Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
Cell Rep ; 43(5): 114232, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38761378

RESUMO

The advent of novel 2D and 3D models for human development, including trophoblast stem cells and blastoids, has expanded opportunities for investigating early developmental events, gradually illuminating the enigmatic realm of human development. While these innovations have ushered in new prospects, it has become essential to establish well-defined benchmarks for the cell sources of these models. We aimed to propose a comprehensive characterization of pluripotent and trophoblastic stem cell models by employing a combination of transcriptomic, proteomic, epigenetic, and metabolic approaches. Our findings reveal that extended pluripotent stem cells share many characteristics with primed pluripotent stem cells, with the exception of metabolic activity. Furthermore, our research demonstrates that DNA hypomethylation and high metabolic activity define trophoblast stem cells. These results underscore the necessity of considering multiple hallmarks of pluripotency rather than relying on a single criterion. Multiplying hallmarks alleviate stage-matching bias.


Assuntos
Trofoblastos , Humanos , Trofoblastos/metabolismo , Trofoblastos/citologia , Metilação de DNA , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Modelos Biológicos , Implantação do Embrião , Diferenciação Celular , Epigênese Genética , Transcriptoma/genética , Proteômica/métodos
2.
Stem Cell Reports ; 18(11): 2016-2037, 2023 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-37863046

RESUMO

For nearly three decades, more than 80 embryonic stem cell lines and more than 100 induced pluripotent stem cell lines have been derived from New World monkeys, Old World monkeys, and great apes. In this comprehensive review, we examine these cell lines originating from marmoset, cynomolgus macaque, rhesus macaque, pig-tailed macaque, Japanese macaque, African green monkey, baboon, chimpanzee, bonobo, gorilla, and orangutan. We outline the methodologies implemented for their establishment, the culture protocols for their long-term maintenance, and their basic molecular characterization. Further, we spotlight any cell lines that express fluorescent reporters. Additionally, we compare these cell lines with human pluripotent stem cell lines, and we discuss cell lines reprogrammed into a pluripotent naive state, detailing the processes used to attain this. Last, we present the findings from the application of these cell lines in two emerging fields: intra- and interspecies embryonic chimeras and blastoids.


Assuntos
Expedições , Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Animais , Chlorocebus aethiops , Macaca mulatta , Linhagem Celular , Células-Tronco Pluripotentes Induzidas/metabolismo , Macaca fascicularis
3.
Development ; 149(17)2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35993311

RESUMO

Despite the growing interest in the rabbit model for developmental and stem cell biology, the characterization of embryos at the molecular level is still poorly documented. We conducted a transcriptome analysis of rabbit preimplantation embryos from E2.7 (morula stage) to E6.6 (early primitive streak stage) using bulk and single-cell RNA-sequencing. In parallel, we studied oxidative phosphorylation and glycolysis, and analysed active and repressive epigenetic modifications during blastocyst formation and expansion. We generated a transcriptomic, epigenetic and metabolic map of the pluripotency continuum in rabbit preimplantation embryos, and identified novel markers of naive pluripotency that might be instrumental for deriving naive pluripotent stem cell lines. Although the rabbit is evolutionarily closer to mice than to primates, we found that the transcriptome of rabbit epiblast cells shares common features with those of humans and non-human primates.


Assuntos
Células-Tronco Pluripotentes , Transcriptoma , Animais , Blastocisto/metabolismo , Epigênese Genética , Camadas Germinativas , Camundongos , Células-Tronco Pluripotentes/metabolismo , Coelhos , Transcriptoma/genética
4.
Blood ; 139(3): 343-356, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34517413

RESUMO

In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.


Assuntos
Embrião de Mamíferos/embriologia , Hemangioblastos/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Animais , Diferenciação Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Hemangioblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Mesonefro/citologia , Mesonefro/embriologia , Mesonefro/metabolismo , Camundongos , Análise de Célula Única , Transcriptoma , Peixe-Zebra
5.
Med Sci (Paris) ; 37(10): 863-872, 2021 Oct.
Artigo em Francês | MEDLINE | ID: mdl-34647874

RESUMO

Inter-species chimeras are both fantastic and monstrous creatures from Greek or Egyptian mythology, and a long-established research tool. Recent advances in the field of pluripotent stem cells have made it possible to extend the repertoire of inter-species chimeras to "systemic" chimeras, in which the mixing of cells from both species involves all organs including the germline. These chimeric embryos and fetuses open up new research avenues and potential medical applications. We will review the latest advances in the field. We will discuss the concepts of developmental complementation and developmental equivalence. We will discuss the methodological hurdles to be unlocked, as well as the biological and ethical limits of these new technologies.


