RESUMEN
Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.
Asunto(s)
Reprogramación Celular , Células Epiteliales/citología , Fibroblastos/citología , Epitelio Pigmentado de la Retina/citología , Factores de Transcripción/genética , Línea Celular , Simulación por Computador , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
DNA methylation at the C-5 position of cytosine (5mC) regulates gene expression and plays pivotal roles in various biological processes. The TET dioxygenases catalyze iterative oxidation of 5mC, leading to eventual demethylation. Inactivation of TET enzymes causes multistage developmental defects, impaired cell reprogramming, and hematopoietic malignancies. However, little is known about how TET activity is regulated. Here we show that all three TET proteins bind to VprBP and are monoubiquitylated by the VprBP-DDB1-CUL4-ROC1 E3 ubiquitin ligase (CRL4(VprBP)) on a highly conserved lysine residue. Deletion of VprBP in oocytes abrogated paternal DNA hydroxymethylation in zygotes. VprBP-mediated monoubiquitylation promotes TET binding to chromatin. Multiple recurrent TET2-inactivating mutations derived from leukemia target either the monoubiquitylation site (K1299) or residues essential for VprBP binding. Cumulatively, our data demonstrate that CRL4(VprBP) is a critical regulator of TET dioxygenases during development and in tumor suppression.
Asunto(s)
Proteínas Portadoras/fisiología , Cromatina/enzimología , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Proteínas de Unión al ADN/genética , Dioxigenasas/metabolismo , Femenino , Células HEK293 , Humanos , Masculino , Ratones Noqueados , Datos de Secuencia Molecular , Mutación Missense , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/genética , Ubiquitina-Proteína LigasasRESUMEN
Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors.
Asunto(s)
Linfocitos B/citología , Linaje de la Célula , Transdiferenciación Celular , Hepatocitos/citología , Células-Madre Neurales/citología , Animales , Linfocitos B/metabolismo , Linaje de la Célula/genética , Transdiferenciación Celular/genética , Reprogramación Celular , Análisis por Conglomerados , Epigénesis Genética , Expresión Génica , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, "naive" state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes/citología , Supervivencia Celular , Cromatina/metabolismo , Elementos de Facilitación Genéticos/genética , Perfilación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Datos de Secuencia Molecular , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , TransgenesRESUMEN
5-hydroxymethylcytosine (5-hmC) is a novel DNA modification that is highly enriched in the adult brain and dynamically regulated by neural activity. 5-hmC accumulates across the lifespan; however, the functional relevance of this change in 5-hmC and whether it is necessary for behavioral adaptation have not been fully elucidated. Moreover, although the ten-eleven translocation (Tet) family of enzymes is known to be essential for converting methylated DNA to 5-hmC, the role of individual Tet proteins in the adult cortex remains unclear. Using 5-hmC capture together with high-throughput DNA sequencing on individual mice, we show that fear extinction, an important form of reversal learning, leads to a dramatic genome-wide redistribution of 5-hmC within the infralimbic prefrontal cortex. Moreover, extinction learning-induced Tet3-mediated accumulation of 5-hmC is associated with the establishment of epigenetic states that promote gene expression and rapid behavioral adaptation.
