Embryonic stem cells are defined by two key characteristics; apparently symmetrical self-renewing cell division and the ability to differentiate into cells of all three germ layers. Self-renewal depends on several extrinsic and intrinsic cues including a gene regulatory network centered around Oct4, Sox2 and Nanog that has been hypothesized to be reinforced by positive reciprocal interactions. Studies measuring Nanog expression by fluorescent reporters and immunoflourescence have shown that some undifferentiated Oct4 positive cells do not express Nanog (Chambers et al., 2007). However, the mechanisms responsible for generating this heterogeneity in Nanog expression are unknown. Here I show that Oct4 heterozygote ES cells lack Nanog-negative cells. Consistent with a model in which ES cell differentiation proceeds effectively through Nanog-negative cells, these Oct4 heterozygotes are retarded in their differentiation kinetics. Importantly, restoring Oct4 levels towards wild type reestablished both heterogenous Nanog expression and rapid differentiation. Analysis of ES cells carrying a mutation in the Oct4 binding site in the proximal Nanog promoter showed that Oct4 acts as a positive activator on the endogenous Nanog. Finally, comparison of gene expression in Nanog expressing and Nanog non-expressing ES cells has identified candidate genes that may be responsible for the switch in Nanog expression.

Prostate cancer is one of the most common cancers affecting one of every six men in United States. It is increasingly appreciated that tumor or cancer stem cells are the cells responsible for initiating tumor formation and therefore should be targeted for eradication in cancer treatment. But the mechanism involved in the acquisition of unlimited self-renewal and tumor initiation by cancer stem cells is unknown. Nanog, along with Oct3/4 and Sox-2, constitute the core transcriptional circuitry for the maintenance of stemness in embryonic stem cells. Herein we report that Nanog expression was detected at mRNA and protein levels in prostate cancer cells. The Nanog-expressing LNCaP-T and DU145 cells were enriched by infection with lentiviruses expressing GFP under the control of Nanog promoter. The Nanog-enriched prostate cancer cells had stronger expressions of stem and progenitor cell surface markers, including CD44 and CD133, when compared with those in the control group. Colony formation assay found that the Nanog-enriched LNCaP-T and DU145 cells formed more holoclones and prosta-spheres, which contained more self-renewing cells, than the control cells did. On the other hand, knockdown of Nanog in DU145 or LNCaP-T cells, via shRNAs, reduced their ability to form holoclones. Instead, most clones derived were meroclone and paraclones as result of increased differentiation and senescence due to knockdown of Nanog. When injected into mice, Nanog-enriched DU145 cells were found to possess increased tumorigenic potentials when compared to the vector controls. On the other hand, LNCaP-T cells with Nanog knocked down did not form tumors, while the vector controls readily formed tumors. Taken together, our data suggest an essential role for Nanog in the self-renewal and tumor initiation of prostate cancer cells. Chemotherapy is the major salvage therapeutic modality available for the patients with advanced cancers. However, drug resistance by some prostate cancer cells is a major barrier to efficacious chemotherapy. It has been increasingly appreciated that cancer stem cells are responsible for resistance to chemo- or radio-therapy, in addition to tumor initiation. However, the mechanisms involved remain unknown. In this study, we examined whether Nanog plays an essential role of Nanog in resistance to chemotherapy. In the surviving fractions of prostate cancer cells, we found increased levels of Nanog protein when compared to the cells treated with solvent control. To determine the role of Nanog in resistance of prostate cancer cells, we marked and enriched Nanog-expressing prostate cancer DU145 and LNCaP-T cells using a reporter gene under control of 2.5 kb hNanog1 promoter. When compared to the control, the prospectively enriched Nanog-expressing cells presented increased resistance to Taxol, vinblastine, and doxorubicin. Profiling of genes in drug resistance and metabolism revealed a marked increase in the mRNA level of ATP-binding cassette (ABC) efflux transporters B1 and G2 in tumor cells enriched with endogenous Nanog expression. The increased expression of ABCB1 and ABCG2 at protein levels in Nanog expressing cells was confirmed by Western blot and immunocytochemistry. Inhibition of ABCB1 activities sensitized Nanog expressing cells toward Taxol and vinblastine, and to less extent, doxorubicin. Blocking of ABCG2 activity sensitized Nanog expressing cells toward doxorubicin, but not Taxol and vinblastine. In addition, the tumor cells enriched with Nanog expression showed reduced apoptosis in response to Taxol treatment. Interestingly, Nanog-enriched prostate carcinoma cells displayed aberrantly activated â-catenin signaling, which is potentially associated with their increased chemo-resistant ability as well as the increased acquisition of epithelial to mesenchymal transition. In summary, Nanog is expressed in prostate cancer cells, especially in those positive for stem/progenitor markers. Enrichment of Nanog expressing cells led to enrichment of tumor cells with increased tumor initiating ability and increased resistance toward chemotherapy. Knockdown of Nanog reduces tumor initiating ability of prostate cancer cells and further sensitizes them toward chemotherapy. The gain-of-function and loss-of-function studies suggest an essential role of Nanog for prostate cancer cells to initiate tumor formation and resist chemotherapy.