TITLE: Les chimères « systémiques ¼ homme/animal. ABSTRACT: Les chimères inter-espèces sont à la fois les créatures fantastiques et monstrueuses des mythologies grecque ou égyptienne, et un outil de recherche établi de longue date. Des avancées récentes dans le domaine des cellules souches pluripotentes ont permis d'élargir le répertoire des chimères inter-espèces aux chimères « systémiques ¼ dans lesquelles le mélange des cellules des deux espèces concerne tous les organes, y compris la lignée germinale. Ces embryons et fœtus chimériques ouvrent de nouvelles voies de recherches et des applications médicales potentielles. Dans cette revue, nous ferons le point sur les dernières avancées dans ce domaine. Nous discuterons les concepts de complémentation et d'équivalence développementale. Nous évoquerons également les verrous méthodologiques à débloquer, ainsi que les limites biologiques et éthiques de ces nouvelles techniques.


Assuntos
Quimera , Células-Tronco Pluripotentes , Células Germinativas , Imunoterapia , Mitologia
6.
Stem Cell Reports ; 16(1): 56-74, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33382978

RESUMO

After reprogramming to naive pluripotency, human pluripotent stem cells (PSCs) still exhibit very low ability to make interspecies chimeras. Whether this is because they are inherently devoid of the attributes of chimeric competency or because naive PSCs cannot colonize embryos from distant species remains to be elucidated. Here, we have used different types of mouse, human, and rhesus monkey naive PSCs and analyzed their ability to colonize rabbit and cynomolgus monkey embryos. Mouse embryonic stem cells (ESCs) remained mitotically active and efficiently colonized host embryos. In contrast, primate naive PSCs colonized host embryos with much lower efficiency. Unlike mouse ESCs, they slowed DNA replication after dissociation and, after injection into host embryos, they stalled in the G1 phase and differentiated prematurely, regardless of host species. We conclude that human and non-human primate naive PSCs do not efficiently make chimeras because they are inherently unfit to remain mitotically active during colonization.


Assuntos
Diferenciação Celular , Quimera/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Células-Tronco Pluripotentes/citologia , Animais , Apoptose , Reprogramação Celular , Transferência Embrionária , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Humanos , Macaca mulatta , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Coelhos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Nat Cell Biol ; 22(10): 1223-1238, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32989249

RESUMO

Pluripotent stem cells (PSCs) transition between cell states in vitro, reflecting developmental changes in the early embryo. PSCs can be stabilized in the naive state by blocking extracellular differentiation stimuli, particularly FGF-MEK signalling. Here, we report that multiple features of the naive state in human and mouse PSCs can be recapitulated without affecting FGF-MEK signalling or global DNA methylation. Mechanistically, chemical inhibition of CDK8 and CDK19 (hereafter CDK8/19) kinases removes their ability to repress the Mediator complex at enhancers. CDK8/19 inhibition therefore increases Mediator-driven recruitment of RNA polymerase II (RNA Pol II) to promoters and enhancers. This efficiently stabilizes the naive transcriptional program and confers resistance to enhancer perturbation by BRD4 inhibition. Moreover, naive pluripotency during embryonic development coincides with a reduction in CDK8/19. We conclude that global hyperactivation of enhancers drives naive pluripotency, and this can be achieved in vitro by inhibiting CDK8/19 kinase activity. These principles may apply to other contexts of cellular plasticity.


Assuntos
Diferenciação Celular , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Metilação de DNA , Elementos Facilitadores Genéticos , Células-Tronco Pluripotentes/citologia , Animais , Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Feminino , Humanos , Camundongos , Fosforilação , Células-Tronco Pluripotentes/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transdução de Sinais
8.
Med Sci (Paris) ; 34(11): 944-953, 2018 Nov.
Artigo em Francês | MEDLINE | ID: mdl-30526839

RESUMO

Since the derivation of the first pluripotent embryonic stem cell lines in mice in the early 1980s, a plethora of lines has been obtained from various mammalian species including rodents, lagomorphs and primates. These lines are distinguished by their molecular and functional characteristics and correspond to the different pluripotency states observed in the developing embryo between the "blastocyst" and "gastrula" stages. These cell lines are positioned along a gradient, or continuum of pluripotency, the ends of which are epitomized by the naïve and primed states, respectively. Conventional human pluripotent stem cells self-renew in the primed state of pluripotency (ie, at the bottom of the gradient), a position that is undoubtedly the cause of their natural instability. Recent studies aim to generate naive human pluripotent stem cells (at the top of the gradient). The importance of this research in the perspective of medical applications will be discussed.


