Terminally differentiated Schwann cells (SCs), the glial cells in the adult peripheral nerves, display a remarkable plasticity by adopting a de-differentiated phenotype following injury and becoming specialised to repair-type cells for promoting nerve regeneration. Adult SC plasticity is also hijacked by leprosy-causing Mycobacterium leprae during peripheral nerve infection, which make SCs susceptible to reprogramming and generation of progenitor/stem-like cells for bacterial advantage. Interestingly, de-differentiated SCs generated during nerve injury and infection reactivated stem cell transcription factor Sox2, which is essential for maintaining pluripotency in embryonic stem cells (ESCs). In this study we address what role Sox2 plays and how it is involved in adult SC plasticity. We identified that Sox2 binds to a network of gene targets in de-differentiated adult SCs across the mouse genome. This Sox2 target network is distinct from Sox2 target genes in core ESC pluripotency, and appears to be modulated by SC microenvironmental changes and pathological conditions, as nerve crush injury and infection-induced reprogramming expanded Sox2 binding to target genes. In vivo knockdown by shRNA of Sox2 in wild type adult nerves demonstrated reduction in SC de-differentiation. Mutant mice defective in natural nerve degeneration, de-differentiation and regeneration (Wallerian degeneration slow mice; Wlds) not only show impaired Sox2 binding to its target genes but also a delay in Sox2 and target gene expression after nerve crush injury. Together, these in vivo data reveal an impact of Sox2 and its target network on SC plasticity. Furthermore, altered expression of many of these target genes after Sox2 knockdown in wild type adult Schwann cells in vitro and in vivo as well as in injured Wlds nerves suggests a functional role of a Sox2 target network in nerve injury-repair processes. This includes Sox2 target genes such as Megf10, Btc, Atf3 and Nestin. By acting on these genes Sox2 may coordinate relevant gene functions ranging from phagocytosis/clearance, proliferation, transcription and cytoskeletal dynamics. Thus, this study proposes a novel concept of how reactivation of an embryonic stem cell regulator like Sox2 in adult tissues coordinates a gene network regulating Schwann cell plasticity and multiple biological functions facilitating the nerve injury-repair process. These findings may aid in developing strategies towards promoting nerve regeneration, or designing treatments for neuropathies in which deregulation of Schwann cell de-differentiation contributes to pathogenesis.

The sensory cells of the inner ear, hair cells, provide vertebrates with the ability to detect auditory stimuli and balance. In mammals, cochlear hair cells, those responsible for hearing, do not regenerate. Zebrafish hair cells do regenerate. Gaining an understanding of the role and regulation of the genes involved in the formation and regeneration of these cells may provide information important for the development of genetic therapies. We show that zebrafish atoh1 acts as the proneural gene responsible for defining the equivalence group from which hair cells form. Expression of atoh1 is dependent upon Fgf and Pax. Atoh1 induces expression of delta, resulting in activation of Notch and subsequent lateral inhibition. Another factor known to be important for hair cell formation in mice is Sox2. In zebrafish, sox2 expression is downstream of Atoh1, Notch and Fgf. Zebrafish Sox2 is not required for hair cell formation, but rather Sox2 is important for hair cell maintenance. In zebrafish, otic hair cell regeneration has not yet been characterized. We show that, following laser ablation, hair cells regenerate by way of transdifferentiation. We further show that this regeneration requires Sox2 activity. These data suggest that Sox2 acts to maintain support cell plasticity. This role is likely conserved because Sox2 is also important for stem cell plasticity in mammals. This new understanding of the role and regulation of both Atoh1 and Sox2 provides essential information that can be used to further efforts to provide genetic therapies for hair cell regeneration in mammals.

Le corépresseur nucléaire NCOR1 est un répresseur transcriptionnel qui régule l’expression génique en s’associant à des récepteurs nucléaires ou à des facteurs de transcription comme AP-1 et NF-B. Ce projet de recherche visait à déterminer comment NCOR1 régule la prolifération et la réponse inflammatoire dans les cellules épithéliales intestinales (CEIs). Nous avons utilisé deux modèles murins de délétion du gène Ncor1 (Ncor1ID et Ncor1Exon11) ainsi que la Cre-recombinase sous le contrôle du promoteur du gène Vil1 (12.4KbVilCre) afin d’invalider le gène Ncor1 au niveau des CEIs (Ncor1IDΔCEI et Ncor1Exon11ΔCEI). Nous avons induit une réponse inflammatoire dans les CEIs en utilisant de l’IL-1 et du LPS. Nous avons observé que l’expression de NCOR1 est augmentée dans les CEIs lors de la réponse inflammatoire. Nous avons également traité les souris invalidées pour le gène Ncor1 avec du DSS afin d’induire une colite chimique expérimentale. Nous avons observé que les animaux Ncor1IDΔCEI et Ncor1Exon11ΔCEI sont plus susceptibles que les témoins. Nous avons analysé l’expression génique chez les animaux Ncor1IDΔCEI et Ncor1Exon11ΔCEI. L’analyse des gènes modulés dans les animaux Ncor1IDΔCEI a révélé que l’expression de la Retnlb est augmentée chez ces animaux, ce qui suggère un dérèglement dans la microflore. Dans les animaux Ncor1Exon11ΔCEI, nous avons noté que l’expression du gène Ido1, un puissant immunosuppresseur, est augmentée et permettrait possiblement à ces animaux de se maintenir dans un état homéostatique en absence de stress. Lorsque nous avons diminué l’expression de NCOR1 dans les CEIs à l’aide de shARN (shNCOR1_655), nous avons observé que celles-ci arrêtent de proliférer et sont sénescentes. De plus, nous avons remarqué une induction de molécules inflammatoires associées au SASP dans ces cellules. Nous avons analysé le transcriptome des cellules shNCOR1_655. Nous avons identifié le facteur de pluripotence SOX2 comme étant induits lorsque l’expression de NCOR1 est diminuée. Finalement, nous avons utilisé la technologie SILAC et la spectrométrie de masse afin de déterminer la composition du complexe de répression de NCOR1 dans les CEIs. Nous avons identifié de nouveaux partenaires d’interaction potentiels de NCOR1.

