the amount of Gata4 protein, and the shRNA4 construct was selected for subsequent experiments due to its specificity in downregulating the expression of this Gata protein in comparison with a negative control shRNA against Luciferase (shLuc). We next assayed the effect of Gata4 downregulation on the efficiency of reprogramming induced by infection of MEF with OKSM using the induction of Alkaline Phosphatase activity and Nanog expression as read outs (Figure 4). In agreement with our previous results, forced expression of Gata4 greatly impaired the appearance of Alkaline Phosphatase-positive colonies (Figure 4A) and Nanog mRNA expression. Compared to the shLuc negative control, downregulation of Gata4 expression during MEF reprogramming (shGata4) accelerated the appearance of colonies during reprogramming. The ratio of colonies for control (shLuc) and shGata4 was 1:3 at day 8 respectively (Figure 4A) and yielded clones with a statistically significant increase in Nanog mRNA (Figure 4B). Altogether, our results suggest that Gata4 prevents Nanog upregulation and impairs MEF reprogramming.

Gata4 and Gata6 play semi-redundant roles in liver and cardiac development partially by regulating the expression of an overlapping set of genes [36-38]. To test whether Gata6 could also limit cell reprogramming, we coexpressed Gata6 with the OKSM reprogramming factors in MEF and assessed the formation of iPS cell-like colonies by both morphology and an Alkaline Phosphatase assay. Gata6 induced the formation of cell-aggregates lacking ES cell morphology which did not express Alkaline Phosphatase (Figure S4), suggesting that both


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Gata factors are negative regulators of somatic cell reprogramming.

Gata4 triggers a selective blockade of reprogramming cornerstones. We reasoned that inclusion of Gata4 in the reprogramming cocktail could induce MEF differentiation into embryonic endoderm. As previously seen with the reprogramming of MEF in the presence of Foxa2 and Gata4 (Figure 1C and S1B), we did not detect expression of critical markers of visceral and parietal endoderm such as Ttr, Sox7 and Afpand the expression of the endodermal markers Lamb1 and Sparc remained below that observed in MEF (Figure S1C). These results indicate that in the presence of Gata4, MEF do not differentiate towards endoderm. On another hand, the expression of Gata4 could limit the G1-S transition during reprogramming and hence proliferation [39]. To investigate this possibility, we performed a BrdU incorporation assay in MEF control and infected with Gata4. We did not observe an effect on BrdU incorporation upon Gata4 expression (Figure S5), excluding a possible G1-S blockade.

Previous reports have characterized the main cornerstones of MEF reprogramming before Nanog is upregulated [40]. Such cornerstones include retroviral silencing, overcoming senescence and initiation of MET. To monitor retroviral silencing, we included a retrovirus expressing GFP in the initial reprogramming cocktail and checked the expression of both GFP and the exogenous factors Oct4, Klf4 and Myc used in the reprogramming at several time points after the viral transduction of MEF. Compared with the OKSM control, the inclusion of Gata4 failed to silence the retroviral GFP reporter (Figure 5A). Also, cultures infected with OKSM+G did not switch off the expression of the exogenous Oct4, Klf4and Myc retroviral constructs after 31 days (Figure 5B). In agreement with the observed failure in retroviral silencing, endogenous pluripotency markers such Oct4, Nanog orLin28 were not induced (Figure 5C). Senescence has been considered an initial barrier that should be overcome to initiate reprogramming [3], therefore we assessed whether Gata4 interfered with this process.

Upon infection, we observed a Gata4-independent upregulation of p16Ink4a and p19Arf41 mRNA between day 6 and 8 (Figure S6). However, we found that the mRNA levels of p16Ink4a were significantly higher at day 17 in the OKSM+G-infected MEF, when reprogramming is already entering its maturation phase. MET is also considered a key event for the initiation of reprogramming [1, 2]. We analyzed the mRNA levels of the epithelial markers Cdh1 and Ocln1 and the mesenchymal markers Snai1 and Zeb1 in the cultures subjected to a reprogramming assay in the absence (OKSM) or presence of Gata4 (OKSM+G). Upregulation of Cdh1 and Ocln1in parallel with downregulation of Snai1 and Zeb1 confirms MET initiation in OKSM and OKSM+G cultured MEF (Figure S7). In summary, Gata4 blocks somatic cell reprogramming without significantly interfering with some of the initial molecular cornerstones of reprogramming such as G1-S transition, senescence or MET.