Assuntos
Células-Tronco Pluripotentes/classificação , Células-Tronco Pluripotentes/fisiologia , Animais , Diferenciação Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Camundongos
9.
Stem Cell Res ; 24: 106-117, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28889080

RESUMO

Rabbit induced pluripotent stem cells (rbiPSCs) possess the characteristic features of primed pluripotency as defined in rodents and primates. In the present study, we reprogrammed rbiPSCs using human Krüppel-like factors (KLFs) 2 and 4 and cultured them in a medium supplemented with fetal calf serum and leukemia inhibitory factor. These cells (designated rbEKA) were propagated by enzymatic dissociation for at least 30 passages, during which they maintained a normal karyotype. This new culturing protocol resulted in transcriptional and epigenetic reconfiguration, as substantiated by the expression of transcription factors and the presence of histone modifications associated with naïve pluripotency. Furthermore, microarray analysis of rbiPSCs, rbEKA cells, rabbit ICM cells, and rabbit epiblast showed that the global gene expression profile of the reprogrammed rbiPSCs was more similar to that of rabbit ICM and epiblast cells. Injection of rbEKA cells into 8-cell stage rabbit embryos resulted in extensive colonization of ICM in 9% early-blastocysts (E3.5), epiblast in 10% mid-blastocysts (E4.5), and embryonic disk in 1.4% pre-gastrulae (E6). Thus, these results indicate that KLF2 and KLF4 triggered the conversion of rbiPSCs into epiblast-like, embryo colonization-competent PSCs. Our results highlight some of the requirements to achieve bona fide chimeric competency.


Assuntos
Reprogramação Celular , Camadas Germinativas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição Kruppel-Like/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Proliferação de Células , Sobrevivência Celular , Quimera/metabolismo , Epigênese Genética , Perfilação da Expressão Gênica , Humanos , Fator 4 Semelhante a Kruppel , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Coelhos , Transdução de Sinais
10.
Mol Cell Biol ; 37(16)2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28584195

RESUMO

Sox2 is known to be important for neuron formation, but the precise mechanism through which it activates a neurogenic program and how this differs from its well-established function in self-renewal of stem cells remain elusive. In this study, we identified a highly conserved cyclin-dependent kinase (Cdk) phosphorylation site on serine 39 (S39) in Sox2. In neural stem cells (NSCs), phosphorylation of S39 enhances the ability of Sox2 to negatively regulate neuronal differentiation, while loss of phosphorylation is necessary for chromatin retention of a truncated form of Sox2 generated during neurogenesis. We further demonstrated that nonphosphorylated cleaved Sox2 specifically induces the expression of proneural genes and promotes neurogenic commitment in vivo Our present study sheds light on how the level of Cdk kinase activity directly regulates Sox2 to tip the balance between self-renewal and differentiation in NSCs.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Neurogênese , Fosfosserina/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , DNA/metabolismo , Regulação da Expressão Gênica , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Células NIH 3T3 , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Fosforilação , Ligação Proteica , Estabilidade Proteica , Fatores de Transcrição SOXB1/química , Serina Proteases/metabolismo
11.
Hum Mol Genet ; 26(2): 367-382, 2017 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-28365779

RESUMO

The cellular and molecular mechanisms underlying neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The ability to generate patient-specific induced pluripotent stem cells (iPSCs) now offers a novel strategy for modelling human diseases. Recent studies have reported the derivation of iPSCs from patients with neurological disorders. The key challenge remains the demonstration of disease-related phenotypes and the ability to model the disease. Here we report a case study with signs of neurodevelopmental disorders (NDDs) harbouring chromosomal rearrangements that were sequenced using long-insert DNA paired-end tag (DNA-PET) sequencing approach. We identified the disruption of a specific gene, GTDC1. By deriving iPSCs from this patient and differentiating them into neural progenitor cells (NPCs) and neurons we dissected the disease process at the cellular level and observed defects in both NPCs and neuronal cells. We also showed that disruption of GTDC1 expression in wild type human NPCs and neurons showed a similar phenotype as patient's iPSCs. Finally, we utilized a zebrafish model to demonstrate a role for GTDC1 in the development of the central nervous system. Our findings highlight the importance of combining sequencing technologies with the iPSC technology for NDDs modelling that could be applied for personalized medicine.