Asunto(s)
Adaptación Fisiológica/fisiología , Citosina/análogos & derivados , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Miedo/fisiología , Neocórtex/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , 5-Metilcitosina/análogos & derivados , Animales , Conducta Animal/fisiología , Condicionamiento Psicológico/fisiología , Citosina/metabolismo , Dioxigenasas , Epigénesis Genética/fisiología , Extinción Psicológica/fisiología , Estudio de Asociación del Genoma Completo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/citología , Neuronas/citología , Neuronas/metabolismoRESUMEN
The Ten-Eleven-Translocation 2 (TET2) gene, which oxidates 5-methylcytosine in DNA to 5-hydroxylmethylcytosine (5hmC), is a key tumor suppressor frequently mutated in hematopoietic malignancies. However, the molecular regulation of TET2 expression is poorly understood. We show that TET2 is under extensive microRNA (miRNA) regulation, and such TET2 targeting is an important pathogenic mechanism in hematopoietic malignancies. Using a high-throughput 3' UTR activity screen, we identify >30 miRNAs that inhibit TET2 expression and cellular 5hmC. Forced expression of TET2-targeting miRNAs in vivo disrupts normal hematopoiesis, leading to hematopoietic expansion and/or myeloid differentiation bias, whereas coexpression of TET2 corrects these phenotypes. Importantly, several TET2-targeting miRNAs, including miR-125b, miR-29b, miR-29c, miR-101, and miR-7, are preferentially overexpressed in TET2-wild-type acute myeloid leukemia. Our results demonstrate the extensive roles of miRNAs in functionally regulating TET2 and cellular 5hmC and reveal miRNAs with previously unrecognized oncogenic potential. Our work suggests that TET2-targeting miRNAs might be exploited in cancer diagnosis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Regiones no Traducidas 3' , 5-Metilcitosina/análogos & derivados , Animales , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas , Regulación hacia Abajo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patología , Hematopoyesis , Humanos , Ratones , Fenotipo , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The recent discovery that normal and neoplastic epithelial cells re-enter the stem cell state raised the intriguing possibility that the aggressiveness of carcinomas derives not from their existing content of cancer stem cells (CSCs) but from their proclivity to generate new CSCs from non-CSC populations. Here, we demonstrate that non-CSCs of human basal breast cancers are plastic cell populations that readily switch from a non-CSC to CSC state. The observed cell plasticity is dependent on ZEB1, a key regulator of the epithelial-mesenchymal transition. We find that plastic non-CSCs maintain the ZEB1 promoter in a bivalent chromatin configuration, enabling them to respond readily to microenvironmental signals, such as TGFß. In response, the ZEB1 promoter converts from a bivalent to active chromatin configuration, ZEB1 transcription increases, and non-CSCs subsequently enter the CSC state. Our findings support a dynamic model in which interconversions between low and high tumorigenic states occur frequently, thereby increasing tumorigenic and malignant potential.
Asunto(s)
Neoplasias de la Mama/patología , Cromatina/metabolismo , Proteínas de Homeodominio/metabolismo , Células Madre Neoplásicas/patología , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Neoplasias de la Mama/genética , Células Epiteliales/patología , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , MicroARNs/metabolismo , Células Madre Neoplásicas/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de ZincRESUMEN
TET dioxygenases successively oxidize 5-methylcytosine (5mC) in mammalian genomes to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC/5caC can be excised and repaired to regenerate unmodified cytosines by thymine-DNA glycosylase (TDG) and base excision repair (BER) pathway, but it is unclear to what extent and at which part of the genome this active demethylation process takes place. Here, we have generated genome-wide distribution maps of 5hmC/5fC/5caC using modification-specific antibodies in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). In wild-type mouse ESCs, 5fC/5caC accumulates to detectable levels at major satellite repeats but not at nonrepetitive loci. In contrast, Tdg depletion in mouse ESCs causes marked accumulation of 5fC and 5caC at a large number of proximal and distal gene regulatory elements. Thus, these results reveal the genome-wide view of iterative 5mC oxidation dynamics and indicate that TET/TDG-dependent active DNA demethylation process occurs extensively in the mammalian genome.
Asunto(s)
5-Metilcitosina/metabolismo , Epigénesis Genética , Técnicas Genéticas , Estudio de Asociación del Genoma Completo , Animales , Citosina/análogos & derivados , Citosina/metabolismo , Metilación de ADN , Reparación del ADN , Dioxigenasas/metabolismo , Células Madre Embrionarias , Heterocromatina/química , Heterocromatina/metabolismo , Ratones , Oxidación-Reducción , Elementos Reguladores de la Transcripción , Timina ADN Glicosilasa/metabolismoRESUMEN
Sox2 is an important transcriptional regulator in embryonic and adult stem cells. Recently, Sox2 was identified as an oncogene in many endodermal cancers, including colon cancer. There is great interest in how Sox2 cooperates with other transcription factors to regulate stem cell renewal, differentiation, and reprogramming. However, we still lack a general understanding of Sox2 transcriptional action. To determine transcriptional partners of Sox2 in adult cells, we generated mice where gene expression could be induced by an externally applied stimulus. We analyzed the consequences in the intestine where cell turnover is rapid. Sox2 expression, but not Oct4, specifically increased the numbers of stem cells and repressed Cdx2, a master regulator of endodermal identity. In vivo studies demonstrated that Sox21, another member of the SoxB gene family, was a specific, immediate, and cell-autonomous target of Sox2 in intestinal stem cells. In vitro experiments showed that Sox21 was sufficient to repress Cdx2 in colon cancer cells and in pluripotent stem cells. Sox21 was also specifically induced by Sox2 in fibroblasts and inhibition of Sox21 blocked reprogramming to the pluripotent state. These results show that transcriptional induction of Sox21 is a rapid and general mediator of the effects of Sox2 on cell identity in a wide range of cell types.