The rat has been an established experimental animal model within many areas of biological investigation for over one hundred years due to its size, breeding characteristics, and knowledge of its physiology and behaviour. In recent years its status as a leading biomedical model has been somewhat surpassed by the mouse. This is largely the result of the isolation and application of mouse embryonic stem (ES) cells. Mouse ES cells have the capacity for unlimited self-renew in vitro whilst maintaining pluripotency and germline competence, and most importantly are amenable to sophisticated reverse genetics strategies such as gene targeting, which have provided a route to germ line modification. Thus far, the derivation of rat ES cells has proved elusive. The generation of rat ES cells would therefore facilitate equivalent applications to rat genetics and significantly strengthen the rat as an experimental model system. Previous attempts to derive rat ES cells led to the isolation of rat ES-like cells. However, whilst these cells exhibit extensive self-renew in vitro, it was known that they fail to maintain significant levels of the key functional ES cell marker Oct4 and do not contribute to chimeras. Rather, these cells express the trophectoderm markers Cdx2 and CyclinD3, and have been termed ExS cells due to their probable extra-embryonic nature. In the work described in this thesis, further investigation of ExS cells revealed the absence of expression of the key pluripotency gene Nanog, although the expression pattern of Nanog in the rat embryo was shown to be similar to that of mouse. It was hypothesised that expression of exogenous Oct4 and Nanog or Sox2 genes could facilitate reprogramming of ExS cells into a 'true' ES cell state. Initial work described in this thesis demonstrated that it was possible to introduce transgenes into rat ExS cells and obtain stable transformants with long term transgene expression. On this basis Oct 4, Nanog and Sox2 transgene expression vectors were constructed and stably integrated into ExS cells, and transgene expression verified. However, no reactivation of an endogenous gene expression profile, characteristic of a true ES cell-like state, was observed in any of the transgenic lines produced. Concurrent with work on ExS cells, investigations by others using chemically defined, serum-free medium containing small molecule inhibitors of MEK and GSK3 (called 3i/2i medium) had demonstrated that it was possible to readily isolate mouse ES cells, even from strains known to be refractory to ES cell isolation. Therefore, the ability of this culture system to facilitate rat ES cell derivation was investigated. Rat 3i/2i cell lines were established from ICM outgrowths of Fischer, DA and Sprague Dawley E4.5 rat embryos. These cells maintained expression of Oct4 and Nanog and could generate complex teratomas consisting of all three germ layers. They were distinct from epiblast stem cells (EpiSC) in that they expressed Klf4, Rex1 and Stella and most importantly, they could contribute to the formation of adult chimaeras and demonstrated germline competency. Isolation of these authentic rat ES cells paves the way for gene targeting in the rat, a development that should greatly facilitate new biomedical discoveries.