Assuntos
Transtorno do Espectro Autista/genética , Glicosiltransferases/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Animais , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Diferenciação Celular/genética , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Genoma Humano , Glicosiltransferases/biossíntese , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Medicina de Precisão , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
12.
Stem Cells ; 34(4): 860-72, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26864965

RESUMO

During development, lineage specification is controlled by several signaling pathways involving various transcription factors (TFs). Here, we studied the RE-1-silencing transcription factor (REST) and identified an important role of this TF in cardiac differentiation. Using mouse embryonic stem cells (ESC) to model development, we found that REST knockout cells lost the ability to differentiate into the cardiac lineage. Detailed analysis of specific lineage markers expression showed selective downregulation of endoderm markers in REST-null cells, thus contributing to a loss of cardiogenic signals. REST regulates cardiac differentiation of ESCs by negatively regulating the Wnt/ß-catenin signaling pathway and positively regulating the cardiogenic TF Gata4. We propose here a new role for REST in cell fate specification besides its well-known repressive role of neuronal differentiation.


Assuntos
Diferenciação Celular/genética , Fator de Transcrição GATA4/biossíntese , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Repressoras/genética , Animais , Linhagem da Célula/genética , Fator de Transcrição GATA4/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Via de Sinalização Wnt
13.
Nat Commun ; 6: 7095, 2015 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-25968054

RESUMO

Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.


Assuntos
Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica/fisiologia , Células-Tronco Pluripotentes/fisiologia , Fator de Transcrição STAT3/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células Alimentadoras , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Camundongos , Análise Serial de Proteínas , Fator de Transcrição STAT3/genética , Transdução de Sinais , Tamoxifeno/farmacologia
14.
Hum Mutat ; 35(11): 1311-20, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25137640

RESUMO

MED13L is a component subunit of the Mediator complex, an important regulator of transcription that is highly conserved across eukaryotes. Here, we report MED13L disruption in a translocation t(12;19) breakpoint of a patient with Pierre-Robin syndrome, moderate intellectual disability, craniofacial anomalies, and muscular defects. The phenotype is similar to previously described patients with MED13L haploinsufficiency. Knockdown of MED13L orthologue in zebrafish, med13b, showed early defective migration of cranial neural crest cells (NCCs) that contributed to cartilage structure deformities in the later stage, recapitulating craniofacial anomalies seen in human patients. Notably, we observed abnormal distribution of developing neurons in different brain regions of med13b morphant embryos, which could be rescued upon introduction of full-length human MED13L mRNA. To compare with mammalian system, we suppressed MED13L expression by short-hairpin RNA in ES-derived human neural progenitors, and differentiated them into neurons. Transcriptome analysis revealed differential expression of components of Wnt and FGF signaling pathways in MED13L-deficient neurons. Our finding provides a novel insight into the mechanism of overlapping phenotypic outcome targeting NCCs derivatives organs in patients with MED13L haploinsufficiency, and emphasizes a clinically recognizable syndromic phenotype in these patients.


Assuntos
Haploinsuficiência , Deficiência Intelectual/genética , Complexo Mediador/genética , Crista Neural/metabolismo , Animais , Diferenciação Celular/genética , Movimento Celular/genética , Pré-Escolar , Pontos de Quebra do Cromossomo , Modelos Animais de Doenças , Células-Tronco Embrionárias/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Humanos , Deficiência Intelectual/diagnóstico , Complexo Mediador/metabolismo , Crista Neural/embriologia , Neurônios/citologia , Neurônios/metabolismo , Fenótipo , RNA Mensageiro/genética , Análise de Sequência de DNA , Transcriptoma , Translocação Genética , Peixe-Zebra
15.
Nat Commun ; 5: 3719, 2014 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-24770696

RESUMO

Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Endoderma/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Mesoderma/embriologia , Animais , Western Blotting , Imunoprecipitação da Cromatina , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real
16.
PLoS One ; 9(6): e90852, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24603971

RESUMO

Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7-11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide (GNAQ), RNA-binding protein, fox-1 homolog (RBFOX3), unc-5 homolog D (C.elegans) (UNC5D), transmembrane protein 47 (TMEM47), and X-linked inhibitor of apoptosis (XIAP). Among them, XIAP is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).