To gain insight into the mechanism of impairment of cell reprogramming by Gata4, we investigated whether Gata4 was also effective when introduced during the maturation phase of reprogramming. OKSM-infected MEF were split as described in detail elsewhere (Figure S8A). Reinfection efficiency was assessed 48 hours later by measuring expression of exogenous Gata4 (Gata4-exo) and GFP/RFP by qRT-PCR and fluorescence microscopy, respectively (Figure S8B). We could not detect expression of Nanog twenty-one days after reinfection (a total of 28 days after the initial transduction of MEF with OKSM) when Nanog mRNA levels are readily detected in control plates (Figure 5D). We conclude that interference of Gata4 with cell reprogramming is effective when introduced at the maturation stage of the process, at approximately day 8 of reprogramming [2].

Rescue of the Gata4-imposed blockade of reprogramming by Nanog. The results shown above suggest that Gata4 exerts a direct effect over an essential step of the maturation phase of reprogramming, not initiation. Acquisition of pluripotency during reprogramming can be monitored by


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measuring the upregulation of pluripotency- and not pluripotency-related genes such as Syne2, Perp, Slc38a5 or Gdf3 [35]. mRNA levels of Syne2, Perp, Slc38a5 and Gdf3 were initially upregulated in OKSM and OKSM+G treated MEF (Figure S9 and 6C lower-left). However, OKSM+G-infected MEF failed to downregulate Slc38a5 or upregulate Gdf3 eight days after the viral transduction of the cultures, as observed in OKSM-infected cultures and in bona fide iPS cells [2]. These results support the notion that Gata4 impairs reprogramming by targeting the maturation stage.

Nanog is initially dispensable during reprogramming but becomes essential for dedifferentiated intermediates to transit to a stable ground state of pluripotency [5, 31].We hypothesized that regulation of Nanog gene expression was the target for the inhibitory effect of Gata4 in the reprogramming of somatic cells. We asked whether exogenous expression of Nanog could reverse the blockade to reprogramming induced by Gata4 expression. To test this, we first infected MEF with the OKSM+G combination in the absence or presence (OKSM+G+N) of Nanog-expressing viruses and monitored colony formation ability and upregulation of pluripotency markers (Figure 6). Initial exogenous Nanog (Nanog-exo) mRNA levels were comparable to that found in undifferentiated ES cells (Figure 6A). Transduction of MEF with the OKSM+G mixture rendered no colonies (Figure 6B, upper panels) and did not upregulate the expression of pluripotency markers (Figure 6C). Remarkably, addition of Nanog to the OKSM+G retroviral cocktail yielded iPS cell-like colonies within 10 days that were able to form embryoid bodies when induced to differentiate as cell aggregates (Figure 6B, lower panels). Moreover, OSKM+G+N-infected MEF upregulated the expression of the pluripotency markers Oct4, Lin28 and Gdf3,and enabled transcriptional activation of the endogenous Nanog locus (Figure 6C). Consistent with Nanog being negatively regulated by Gata4, Nanog expression rescued OKSM+G infected fibroblasts.

Gata4 represses Nanog transcription by binding to a distal enhancer We searched for Evolutionary Conserved Regions (ECR) within the mouse Nanog gene and identified 4 ECRs located upstream of the transcription start site (Figure S10A). ECRs I to III are conserved in mouse, human and monkey. ECR I (97 bp in length) contains a composite Oct-Sox cis-regulatory element essential for Nanog expression in pluripotent cells [20]. In silico analysis of the ECR II (180bp) and III (635bp) regions revealed a scarcity of known transcription binding sites. ECR IV (732bp), located approximately 9000 bp upstream of the transcription start site is conserved among all species included in the analysis. Analysis of the ECR IV region using the rVista2.0 software revealed the presence of 66 transcription factor binding motifs, suggesting a possible role as a distal enhancer. Among these binding motifs, we identified a consensus binding site for Gata factors, core sequence (A/T)GATA(A/G), located at -9180bp from the transcription start site, within the longest uninterrupted homologous sequence of 14bp (Figure S10B).