A 3.5 jours de développement (J3.5), l'embryon de souris est constitué d'un épithélium externe, le trophectoderme, et d'une masse cellulaire interne (MCI). La MCI est hétérogène, constituée des précurseurs de l'épiblaste (Epi) et de l'endoderme primitif (EPr), représentée par l'expression exclusive de Nanog et de Gata6 respectivement. Lors de l'implantation à E4.5, l'EPr forme un épithélium à la surface de la MCI, en regard de la cavité blastocoelique. L'Epi donnera tous les tissus du nouveau-né. L'EPr permet les premiers échanges nutritionnels entre l'embryon et la mère. Je m'intéresse au rôle de Nanog et de Gata6 dans la détermination et la différenciation de l'Epi et de l'EPr. De plus, je m'intéresse à l'implication de la signalisation RTK dans l'expression de ces deux gènes. Enfin, je cherche à comprendre les interrelations entre Gata6 et Nanog. A l'aide des modèles de souris KO, des modèles in vitro ainsi que des techniques innovantes développées au sein du laboratoire, nous avons mis en évidence que la modulation de l'expression de Nanog, Gata6, Fgf4 et Fgfr2 semble suffisante pour l'engagement des cellules vers un devenir Epi ou EPr. De plus, ces résultats permettent de proposer un nouveau modèle expliquant le rôle de Gata6 et de Nanog dans la spécification des cellules Epi et EPr. / At 3.5 days of development (E3.5), the mouse embryo consists of an outer epithelium, the trophectoderm, and an inner cell mass (ICM). The ICM is heterogeneous, composed of the precursors of the epiblast (Epi) and the primitive endoderm (PrE), expressing either Nanog or Gata6 respectively. Upon implantation at E4.5 the EPr forms an epithelium on the surface of the ICM, facing the blastocoelic cavity. The Epi give rise all tissues of the newborn. The PrE allows the first nutritional exchanges between the embryo and the mother. I focus on the role of Nanog and Gata6 in the determination and differentiation of Epi and PrE. In addition I am interested in the involvement of RTK signaling in the expression of both genes. Finally, I seek to understand the relationships between Gata6 and Nanog. Using the transgenic mouse models, in vitro models as well as innovative techniques developed in the laboratory, we have demonstrated that modulating the expression of Nanog, Gata6, FGF4 and FGFR2 seems sufficient for commitment of cells to become an Epi or EPr. Furthermore, these results allow proposing a new model explaining the role of Gata6 and Nanog in the determination and differentiation of Epi and PrE cells.

Células-tronco embrionárias (CTE) são importantes para estudos de desenvolvimento embrionário, diferenciação e manipulação genética. Além disso, essas células podem ser utilizadas na terapia celular e organogênese in vitro. Na pesquisa sobre terapia celular a partir de CTE oriundas de embriões humanos, considerações éticas, morais e religiosas têm sido feitas por pesquisadores e leigos. Portanto, um modelo animal como o suíno (Sus scrofa) será bastante válido por transpor tais barreiras, visto que o suíno possui parâmetros fisiológicos semelhantes aos humanos. Apesar do alto potencial biomédico das CTE, existem dificuldades na manutenção da pluripotência in vitro dessas células em suínos. Portanto, estudos que visam elucidar os mecanismos de manutenção da pluripotência de CTE in vitro são necessários para viabilizar o cultivo dessas células. Os objetivos do presente estudo foram (1) isolar células-tronco embrionárias suínas a partir de blastocistos produzidos in vitro e in vivo; (2) comparar dois sistemas de cultivo in vitro das massas celulares internas (MCI) isoladas, MEF ou Matrigel e (3) identificar e comparar a expressão dos fatores de transcrição Nanog, Sox2 e FoxD3 em CTE e blastocistos suínos produzidos in vitro e in vivo. Assim, blastocistos