Assuntos
Pontos de Quebra do Cromossomo , Deficiências do Desenvolvimento/genética , Transtornos do Desenvolvimento da Linguagem/genética , Sequência de Bases , Inversão Cromossômica , Variações do Número de Cópias de DNA , Feminino , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Análise de Sequência de DNA , Translocação Genética
17.
Stem Cells ; 31(12): 2632-46, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23963638

RESUMO

The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs, but Sox4, Sox5, Sox6, Sox8, Sox9, Sox11, Sox12, Sox13, and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover, the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming, albeit at low efficiency. By molecular dissection of the C-terminus of Sox17, we found that the ß-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK, we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2.


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXF/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Técnicas de Cultura de Células , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Camundongos SCID , Fator 3 de Transcrição de Octâmero/genética , Mutação Puntual , Fatores de Transcrição SOXF/genética , Ativação Transcricional , beta Catenina/metabolismo
18.
EMBO J ; 32(7): 938-53, 2013 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-23474895

RESUMO

How regulatory information is encoded in the genome is poorly understood and poses a challenge when studying biological processes. We demonstrate here that genomic redistribution of Oct4 by alternative partnering with Sox2 and Sox17 is a fundamental regulatory event of endodermal specification. We show that Sox17 partners with Oct4 and binds to a unique 'compressed' Sox/Oct motif that earmarks endodermal genes. This is in contrast to the pluripotent state where Oct4 selectively partners with Sox2 at 'canonical' binding sites. The distinct selection of binding sites by alternative Sox/Oct partnering is underscored by our demonstration that rationally point-mutated Sox17 partners with Oct4 on pluripotency genes earmarked by the canonical Sox/Oct motif. In an endodermal differentiation assay, we demonstrate that the compressed motif is required for proper expression of endodermal genes. Evidently, Oct4 drives alternative developmental programs by switching Sox partners that affects enhancer selection, leading to either an endodermal or pluripotent cell fate. This work provides insights in understanding cell fate transcriptional regulation by highlighting the direct link between the DNA sequence of an enhancer and a developmental outcome.


Assuntos
Embrião de Mamíferos/embriologia , Endoderma/embriologia , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas HMGB/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXF/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Diferenciação Celular/fisiologia , Linhagem Celular , Embrião de Mamíferos/citologia , Endoderma/citologia , Proteínas HMGB/genética , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXF/genética , Transcrição Gênica/fisiologia
19.
Stem Cells ; 29(6): 940-51, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21472822

RESUMO

Very few proteins are capable to induce pluripotent stem (iPS) cells and their biochemical uniqueness remains unexplained. For example, Sox2 cooperates with other transcription factors to generate iPS cells, but Sox17, despite binding to similar DNA sequences, cannot. Here, we show that Sox2 and Sox17 exhibit inverse heterodimerization preferences with Oct4 on the canonical versus a newly identified compressed sox/oct motif. We can swap the cooperativity profiles of Sox2 and Sox17 by exchanging single amino acids at the Oct4 interaction interface resulting in Sox2KE and Sox17EK proteins. The reengineered Sox17EK now promotes reprogramming of somatic cells to iPS, whereas Sox2KE has lost this potential. Consistently, when Sox2KE is overexpressed in embryonic stem cells it forces endoderm differentiation similar to wild-type Sox17. Together, we demonstrate that strategic point mutations that facilitate Sox/Oct4 dimer formation on variant DNA motifs lead to a dramatic swap of the bioactivities of Sox2 and Sox17.


Assuntos
DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas HMGB/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXF/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , Simulação por Computador , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Endoderma/metabolismo , Proteínas HMGB/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Engenharia de Proteínas , Multimerização Proteica , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXF/metabolismo , Alinhamento de Sequência
20.
PLoS One ; 4(9): e6804, 2009 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-19727443

RESUMO

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the "Functional Genomics in Embryonic Stem Cells" consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in "Expression Waves" and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.


Assuntos
Bases de Dados Genéticas , Genômica , Células-Tronco/citologia , Animais , Diferenciação Celular , Linhagem Celular , Análise por Conglomerados , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Camundongos , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Software
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA