RESUMEN
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common genetic enzyme-defect disorder. In this study, CHULAi001-A was established from peripheral blood mononuclear cells of a G6PD-deficient subject who carried the Viangchan variant (871 G > A). Episomal plasmids expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA against p53 were introduced into parental cells by electroporation and cultured under feeder-free conditions to reprogram iPSCs. Embryonic pluripotency, in vitro differentiation capacity, and episomal vector integration of the established CHULAi001-A were verified. The CHULAi001-A iPSCs retained a normal karyotype.
Asunto(s)
Glucosafosfato Deshidrogenasa , Células Madre Pluripotentes Inducidas , Humanos , Glucosafosfato Deshidrogenasa/genética , Leucocitos Mononucleares , Mutación/genéticaRESUMEN
Gene screenings have identified a number of reprogramming factors that induce pluripotency from somatic cells. However, the screening methods have mostly considered only factors that maintain pluripotency in embryonic stem cells, ignoring a potentially long list of other contributing factors involved. To expand the search, we developed a new screening method that examined 2,008 human genes in the generation of human induced pluripotent stem cells (iPSCs), including not only pluripotent genes but also differentiation-related genes that suppress pluripotency. We found the top 100 genes that increased reprogramming efficiency and discovered they contained many differentiation-related genes and homeobox genes. We selected two, HHEX and HLX, for further analysis. These genes enhanced the appearance of premature reprograming cells in the early phase of human iPSC induction, but had inhibitory effect on the late phase. In addition, when expressed in human iPSCs, HHEX and HLX interfered with the pluripotent state, indicating inverse effects on somatic reprograming and pluripotent maintenance. These results demonstrate that our screening is useful for identifying differentiation-related genes in somatic reprograming. Stem Cells 2016;34:2661-2669.
Asunto(s)
Reprogramación Celular , Fibroblastos/metabolismo , Biblioteca de Genes , Proteínas de Homeodominio/genética , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción/genética , Animales , Diferenciación Celular , Línea Celular , Fibroblastos/citología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.
Asunto(s)
Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Procesamiento Proteico-Postraduccional , Procesos de Determinación del Sexo/genética , Animales , Epidídimo/anomalías , Femenino , Histona Demetilasas con Dominio de Jumonji/genética , Masculino , Metilación , Ratones , Ratones Mutantes , Ratones Transgénicos , Ovario/anomalías , Ovario/enzimología , Testículo/anomalías , Testículo/enzimología , Útero/anomalíasRESUMEN
The generation of induced pluripotent stem cells (iPSCs) provides the opportunity to use patient-specific somatic cells, which are a valuable source for disease modeling and drug discovery. To promote research involving these cells, it is important to make iPSCs from easily accessible and less invasive tissues, like blood. We have recently reported the efficient generation of human iPSCs from adult fibroblasts using a combination of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. We herein report a modified protocol enabling efficient iPSC induction from CD34+ cord blood cells and from peripheral blood isolated from healthy donors using these plasmid vectors. The original plasmid mixture could induce iPSCs; however, the efficiency was low. The addition of EBNA1, an essential factor for episomal amplification of the vectors, by an extra plasmid greatly increased the efficiency of iPSC induction, especially when the induction was performed from αßT cells. This improvement enabled the establishment of blood-derived iPSCs from seven healthy donors ranging in age from their 20s to their 60s. This induction method will be useful for the derivation of patient-specific integration-free iPSCs and would also be applicable to the generation of clinical-grade iPSCs in the future.