suínos foram produzidos in vitro a partir da maturação e fecundação in vitro de oócitos de ovários obtidos em matadouro. Os embriões foram cultivados in vitro por 7 dias, até atingirem o estágio de blastocisto. Blastocistos suínos também foram produzidos in vivo, através de superovulação seguida de inseminação artificial de marrãs com 150 dias de idade. Para a colheita dos embriões, foi realizada lavagem dos cornos uterinos post-mortem cinco dias após a ovulação. Tanto blastocistos produzidos in vitro quanto os produzidos in vivo foram submetidos à imunocirurgia para isolamento da MCI. Brevemente, a zona pelúcida foi digerida com solução de pronase e os embriões incubados com soro de coelho anti-suíno para remoção das células do trofoectoderma e soro complemento de cobaia. A MCI resultante foi cultivada em meio para células-tronco (GMEM acrescido de 15% SFB, 0,1 mM ß-mercaptoetanol, 1% aminoácidos não essenciais e 4 ng/mL de bFGF) sobre monocamada de fibroblastos fetais murinos (MEF) inativados por radiação ou sobre Matrigel. Não foi observada diferença entre os dois sistemas de cultivo in vitro (MEF e Matrigel) na adesão das MCI isoladas. Também não foi verificada diferença entre os grupos de blastocistos, produzidos in vitro e in vitro, nas taxas de adesão das MCI cultivadas. Contudo, nenhuma colônia de CTE suínas foi obtida. A análise da expressão gênica em blastocistos produzidos in vitro e in vitro demonstrou que os genes Nanog e Sox2 são menos expressos em blastocistos produzidos in vitro. Contudo, a expressão do gene FoxD3, demonstrada pela primeira vez em suínos no presente trabalho, se mostrou semelhante entre os dois grupos de embriões. Visto que nenhuma linhagem de CTE legítima foi isolada em suínos até o momento, sugere-se que esta espécie possua requerimentos diferentes dos já conhecidos para as espécies murina e humana. Portanto, novos estudos são necessários para o estabelecimento de protocolos mais efetivos para o isolamento de CTE de suínos. / Embryonic stem cells (ESC) represent a useful tool to study embryonic development, cell differentiation and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral and religious considerations by scientists and laymen alike. Therefore, an animal model like the pig (Sus scrofa) is valuable by overcoming such hurdles, since this species holds physiologic parameters similar to humans. In spite of the high biomedical potential of ESC, many difficulties have been faced to maintain these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro and in vitro produced swine blastocysts, (2) to compare two in vitro culture conditions to maintain isolated inner cell masses (ICM), MEF or Matrigel and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2 and FoxD3 at ESC and in vitro and in vitro produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vitro produced blastocysts were obtained by superovulation followed by artificial insemination of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing five days after ovulation. in vitro and in vitroproduced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. The resultant ICM was cultured in stem cells media (GMEM added by 15% SFB, 0.1 mM ß-mercaptoethanol, 1% non essential amino acids and 4 ng/mL of bFGF) over monolayer of irradiated murine fetal fibroblasts (MEF) or Matrigel. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro and in vitro produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis of in vitro and in vitro produced blastocysts showed that Nanog and Sox2 are less expressed in in vitro produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Since no ESC lineage was obtained in swine until now, we believe this species have different requirements compared to murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC.