Asunto(s)
Células Madre Pluripotentes/metabolismo , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB2/metabolismo , Activación Transcripcional , Animales , Factor de Transcripción CDX2 , Diferenciación Celular/genética , Línea Celular , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/antagonistas & inhibidores , Mucosa Intestinal/metabolismo , Intestinos/citología , Ratones , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Células Madre Pluripotentes/citología , Factores de Transcripción SOXB2/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Transcripción GenéticaRESUMEN
Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. The recent demonstration that members of the Ten-eleven translocation (Tet) family of proteins can convert 5-methylcytosine to 5-hydroxymethylcytosine raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, here we show in mouse ES cells that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite an increase in levels of DNA methylation at many Tet1-binding sites, Tet1 depletion does not lead to downregulation of all the Tet1 targets. Interestingly, although Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also involved in repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators.
Asunto(s)
Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Silenciador del Gen , Proteínas Proto-Oncogénicas/metabolismo , Transcripción Genética , 5-Metilcitosina/análogos & derivados , Animales , Línea Celular , Cromatina/metabolismo , Islas de CpG/genética , Citosina/análogos & derivados , Citosina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genoma/genética , Ratones , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas/genética , Proteínas Represoras/metabolismoRESUMEN
Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5hmC in mouse embryonic stem (ES) cells. A combined analysis of global 5hmC distribution and gene expression profile in wild-type and Tet1-depleted ES cells suggests that 5hmC is enriched at both gene bodies of actively transcribed genes and extended promoter regions of Polycomb-repressed developmental regulators. Thus, our study reveals the first genome-wide 5hmC distribution in pluripotent stem cells, and supports its dual function in regulating gene expression.
Asunto(s)
Citosina/análogos & derivados , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estudio de Asociación del Genoma Completo , 5-Metilcitosina/análogos & derivados , Animales , Línea Celular , Cromatina/metabolismo , Citosina/química , Citosina/metabolismo , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
DNA methylation is one of the best-characterized epigenetic modifications. Although the enzymes that catalyse DNA methylation have been characterized, enzymes responsible for demethylation have been elusive. A recent study indicates that the human TET1 protein could catalyse the conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC), raising the possibility that DNA demethylation may be a Tet1-mediated process. Here we extend this study by demonstrating that all three mouse Tet proteins (Tet1, Tet2 and Tet3) can also catalyse a similar reaction. Tet1 has an important role in mouse embryonic stem (ES) cell maintenance through maintaining the expression of Nanog in ES cells. Downregulation of Nanog via Tet1 knockdown correlates with methylation of the Nanog promoter, supporting a role for Tet1 in regulating DNA methylation status. Furthermore, knockdown of Tet1 in pre-implantation embryos results in a bias towards trophectoderm differentiation. Thus, our studies not only uncover the enzymatic activity of the Tet proteins, but also demonstrate a role for Tet1 in ES cell maintenance and inner cell mass cell specification.
Asunto(s)
5-Metilcitosina/metabolismo , Masa Celular Interna del Blastocisto/metabolismo , Citosina/análogos & derivados , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Proteínas Proto-Oncogénicas/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Proliferación Celular , Citosina/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/metabolismo , Ratones , Proteína Homeótica Nanog , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Maternal care influences hypothalamic-pituitary-adrenal (HPA) function in the rat through epigenetic programming of glucocorticoid receptor expression. In humans, childhood abuse alters HPA stress responses and increases the risk of suicide. We examined epigenetic differences in a neuron-specific glucocorticoid receptor (NR3C1) promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls. We found decreased levels of glucocorticoid receptor mRNA, as well as mRNA transcripts bearing the glucocorticoid receptor 1F splice variant and increased cytosine methylation of an NR3C1 promoter. Patch-methylated NR3C1 promoter constructs that mimicked the methylation state in samples from abused suicide victims showed decreased NGFI-A transcription factor binding and NGFI-A-inducible gene transcription. These findings translate previous results from rat to humans and suggest a common effect of parental care on the epigenetic regulation of hippocampal glucocorticoid receptor expression.
Asunto(s)
Adultos Sobrevivientes del Maltrato a los Niños/psicología , Epigénesis Genética/fisiología , Hipocampo/fisiología , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Adulto , Secuencia de Bases , Línea Celular , Metilación de ADN/genética , Femenino , Hipocampo/patología , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/fisiología , Receptores de Glucocorticoides/biosíntesis , Receptores de Glucocorticoides/fisiología , Suicidio/psicología , Adulto JovenRESUMEN
Increasing evidence suggests that islet cell transplantation for patients with type I diabetes holds great promise for achieving insulin independence. However, the extreme shortage of matched organ donors and the necessity for chronic immunosuppression has made it impossible for this treatment to be used for the general diabetic population. Recent success in generating insulin-secreting islet-like cells from human embryonic stem (ES) cells, in combination with the success in deriving human ES cell-like induced pluripotent stem (iPS) cells from human fibroblasts by defined factors, have raised the possibility that patient-specific insulin-secreting islet-like cells might be derived from somatic cells through cell fate reprogramming using defined factors. Here we confirm that human ES-like iPS cells can be derived from human skin cells by retroviral expression of OCT4, SOX2, c-MYC, and KLF4. Importantly, using a serum-free protocol, we successfully generated insulin-producing islet-like clusters (ILCs) from the iPS cells under feeder-free conditions. We demonstrate that, like human ES cells, skin fibroblast-derived iPS cells have the potential to be differentiated into islet-like clusters through definitive and pancreatic endoderm. The iPS-derived ILCs not only contain C-peptide-positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Thus, our study provides evidence that insulin-secreting ILCs can be generated from skin fibroblasts, raising the possibility that patient-specific iPS cells could potentially provide a treatment for diabetes in the future.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Piel/metabolismo , Diferenciación Celular , Línea Celular , Fibroblastos , Humanos , Células Secretoras de Insulina/citología , Factor 4 Similar a Kruppel , Piel/citologíaRESUMEN
Paired-like homeodomain transcription factors (PITX) regulate the activity of pituitary hormone-encoding genes. Here, we examined mechanisms through which the family of PITX proteins control murine FSH beta-subunit (Fshb) transcription. We observed that endogenous PITX1 and PITX2 isoforms from murine LbetaT2 gonadotrope cells could bind a highly conserved proximal cis-element. Transfection of PITX1 or PITX2C in heterologous cells stimulated both murine and human Fshb/FSHB promoter-reporter activities, and in both cases, mutation of the critical cis-element abrogated these effects. In homologous LbetaT2 cells, the same mutation decreased basal reporter activity and greatly reduced activin A-stimulated transcription from murine and human promoter-reporters. Transfecting dominant-negative forms of PITX1 or PITX2C or knocking down PITX1 or -2 expression by RNA interference in LbetaT2 cells inhibited murine Fshb transcription, confirming roles for endogenous PITX proteins. Both PITX1 and PITX2C interacted with Smad3 (an effector of the activin signaling cascade in these cells) in coprecipitation experiments, and the PITX binding site mutation greatly inhibited Smad2/3/4-stimulated Fshb transcription. In summary, both PITX1 and PITX2C regulate murine and human Fshb/FSHB transcription through a conserved cis-element in the proximal promoter. Furthermore, the data indicate both common and distinct mechanisms of PITX1 and PITX2C action.
Asunto(s)
Hormona Folículo Estimulante de Subunidad beta/genética , Proteínas de Homeodominio/fisiología , Factores de Transcripción Paired Box/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Bovinos , Cromatina/genética , Cromatina/fisiología , Secuencia Conservada , Genes Reporteros , Proteínas de Homeodominio/genética , Humanos , Luciferasas/genética , Ratones , Factores de Transcripción Paired Box/genética , ARN Interferente Pequeño/genética , Ovinos , Porcinos , Factores de Transcripción/genética , Transcripción Genética , Transfección , Proteína del Homeodomínio PITX2RESUMEN
A hallmark of vertebrate genes is that actively transcribed genes are hypomethylated in critical regulatory sequences. However, the mechanisms that link gene transcription and DNA hypomethylation are unclear. Using a trichostatin A (TSA)-induced replication-independent demethylation assay with HEK 293 cells, we show that RNA transcription is required for DNA demethylation. Histone acetylation precedes but is not sufficient to trigger DNA demethylation. Following histone acetylation, RNA polymerase II (RNAP II) interacts with the methylated promoter. Inhibition of RNAP II transcription with actinomycin D, alpha-amanitin, or CDK7-specific small interfering RNA inhibits DNA demethylation. H3 trimethyl lysine 4 methylation, a marker of actively transcribed genes, was associated with the cytomegalovirus promoter only after demethylation. TSA-induced demethylation of the endogenous cancer testis gene GAGE follows a similar sequence of events and is dependent on RNA transcription as well. These data suggest that DNA demethylation follows rather than precedes early transcription and point towards a novel function for DNA demethylation as a memory of actively transcribed genes.
Asunto(s)
Metilación de ADN , Silenciador del Gen , Transcripción Genética , Acetilación/efectos de los fármacos , Antígenos de Neoplasias/genética , Línea Celular , Quinasas Ciclina-Dependientes/metabolismo , Citomegalovirus/efectos de los fármacos , Metilación de ADN/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Modelos Biológicos , Proteínas de Neoplasias/genética , Sistemas de Lectura Abierta/genética , Regiones Promotoras Genéticas/genética , ARN/genética , ARN/metabolismo , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Maternal care alters epigenetic programming of glucocorticoid receptor (GR) gene expression in the hippocampus, and increased postnatal maternal licking/grooming (LG) behavior enhances nerve growth factor-inducible protein A (NGFI-A) transcription factor binding to the exon 1(7) GR promoter within the hippocampus of the offspring. We tested the hypothesis that NGFI-A binding to the exon 1(7) GR promoter sequence marks this sequence for histone acetylation and DNA demethylation and that such epigenetic alterations subsequently influence NGFI-A binding and GR transcription. We report that (1) NGFI-A binding to its consensus sequence is inhibited by DNA methylation, (2) NGFI-A induces the activity of exon 1(7) GR promoter in a transient reporter assay, (3) DNA methylation inhibits exon 1(7) GR promoter activity, and (4) whereas NGFI-A interaction with the methylated exon 1(7) GR promoter is reduced, NGFI-A overexpression induces histone acetylation, DNA demethylation, and activation of the exon 1(7) GR promoter in transient transfection assays. Site-directed mutagenesis assays demonstrate that NGFI-A binding to the exon 1(7) GR promoter is required for such epigenetic reprogramming. In vivo, enhanced maternal LG is associated with increased NGFI-A binding to the exon 1(7) GR promoter in the hippocampus of pups, and NGFI-A-bound exon 1(7) GR promoter is unmethylated compared with unbound exon 1(7) GR promoter. Knockdown experiments of NGFI-A in hippocampal primary cell culture show that NGFI-A is required for serotonin-induced DNA demethylation and increased exon 1(7) GR promoter expression. The data are consistent with the hypothesis that NGFI-A participates in epigenetic programming of GR expression.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Epigénesis Genética/fisiología , Regulación de la Expresión Génica/fisiología , Genes Inmediatos-Precoces/fisiología , Animales , Conducta Animal , Células Cultivadas , Inmunoprecipitación de Cromatina/métodos , Mapeo Cromosómico , Metilación de ADN , Ensayo de Cambio de Movilidad Electroforética/métodos , Embrión de Mamíferos , Exones/fisiología , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Conducta Materna/fisiología , Regiones Promotoras Genéticas/fisiología , Unión Proteica/fisiología , Ratas , Ratas Long-Evans , Receptores de Glucocorticoides/metabolismo , Serotonina/metabolismo , Transfección/métodosRESUMEN
2-Cys peroxiredoxin (2-Cys Prx) is a large group of proteins that participate in cell proliferation, differentiation, apoptosis, and photosynthesis. In the prevailing view, this ubiquitous peroxidase poises the concentration of H2O2 and, in so doing, regulates signal transduction pathways or protects macromolecules against oxidative damage. Here, we describe the first purification of 2-Cys Prx from higher plants and subsequently we show that the native and the recombinant forms of rapeseed leaves stimulate the activity of chloroplast fructose-1,6-bisphosphatase (CFBPase), a key enzyme of the photosynthetic CO2 assimilation. The absence of reductants, the strict requirement of both fructose 1,6-bisphosphate and Ca2+, and the response of single mutants C174S and C179S CFBPase bring forward clear differences with the well-known stimulation mediated by reduced thioredoxin via the regulatory 170's loop of CFBPase. Taken together, these findings provide an unprecedented insight into chloroplast enzyme regulation wherein both 2-Cys Prx and the 170's loop of CFBPase exhibit novel functions.