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.

Pluripotency is defined as the capacity to differentiate into cells from each of the three primary germ layers, the ectoderm, mesoderm and endoderm. This is a property of cells located in the inner cell mass (ICM) of preimplantation blastocysts and in the epiblast layer of postimplantation, presomite embryos. Preimplantation and postimplantation pluripotency can be captured indefinitely in cultured embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively. Preimplantation pluripotency in ES cells is regulated by a network of genes centred on three transcription factors (TFs) Oct4, Sox2 and Nanog. Oct4 and Sox2 form a mutually-reinforcing circuit and cooperatively stimulate transcription of downstream genes, including Nanog. All three TFs are expressed in EpiSCs and in the postimplantation epiblast. Functional studies established a role for Oct4 and Nanog in the specification of ICM cell identity, and a role for Oct4 in the maintenance of postimplantation pluripotency. Although the role of Sox2 in preimplantation ICM cells is unclear, it is critical for the establishment of egg cylinder following implantation and indispensable for ES cell pluripotency. However, despite the presence of Sox2 in postimplantation pluripotent cells the role of Sox2 in postimplantation pluripotency is unknown. In this thesis the role of Sox2 in the regulation of postimplantation pluripotency was examined. In contrast to the situation in the preimplantation ICM, Sox2 and Nanog are expressed in opposing gradients in the gastrulation-stage postimplantation epiblast, with Sox2 highest anteriorly and Nanog highest posteriorly. Interestingly the posterior epiblast of neural-plate (NP)-staged embryos was shown not to be pluripotent. Furthermore, forced expression of Sox2 but not Oct4 in this region rescued pluripotency. The ability of Oct4 to reinstate pluripotency in the somitogenesis-stage embryo is limited to Sox2-positive tissues. This strongly suggests that coexpression of Sox2 and Oct4 is important for establishing postimplantation pluripotent identity. Sox2HIGH cultured EpiSCs were not positively correlated with NanogHIGH cells. This reciprocal relationship emerged during the transition from ES cells to EpiSCs in culture. Using mutant cells with reduced levels of Sox2 or Nanog, Sox2 positively influences Nanog but Nanog negatively influences Sox2 expression post-transcriptionally. The negative influence of Nanog on Sox2 protein level was confirmed using doxycycline-inducible Nanog overexpressing EpiSCs. This negative relationship indicates that the regulation of Sox2 expression is different in postimplantation pluripotency and that Nanog may negatively regulate Sox2 on the protein level in the posterior epiblast. Sox2 is expressed at a lower level in EpiSCs than ES cells and the significance of this was further investigated by microarray transcription profiling using cells in which a fluorescent reporter (tdTomato) was knocked in to the Sox2 gene. Sox2- tdTomatoHIGH cells cultured in LIF/FCS/GMEMβ correlate with an undifferentiated cell identity and Sox2-tdTomatoLOW cells are associated with non-neural differentiation. Interestingly the global profile of ES cells and EpiSCs that share similar Sox2-tdTomato signal are non-identical. This suggests that Sox2 has different roles in different pluripotent states. ES cells with enforced Sox2 expression were unable to enter the EpiSC state, while ES cells with lowered Sox2 levels were inefficient in neural differentiation. Therefore, levels of Sox2 are critical for cell fate decisions. Strikingly, given the apparent requirement for Sox2 during Oct4-induced reinstatement of post-implantation pluripotency, deletion of Sox2 had no effect on the maintenance of EpiSC pluripotency. This is likely due to the presence of redundant Sox factors and indeed Sox3 is able to rescue the Sox2-null phenotype in ES cells. Taken together, these results suggest the hypothesis that postimplantation pluripotency is maintained by multiple Sox factors, while Nanog negatively regulates Sox2 post-transcriptionally to repress neural specification in the posterior epbilast. The positive influence of Sox2 on Nanog protein level suggests a possible negative feedback loop to balance the proneural and pluripotent properties of Sox2 in postimplantation pluripotency.

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.

Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.

Sox2 is the most frequently amplified oncogene in lung squamous cell carcinoma (SCC). Lung SCC arises in the proximal to central airways and is thought to originate from the p63-positive basal progenitor cells. Since Sox2 amplification occurs early in SCC pathogenesis, we investigated the oncogenic role of Sox2 using normal primary human lung basal progenitor cells. Although Sox2 is highly expressed in normal basal progenitors in a quiescent tracheal epithelium in vivo, we found that Sox2 expression decreases substantially during in vitro proliferation. When Sox2 expression is elevated in the proliferating basal cells in vitro to a level clinically observed in lung SCCs, Sox2 causes hyperplasia and promotes both squamous and Mucin16-positive glandular lineages at the expense of ciliated cell differentiation. Furthermore, our data suggest that the squamous and glandular-differentiating activity of Sox2 is differentially modulated by Receptor tyrosine kinase (RTK) and/or PI3-kinase signaling to promote squamous metaplasia of basal progenitor cells during SCC development.