Células-tronco embrionárias (CTE) são importantes para estudos de desenvolvimento embrionário, diferenciação e manipulação genética. Além disso, essas células podem ser utilizadas na terapia celular e organogênese in vitro. Na pesquisa sobre terapia celular a partir de CTE oriundas de embriões humanos, considerações éticas, morais e religiosas têm sido feitas por pesquisadores e leigos. Portanto, um modelo animal como o suíno (Sus scrofa) será bastante válido por transpor tais barreiras, visto que o suíno possui parâmetros fisiológicos semelhantes aos humanos. Apesar do alto potencial biomédico das CTE, existem dificuldades na manutenção da pluripotência in vitro dessas células em suínos. Portanto, estudos que visam elucidar os mecanismos de manutenção da pluripotência de CTE in vitro são necessários para viabilizar o cultivo dessas células. Os objetivos do presente estudo foram (1) isolar células-tronco embrionárias suínas a partir de blastocistos produzidos in vitro e in vivo; (2) comparar dois sistemas de cultivo in vitro das massas celulares internas (MCI) isoladas, MEF ou Matrigel e (3) identificar e comparar a expressão dos fatores de transcrição Nanog, Sox2 e FoxD3 em CTE e blastocistos suínos produzidos in vitro e in vivo. Assim, blastocistos suínos foram produzidos in vitro a partir da maturação e fecundação in vitro de oócitos de ovários obtidos em matadouro. Os embriões foram cultivados in vitro por 7 dias, até atingirem o estágio de blastocisto. Blastocistos suínos também foram produzidos in vivo, através de superovulação seguida de inseminação artificial de marrãs com 150 dias de idade. Para a colheita dos embriões, foi realizada lavagem dos cornos uterinos post-mortem cinco dias após a ovulação. Tanto blastocistos produzidos in vitro quanto os produzidos in vivo foram submetidos à imunocirurgia para isolamento da MCI. Brevemente, a zona pelúcida foi digerida com solução de pronase e os embriões incubados com soro de coelho anti-suíno para remoção das células do trofoectoderma e soro complemento de cobaia. A MCI resultante foi cultivada em meio para células-tronco (GMEM acrescido de 15% SFB, 0,1 mM ß-mercaptoetanol, 1% aminoácidos não essenciais e 4 ng/mL de bFGF) sobre monocamada de fibroblastos fetais murinos (MEF) inativados por radiação ou sobre Matrigel. Não foi observada diferença entre os dois sistemas de cultivo in vitro (MEF e Matrigel) na adesão das MCI isoladas. Também não foi verificada diferença entre os grupos de blastocistos, produzidos in vitro e in vitro, nas taxas de adesão das MCI cultivadas. Contudo, nenhuma colônia de CTE suínas foi obtida. A análise da expressão gênica em blastocistos produzidos in vitro e in vitro demonstrou que os genes Nanog e Sox2 são menos expressos em blastocistos produzidos in vitro. Contudo, a expressão do gene FoxD3, demonstrada pela primeira vez em suínos no presente trabalho, se mostrou semelhante entre os dois grupos de embriões. Visto que nenhuma linhagem de CTE legítima foi isolada em suínos até o momento, sugere-se que esta espécie possua requerimentos diferentes dos já conhecidos para as espécies murina e humana. Portanto, novos estudos são necessários para o estabelecimento de protocolos mais efetivos para o isolamento de CTE de suínos. / Embryonic stem cells (ESC) represent a useful tool to study embryonic development, cell differentiation and genetic manipulation. Moreover, these cells can be applied in cell-based therapies and in vitro organogenesis. The research conducted with human ESC has generated many ethical, moral and religious considerations by scientists and laymen alike. Therefore, an animal model like the pig (Sus scrofa) is valuable by overcoming such hurdles, since this species holds physiologic parameters similar to humans. In spite of the high biomedical potential of ESC, many difficulties have been faced to maintain these cells in a pluripotent state in vitro. For this reason, studies to elucidate the mechanisms of in vitro maintenance of undifferentiated ESC are needed to improve the culture of these cells. The objectives of this study were (1) to isolate ESC from in vitro and in vitro produced swine blastocysts, (2) to compare two in vitro culture conditions to maintain isolated inner cell masses (ICM), MEF or Matrigel and (3) to identify and to compare the expression of the pluripotency markers Nanog, Sox2 and FoxD3 at ESC and in vitro and in vitro produced swine blastocysts. In this manner, swine blastocysts were obtained by in vitro maturation and fertilization of oocytes from ovaries collected in abattoirs. Embryos were in vitro cultured for 7 days until blastocyst stage. In addition, in vitro produced blastocysts were obtained by superovulation followed by artificial insemination of gilts (150 days of age). Embryos were collected by post-mortem uterus flushing five days after ovulation. in vitro and in vitroproduced blastocysts were submitted to immunosurgery to isolate the ICM. Briefly, zona pellucida was digested with pronase solution and embryos were incubated with anti-swine rabbit serum to remove trophoectoderm cells and with guinea-pig complement serum. The resultant ICM was cultured in stem cells media (GMEM added by 15% SFB, 0.1 mM ß-mercaptoethanol, 1% non essential amino acids and 4 ng/mL of bFGF) over monolayer of irradiated murine fetal fibroblasts (MEF) or Matrigel. No difference was observed between the in vitro culture conditions (MEF and Matrigel) on isolated ICM adhesion. In addition, no difference was verified between in vitro and in vitro produced blastocysts on adhesion of cultured ICM. However, no swine ESC was obtained. Gene expression analysis of in vitro and in vitro produced blastocysts showed that Nanog and Sox2 are less expressed in in vitro produced blastocysts. However, the expression of FoxD3, demonstrated in this study for the first time, was similar between groups. Since no ESC lineage was obtained in swine until now, we believe this species have different requirements compared to murine and human. Therefore, more studies are necessary to establish protocols to isolate porcine ESC.

Lors de la formation du blastocyste, l'embryon de souris est constitué d'un épithélium externe, le trophectoderme (TE), et d'une masse cellulaire interne (MCI). L’épiblaste (EPI) et l’endoderme primitif (EPr) se spécifient au sein de la MCI sous un patron de « sel et poivre » caractérisé par l’expression complémentaire de NANOG, marqueur de l’EPI et de GATA6, marqueur de l’EPr. Nanog est nécessaire pour l’acquisition d’une identité EPI et Gata6 induit le devenir en EPr. La voie FGF/MAPK joue un rôle critique dans l’acquisition de l’identité EPr et la perturbation de son activité impacte directement sur le ratio EPr/EPI dans la MCI. Je recherche des facteurs qui serait exprimés de manière hétérogène avant la spécification des cellules internes et pourraient faire pencher la balance vers un destin ou l’autre. Pour cela, j’ai disséqué l’évolution des cellules de la MCI au sein des embryons Nanog-/- et Gata6-/-. Ces embryons forment correctement le TE et la MCI qui ne se spécifie ni en EPI ni en EPr. En effet, les cellules internes des embryons Nanog-/- ; Gata6-/- restent bloquées autour du stade E3.25. De manière étonnante, dans les cellules de la MCI, le facteur de transcription SOX2 est présent et ce, de manière hétérogène. De plus, grâce à des traitements inhibiteurs de la voie FGF/MAPK, je montre que cette voie n’est pas responsable de l’hétérogénéité d’expression de SOX2. Ainsi, l’expression hétérogène de SOX2 dans les cellules internes des embryons est donc indépendante de Nanog, de Gata6 et de la voie FGF/MAPK. / During mouse blastocyst formation, the embryo consists of an outer epithelium, the trophectoderm (TE), and the inner cell mass (ICM). The epiblast (EPI) and the primitive endoderm (PrE) are specified within the MCI in a "salt and pepper" pattern characterized by the complementary expression of NANOG, marker of EPI and gata6, marker of PrE. Nanog is mandatory to acquire an EPI identity and Gata6 induces the PrE identity. FGF /MAPK pathway plays a critical role in the acquisition of a PrE identity and disruption of its activity directly impacts the PrE/Epi ratio within the ICM. I’m looking for factors that would be expressed heterogeneously before the specification of internal cells and might tilt the balance towards one fate or the other. For this, I dissected the evolution of ICM cells within Nanog-/- ; Gata6-/- embryos. These embryos form properly the TE and MCI that specifies neither EPI nor PrE. Indeed, the internal cells of Nanog-/- ; Gata6-/- embryos remain stuck around the stage of E3.25. Surprisingly, in the MCI cells, the transcription factor SOX2 is present and this, heterogeneously. Moreover, using inhibitors treatments of the FGF/MAPK pathway, I show that this pathway is not responsible for the heterogeneity of expression of SOX2. Thus, the heterogeneous expression of SOX2 in the inner cells of the embryos is therefore independent of Nanog, Gata6 and the FGF/MAPK pathway.

Embryonic stem (ES) cells are maintained in an undifferentiated state by a gene regulatory network centred on the triumvirate of transcription factors Nanog, Oct4 and Sox2. Genome-wide chromatin immunoprecipitation studies indicate that in many cases target genes contain closely localised binding sites for each of these proteins, as well as additional members of the extended pluripotency transcription factor network. However, the biochemical basis of the interactions between these proteins is largely unknown, as are the mechanisms by which these interactions control ES cell identity. By purifying Nanog from ES cells and identifying co-purified proteins, we determined a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members and RNA processing factors. Validation of interactions was obtained by co-immunoprecipitation of Nanog with putative partners. Sox2 was identified as a robust interacting partner of Nanog and the interaction was investigated further. We show that the interaction is independent of DNA binding and that a region of Nanog known as tryptophan repeat, in which tryptophan is present every 5th residue is necessary and sufficient for the binding of Sox2. Furthermore, mutation of tryptophan residues within the Nanog tryptophan repeat (WR) abolishes the interaction with Sox2. A region of Sox2 known as serine rich region, a triple-repeat motif (S X T/S Y) within a stretch of 21 residues is required for the interaction with Nanog. Mutation of tyrosines to alanine within the three motifs (S X T/S Y) abrogates the Nanog–Sox2 interaction. The disruption of the Nanog-Sox2 interaction results in the alteration of expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines of the motif with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal. Together these data shed light on the extent of the interactions of Nanog with protein partners as well as the biochemical nature of the interaction between Nanog and one of the most important partners Sox2, an interaction crucial for maintaining optimal mouse ES cell self-renewal efficiency.

Pluripotent cells have the dual abilities to self-renewal and to differentiate into all three germ layers. Pluripotent cells can be isolated from two different stages of mouse embryogenesis. Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of the pre-implantation embryo and are considered to be in a naïve state. On the other hand, cells isolated from epiblast of the post-implantation embryo are referred as epiblast stem cells (EpiSC) and are representative of primed pluripotency. ESCs and EpiSCs are distinct from each other in terms of the morphology, the gene regulatory network and the signalling pathways regulating self-renewal. Under certain conditions, ESCs and EpiSCs can be transitioned into each other. However, the mechanism that regulates this transition from naïve to primed pluripotent state remains to be solved. Nanog, Oct4 and Sox2 form the core gene regulatory network of pluripotency. Additionally, the Id protein family is also important in the maintenance of pluripotency in ESCs. Id proteins function by inhibiting the activity of pro-differentiation factors. Tcf15 is identified as one of the targets of Id inhibition in ESCs. Moreover, Tcf15 has been identified as a repression target of Nanog. In this study, to understand the function of Tcf15, the expression of Tcf15 was characterized in differentiating ESCs. The transient upregulation of Tcf15 mRNA and protein was detected at early stages of differentiation before lineage commitment. Furthermore, Tcf15 protein was heterogeneously expressed in differentiating cells. Mutually exclusive expression of Nanog and Tcf15 proteins were demonstrated in both self-renewing and differentiating ESCs. Further characterization of the effect of Nanog on Tcf15 transcription showed that Tcf15 pre-mRNA was downregulated within 20 minute of Nanog induction. A Nanog binding site was identified at +32kb relative to the Tcf15 transcription start site (TSS). Initially, Nanog binding at this region was confirmed by performing ChIP-PCR experiments. Then, this Nanog binding region was further analysed for its enhancer activity related to the Tcf15 gene. Deletion of the Nanog binding region using CRISPR-Cas9 confirmed that this region acts as Tcf15 enhancer; it was shown that this region was required for the activation of Tcf15 transcription during differentiation. Tcf15 induction experiments were performed in order to the check whether Tcf15 affects Nanog transcription. The results indicate that Nanog is not a direct target of Tcf15, but Tcf15 contributes indirectly to the repression of Nanog. Additional analysis with the Tcf15 enhancer deletion cells showed that Tcf15 is not required for efficient downregulation of naïve markers and the upregulation of primed markers. However, the genes related to the regulation of adhesion properties of cells such as Zyc, Itga3 were induced with lower efficiency in the absence of Tcf15 compared to the wild type cells. In summary, I investigated the reciprocal regulation of Tcf15 and Nanog and the role of Tcf15 for the differentiation. My results suggest that Tcf15 is expressed in the cells that have initiated differentiation but are not lineage-committed. Additionally, Tcf15 can contribute to the regulation of adhesion related genes in order to help the epithelisation of the cells required during the differentiation from naïve to the primed pluripotent state. As a conclusion, Nanog is proposed to help to prevent certain aspects of ESCs differentiation by repressing Tcf15.