Asunto(s)
Células Sanguíneas/citología , Técnicas de Cultivo de Célula/métodos , Sangre Fetal/citología , Células Madre Pluripotentes Inducidas/citología , Adulto , Femenino , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Masculino , Persona de Mediana EdadRESUMEN
We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match â¼20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Pueblo Asiatico/genética , Electroporación , Perfilación de la Expresión Génica , Frecuencia de los Genes , Vectores Genéticos , Antígenos HLA/genética , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Células Madre Pluripotentes Inducidas/metabolismo , Plásmidos/genética , Donantes de TejidosRESUMEN
Methylation of DNA and lysine 9 of histone H3 (H3K9) are well-conserved epigenetic marks for transcriptional silencing. Although H3K9 methylation directs DNA methylation in filamentous fungi and plants, this pathway has not been corroborated in mammals. G9a and GLP/Eu-HMTase1 are two-related mammalian lysine methyltransferases and a G9a/GLP heteromeric complex regulates H3K9 methylation of euchromatin. To elucidate the function of G9a/GLP-mediated H3K9 methylation in the regulation of DNA methylation and transcriptional silencing, we characterized ES cells expressing catalytically inactive mutants of G9a and/or GLP. Interestingly, in ES cells expressing a G9a-mutant/GLP complex that does not rescue global H3K9 methylation, G9a/GLP-target genes remain silent. The CpG sites of the promoter regions of these genes were hypermethylated in such mutant ES cells, but hypomethylated in G9a- or GLP-KO ES cells. Treatment with a DNA methyltransferase inhibitor reactivates these G9a/GLP-target genes in ES cells expressing catalytically inactive G9a/GLP proteins, but not the wild-type proteins. This is the first clear evidence that G9a/GLP suppresses transcription by independently inducing both H3K9 and DNA methylation.
Asunto(s)
Metilación de ADN , Regulación de la Expresión Génica , Silenciador del Gen , N-Metiltransferasa de Histona-Lisina/fisiología , Histonas/química , Transcripción Genética , Animales , Catálisis , Islas de CpG , Células Madre Embrionarias/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , MutaciónRESUMEN
A novel cDNA-encoding polypeptide of 545 amino acid residues was identified from a mouse testis cDNA library. The transcript of this gene, p59(scr), was predominantly expressed in the testis and was developmentally regulated during spermatogenesis. The encoded protein was expressed in spermatocytes and round spermatids within seminiferous tubules of the adult testis. Using an adult-mouse model of experimental unilateral cryptorchidism, it was observed that the expression of the p59(scr) mRNA was reduced in the cryptorchid testes in association with destruction of spermatogenesis. In vitro heat stress experiment further demonstrated that p59(scr) mRNA was more sensitive to heat stress than the other mRNA species, such as germ-cell-specific meiosis-activating kinase (mak) gene and a housekeeping beta-actin gene. Our results suggest that this novel p59(scr) gene is involved in spermatogenesis and may play an important role in development of testicular germ cells.
Asunto(s)
Criptorquidismo/metabolismo , Proteínas/metabolismo , Espermatogénesis/fisiología , Testículo/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Clonación Molecular , Criptorquidismo/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Células Germinativas/citología , Células Germinativas/metabolismo , Calor , Hibridación in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Proteínas Quinasas , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/genética , ARN Mensajero/metabolismo , Espermátides/citología , Espermátides/metabolismo , Espermatocitos/citología , Espermatocitos/metabolismo , Testículo/citología , Testículo/patologíaRESUMEN
Tumor suppressor p53 has been shown to repress expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a key mediator of tumor angiogenesis. The p63 gene, recently identified as a p53-relative, encodes multiple isoforms with structural and functional similarities and differences from p53. In this study, we show the evidence that the two major isoforms of the p63 gene, TAp63gamma (p51A) and dNp63alpha (p73L), represses and upregulates VEGF expression, respectively, on transcription and protein levels. Transient transfection assays show that a hypoxia-inducible factor (HIF) 1 binding site within the VEGF promoter region is responsible for both upregulation and repression by dNp63alpha and by TAp63gamma, respectively, of the VEGF promoter activity. We also show that TAp63gamma targets HIF1alpha for promoting proteasomal degradation but that dNp63alpha targets HIF1alpha for stabilization. Mammalian two-hybrid assays show that HIF1alpha-dependent transcription is repressed by TAp63gamma as well as by p53, whereas it is upregulated by dNp63alpha in collaboration with a transcription coactivator p300. Our data also show that dNp63alpha acts as a dominant-negative reagent toward both p53- and TAp63gamma-mediated degradation of HIF1alpha and repression of HIF1alpha-dependent transcription. These results suggest that p63 is involved in the regulation of the VEGF gene expression and that modulation of VEGF expression by TAp63gamma and dNp63alpha is closely correlated with their distinct roles on the regulation of HIF1alpha stability.