We investigated whether Gata4 binds to this region of the Nanog gene and regulates its transcription by using luciferase and electrophoretic mobility shift (EMSA) assays. We constructed a luciferase reporter plasmid containing the complete sequence of the distal enhancer ECR IV upstream the Nanog proximal promoter (ECR I). The chimeric Nanog promoter/enhancer was active in mouse ES cells and its activity decreased in a dose-dependent manner to 50% when cotransfected with an episomal vector expressing Gata4 (pPYCAG-HAGata4) (Figure 7A).

In vitro binding of Gata4 to the ECR IV region was assessed using a 33bp probe spanning the Gata4 binding site in the Nanog putative enhancer by an EMSA assay using nuclear extracts from control-transfected or Gata4-transfected ES cells (Figure 7B). Two complexes could be detected: an upper complex (more retarded), which is more intense in Gata4-expressing ES cells and an unspecific lower complex (less retarded) that was equally present in control and Gata4-


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expressing ES cells. The intensity of the slower migrating complex was reduced to control levels when incubated with the unlabeled probe (lane 3), but not with an unlabeled mutated probe, where Gata4 binding site TGATAG was changed to GCGCGC (lane 4). To gain insight into the nature of the weaker upper complex present in control ES cells (lane 1), we conducted similar competition experiments using nuclear extracts from control-transfected ES cells (lanes 6-9).The upper complex disappeared when the extracts were incubated with unlabelled probe (lane 7) or with an anti-Gata4 antibody (lane 9), suggesting that it might correspond to the binding of an endogenous Gata factor expressed in ES cells. Finally, a probe designed in the proximal promoter of the atrial natriuretic factor was used as a positive control for Gata4 binding (lanes 10-12 [42]). Our in silico analysis revealed the presence of a novel and evolutionary conserved enhancer in the Nanog gene located 9 kbps upstream from the transcription start site. Also, our in vitro results indicate that this distal enhancer contains a functional Gata binding site and that binding of Gata4 to this sequence negatively regulates gene expression.

Finally we evaluated whether Gata4 could bind to the identified Nanog distal enhancer in intact cells. We carried out chromatin immunoprecipitation assays in non transfected and HA-Gata4 transfected ES cells. First we validated the antibodies used for the immunoprecipitation and confirmed their specificity by immunoblotting (Figure S11). Immunoprecipitated DNA fragments were quantitated by qRT-PCR. Three different genomic loci were analyzed, a 149bp region located in ECR III, a 143bp domain containing the Gata4 binding motif in ECR IV and a 269bp stretch located in the Cyp17a1gene as a positive control [43]. We observed an approximate 20-fold and 4-fold enrichment of the ECR IV enhancer in Gata4-expressing ES cell samples precipitated with the anti-Gata4 and anti-HA antibodies respectively, as opposed to the IgG controls (Figure 7C, middle bar diagram). We did not detect amplification of the ECR III control region in the anti-Gata4 or anti-HA precipitates. In agreement with

previously published results [43], immunoprecipitation with anti-HA or anti-Gata4 antibodies resulted in a 28-fold enrichment of the Cyp17a1 promoter region relative to the IgG controls. In conclusion, our data indicate that Gata4 binds to a distal enhancer of the Nanog gene and negatively regulates the expression of this pluripotency gene.

DISCUSSION

In this paper, we have shown that Gata4 blocks OKSM-induced cell reprogramming by repressing Nanog gene expression through its direct binding to a novel distal enhancer (Figure S12). When Gata4 is expressed together with OKSM in MEF, cells become trapped into a metastable cell type that is unable to both activate the robust expression of pluripotent genes such Nanog and Lin28 and form ES cell-like colonies in 2i+LIF medium. Similar results were obtained when the closely related factor Gata6 was expressed in the reprogramming assays instead of Gata4. Gata4 does not interfere with the initial phase of reprogramming but rather with the maturation phase of the process, when Nanog expression becomes essential to reach pluripotency. This phenotype is highly similar to that observed in the reprogramming of Nanog-deficient cells [5] and, in fact, the blockade of cell reprogramming imposed by Gata4 overexpression was rescued by the introduction of exogenous Nanog.

While we have observed that endodermal transcription factors, such as Hhex or Foxa2 may facilitate iPS cell formation (unpublished observations), the expression of Gata4 caused a severe impairment of the reprogramming process, rendering loose colonies that did not upregulate Nanog, a hallmark of full reprogramming [5]. Conversely, knockdown of Gata4 increased the number of early iPS cell colonies and favored Nanog upregulation in MEF transduced with OKS/M (Figure 4). When Foxa2 was coexpressed with Gata4, colonies could be isolated and clonally expanded, but still Nanog mRNA levels remained low (Figure 1A and 2C). This partial rescue of the negative effect of Gata4 in cell


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reprogramming by its coexpression with Foxa2 is not completely unexpected, since it has been suggested that Foxa2 promotes iPS cell generation in cooperation with Glis1 [8].

Reprogramming is a gradual process that can be divided in three stages based on the expression of molecular markers [2]: initiation (0-8 days), maturation (8-16 days) and stabilization phases (16-21 days). The fully reprogrammed phenotype is adopted when senescence is overcame, the cells enter MET, upregulate initial pluripotent genes, silence the retroviral constructs and show a robust expression of Nanog (reviewed in [44]). Gata4 expression did not hinder the initial upregulation of senescence or MET markers such p16Ink4a and p19Arf or Cdh1 and Ocln1,respectively (Figure S6-S7). Similarly, markers of the first stage of reprogramming such Gdf3,Slc38a5, Syne2 or PERP were upregulated in the presence of Gata4. However, the downregulation of Slc38a5 and the maintenance of Gdf3 mRNA levels during the maturation phase of the process were impaired. These data indicate that Gata4 does not block reprogramming at the initiation phase, but it impairs the transition of the cells to the maturation phase. In agreement with this possibility, expression of Gata4 during the maturation stage (eight days after the initial transduction of MEF with the OKSM viral mixture) also resulted in a lack of Nanog expression in the cultures and rendered no iPS cell colonies (Figure 5D).

We therefore speculated that Gata4 directly limited Nanog induction during reprogramming based in the following observations: 1) expression of Gata4 together with the Oct4, Klf4, Sox2 and Myc (OKSM) induced the formation of colonies that resembled those obtained with the reprogramming of Nanog-null Neural Stem cells [5]; 2) the initiation phase of reprogramming was not altered, similarly to the published results obtained when reprogramming assays were carried out using Nanog-defficient MEF [2]; 3) addition of Gata4 after the initiation phase of the reprogramming impaired the generation of iPS cells and Nanog mRNA induction during the

process; 4) knockdown of Gata4 rendered iPS cell clones that expressed significantly higher levels of Nanog mRNA than the reprogrammed control clones; and 5) forced Nanog expression rescued the failure to obtain iPS cells in the presence of Gata4 (Figure 6).

In silico search for Gata4 binding sites in evolutionary conserved regions present in the Nanog gene led us to the identification of a Gata binding site located in a novel distal enhancer in this gene, ECR IV. Using EMSA and ChIP assays we found that Gata4 was able to bind in vitro in intact cells to this site. In addition, we found that Gata4 inhibited the expression of a luciferase reporter driven by the distal enhancer ECR IV, suggesting that the upregulation of the Nanog gene during cell reprogramming in the presence of Gata4 is inhibited by the binding of this transcription factor to the ECR IV enhancer. Then, the negative regulation of the Nanog gene by Gata4 will disable the molecular mechanisms necessary to acquire induced pluripotency. Interestingly, EMSA assays suggested that an endogenous Gata4 [45, 46] is bound to this site (Figure 7C, lanes 6-9). The physiological significance of the binding of Gata factors to the distal enhancer in the Nanog gene is currently unknown, but given the roles of Gata4 as a pioneering factor in different cell types [47-49], we hypothesize that binding of Gata4 to the ECR IV enhancer might contribute to the differentiation of the inner cell mass into extraembryonic endoderm when other factors or increased Gata4 levels, are into play.

Exogenous expression of Oct4, Klf4, Sox2 and Myc are necessary and sufficient for triggering the initial steps of reprogramming causing global gene expression changes that will eventually lead to pluripotency, successfully achieved by the action of a Nanog-dependent transcriptional network [31]. By introducing Gata4 together with OKSM (OKSM+G) or eight days after OKSM infection, Nanogexpression was not induced and cell reprogramming was impaired. OKSM+G-infected cells could not grow and expand in 2i+LIF medium and lacked the ability to form embryoid bodies during aggregation-induced


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differentiation and they cannot therefore be considered pr-iPS cells. Our findings indicate that the lack of reprogramming observed by forced expression of Gata4 together with the four reprogramming factors was not the final result, but rather the consequence of the repression of the Nanog gene caused by the direct binding of Gata4 to the novel distal enhancer ECR IV that we have identified in this study. Given the important role of Gata4 and Nanog in cell differentiation, the results presented here have broad implications for deciphering the inter-regulatory networks controlling cell fate during the segregation of the primitive endoderm and the epiblast from the inner cell mass during early embryogenesis.

ACKNOWLEDGMENTS

We thank Cristina Corchero for assistance in qRT-PCR, Eva Serna and Juan José Lozano for microarray analysis. We are indebted to Dr Lisa Sevilla for critical reading of the manuscript. This study was supported by the Ministry of Science and Innovation, grants SAF2010-15376 and SAF2011-29718 to RB and the Instituto de Salud Carlos III, grant PS09/00248 to JT. CIBEREHD (CIBER de Enfermedades Hepaticas y Digestivas) is funded by the Instituto de Salud Carlos III, Spain.

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Figure 1. Gata4 and Foxa2 block Nanog upregulation during reprogramming without alternative cell commitment. (A) MEF were subjected to a reprogramming protocol using the indicated combinations of transcription factors (Table S1). Nanog mRNA levels were quantified by qRT-PCR in three clones isolated from each group. (B) MEF were infected with the indicated combination of transcription factors and the levels of Nanog mRNA measured at specific time points by qRT-PCR. Data are represented as the average ± SD of three replicates relative to the Nanog mRNA levels found in clon #1 from OKSM in (A) and in OKSM after 28 days in (B). (C) Heat map depicting relative expression levels of selected mRNAs across mouse embryonic fibroblast (MEF), mouse ES cells (mESC) and five clones isolated from MEF reprogrammed with OKS in the presence of Foxa2- and Gata4-expressing retroviruses. Genes were selected based on their expression profiles in specific embryonic tissue layers. Black: not expressed; red: higher expression; green: lower expression. Microarrays data have been deposited in NCBI's Gene Expression Omnibus [50]and are accessible through GEO Series accession number GSE37548 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37548).


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Figure 2. MEF reprogrammed in the presence of Gata4 and Foxa2 are not partially reprogrammed iPS cells. (A) Heat map depicting relative expression levels of selected mRNAs across MEF, mouse ES cells (mESC), Nanog-negative clones from OKSM-reprogrammed MEF and five clones isolated from MEF reprogrammed with OKS in the presence of Foxa2- and Gata4-expressing viruses (OKS+F+G). Genes were selected based on a previous report [35]. (B) Dendogram plot based on Euclidean distance and average linkage showing hierarchical clustering or closeness in expressed mRNAs shown in (A) (52 probes). (C) Phase contrast images of a representative clone isolated from MEF reprogrammed with the indicated combination of transcription factors growing for 5 and 10 days in 2i+LIF medium (original magnification x10) and induced to differentiate as embryoid bodies (EB) (original magnification x20).


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Figure 3. Foxa2 significantly increases Nanog mRNA levels during reprogramming. (A) MEF were infected with OKSM together with Foxa2- (OKSM+F) or Gata4-expressing viruses (OKSM+G) and Nanog mRNA levels were analyzed at the specified time points by qRT-PCR. Data are represented as the average ± SD of three replicates relative to the Nanog mRNA levels found in OKSM at day 31 of the procedure. (B) MEF from Tg(Nanog-GFP,Puro) transgenic reporter mice [25] were infected with OKSM+F or OKSM+G. Phase-contrast (left panels) and fluorescence (right panels) images of representative colonies are shown (original magnification x10). No GFP-positive colonies were observed upon OKSM+G infection of Nanog-GFP:puro MEF. (C) Phase-contrast images of a representative clone isolated from MEF reprogrammed with OKSM+F or OKSM+G grown for the indicated time points in 2i+LIF selective medium (original magnification x10).


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Figure 4. Downregulation of Gata4 favors cell reprogramming. (A) MEF were infected with the indicated combination of transcription factors and lentiviral vectors expressing shRNAs for Luciferase (shLuc) or Gata4 (shGata4) and stained for Alkaline Phosphatase activity. Photographs show an increase in the number of colonies positive for Alkaline Phosphatase staining in plates that included lentiviruses expressing shGata4. (B) Bar diagram showing the quantitation of Nanog mRNA levels in isolated clones (3 from each condition) from MEF transduced as indicated in (A). Downregulation of Gata4 during reprogramming significantly increased Nanog expression. Data are represented as the average ± SD of three replicates relative to the Nanog mRNA levels found in in OKSM-derived clones.


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Figure 5. Gata4 blocks somatic cell reprogramming. (A) MEF were infected with OKSM in the absence or in the presence of Gata4-expressing virus (OKSM+G) and a retrovirus expressing GFP to monitor retroviral silencing. Representative fluorescence (GFP) and phase-contrast (PhC) images of the cultures at the indicated time points are shown (original magnification x10). Retroviral silencing (GFP-negative) in the OKSM-infected cultures is already visible in some colonies at day 8 (red arrowhead). No GFP-negative colonies were observed in OKSM+G-infected MEF. (B) Bar diagrams showing transcript levels of the indicated exogenous reprogramming factors in MEF reprogrammed in the absence (OKSM) or presence (OKSM+G) of Gata4-expressing virus. mRNA levels in MEF infected with OKSM and cultured for 5 days were set to 1. (C) Bar diagrams displaying the transcript levels of the indicated endogenous pluripotency genes by qRT-PCR in MEF reprogrammed as in (B). (D) Bar diagrams showing the analysis by qRT-PCR of Nanog mRNA levels in MEF reprogrammed with OKSM and reinfected with GFP- or Gata4-expressing viruses as specified in the text. Data are represented as the average ± SD of three replicates. Values are relative to the transcript levels of the indicated exogenous cDNAs found in MEF infected with OKSM and cultured for 5 days in (B), the specified endogenous genes in iPS cells in (C) and the Nanog gene in MEF infected with OKSM and cultured for 31 days in (D).


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Figure 6. Gata4 inhibition of cell reprogramming is rescued by forced expression of Nanog. (A) Bar diagram showing the time-course expression of exogenous Nanog mRNA in MEF infected with OKSM together with Gata4- and Nanog-expressing viruses (OKSM+G+N). (B) Phase-contrast images of representative clones isolated from cells transduced with the indicated combination of viruses and grown for the indicated time points in 2i+LIF medium and induced to differentiate as embryoid bodies (EB, right most panels) (original magnification x10). (C) Bar diagram showing the qRT-PCR analysis of the mRNA levels of the indicated endogenous pluripotency genes in Nanog-rescued OKSM+G cells. Data are represented as the average ± SD of three replicates relative to the transcript levels of Nanog found in undifferentiated ES cells in (A) and the specified endogenous genes found in iPS cells (C).


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Figure 7. Gata4 binds to a novel distal enhancer in the Nanog gene and represses its transcription. (A) Luciferase assays in undifferentiated ES cells transfected with the pGL3-Nanog-enh/prom reporter and empty vector or pPYCAG-HAGata4. Data are represented as the means ± s.e.m. of the firefly/renilla ratios of 3 experiments conducted in duplicate relative to cells transfected with the empty vector. Upper diagram, representative immunoblot analysis of the expression of Gata4 in cell extracts used for the luciferase experiments. (B) Electrophoretic mobility shift assay (EMSA) using nuclear extracts from ES cells transfected with pPYCAG (Cont) or pPYCAG-HAGata4 (Gata4). Lanes 1-5 show the binding of Gata4 to the Nanog enhancer identified in this study. Lanes 6-9 display the weak binding of an endogenous Gata factor to the same enhancer sequence. Lanes 10-12 show the binding of Gata4 to an oligo based on the proximal promoter of the atrial natriuretic factor included as a positive control. A representative experiment is shown. (C) Bar diagrams of ChIP assays showing the analysis by qRT-PCR of the binding of Gata4 to the indicated DNA regions using the specified antibodies. ECR III and Cyp17a1 promoter were used as negative and positive control, respectively. Data are represented as the average of 4 independent experiments conducted in duplicate. (D) A representative immunoblot to detect Gata4 expression in nuclear extracts and cytoplasmic extracts from the samples used in EMSA and ChIP experiments. Tubulin is only detected in cytoplasmic extracts. Coomassie blue staining of the membrane is shown below.

gata4 blocks somatic cell reprogramming by directly repressing nanog

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