Nanog is a transcription factor that plays a central part in the gene regulatory network that maintains and induces pluripotency of embryonic stem cells (ESCs). However, the molecular basis by which Nanog achieves its functions is not fully understood. At the centre of C-terminal domain of Nanog a tryptophan repeat (WR) is located, comprising 10 penta-peptide repeats each starting with a tryptophan. A mutant form of Nanog (Nanog-W10A) in which all 10 tryptophan residues have been substituted by alanine has an impaired capacity to drive LIF-independent self-renewal and a reduced efficiency in reprogramming primed epiblast stem cells to naïve pluripotency. To understand how the WR contributes to Nanog function, Nanog-W10A-ERT2 was introduced into Nanog null cells. Upon hydroxytamoxifen addition, the Nanog-ERT2 fusion proteins were detected on chromatin within 1 hour, allowing a comparison of genome-wide transcriptional responses to Nanog and Nanog-W10A by microarray. When treated with LIF, Nanog-W10A can activate most of Nanog targets as efficiently as Nanog. In contrast, Nanog-W10A did not efficiently repress most Nanog targets, including Otx2 and Tcf15 that were previously suggested to prime ESCs for differentiation. The microarray experiments performed in the absence of LIF signalling showed that Nanog and LIF co-regulate an extensive list of targets, including Klf4 and Mras. When LIF is absent, wildtype Nanog can still activate pro-self-renewal factors, including Esrrb and repress differentiation-priming factor, such as Tcf15 and Otx2. In contrast, in the absence of LIF, the activation of pro-self-renewal factors Klf4 and Mras is reduced. In addition, activation of Esrrb by Nanog-W10A induction delays but does not prevent differentiation. These effects allow the de-repression of Otx2 and Tcf15 by Nanog-W10A to dominate. Therefore, the function of Nanog is not only mediated by the activation of pro-self-renewal genes, but also repression of pro-differentiation signals. The functional significance of the repression of Nanog targets was further exemplified by the robust capacity of Otx2 to dominate over the self-renewal signals and to drive differentiation. The Otx2 protein is a direct interacting partner of Nanog that binds the Nanog WR tryptophan residues. The previously identified Otx2 “tail domain” comprises two imperfectly aligned repeats and aromatic residues of each repeat align with aromatic residues of the Sox2 “SXS/TY” motif previously identified to mediate the interaction between Sox2 and Nanog. Aromatic residues of Otx2 were demonstrated to directly interact with both Nanog and Sox2. The interactions between Otx2, Nanog and Sox2 are essential for Otx2 functions in driving ESCs differentiation, as Otx2 mutants with alanine substitutions of the aromatic residues in both or either of the repeats have reduced efficiency to drive differentiation. As Nanog and Sox2 may co-occupy many loci important in maintaining ESC self-renewal, Otx2 may be able to “read” the Nanog/Sox2 co-binding sites to dissolve the pluripotent networks. In summary, the repression function of Nanog is located within the Nanog WR region and represents an important module of Nanog in fine-tuning the balance between self-renewal and differentiation. This module involving Nanog WR can also be recognised by differentiation-priming factor Otx2 and may represent an initial step during the exit of differentiation.