RESUMEN
The acquisition of cell identity is associated with developmentally regulated changes in the cellular histone methylation signatures. For instance, commitment to neural differentiation relies on the tightly controlled gain or loss of H3K27me3, a hallmark of polycomb-mediated transcriptional gene silencing, at specific gene sets. The KDM6B demethylase, which removes H3K27me3 marks at defined promoters and enhancers, is a key factor in neurogenesis. Therefore, to better understand the epigenetic regulation of neural fate acquisition, it is important to determine how Kdm6b expression is regulated. Here, we investigated the molecular mechanisms involved in the induction of Kdm6b expression upon neural commitment of mouse embryonic stem cells. We found that the increase in Kdm6b expression is linked to a rearrangement between two 3D configurations defined by the promoter contact with two different regions in the Kdm6b locus. This is associated with changes in 5-hydroxymethylcytosine (5hmC) levels at these two regions, and requires a functional ten-eleven-translocation (TET) 3 protein. Altogether, our data support a model whereby Kdm6b induction upon neural commitment relies on an intronic enhancer the activity of which is defined by its TET3-mediated 5-hmC level. This original observation reveals an unexpected interplay between the 5-hmC and H3K27me3 pathways during neural lineage commitment in mammals. It also questions to which extent KDM6B-mediated changes in H3K27me3 level account for the TET-mediated effects on gene expression.
Asunto(s)
Dioxigenasas/metabolismo , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , Neurogénesis , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Células Cultivadas , Dioxigenasas/genética , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Técnicas de Silenciamiento del Gen , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Regulación hacia ArribaRESUMEN
Parental allele-specific expression of imprinted genes is mediated by imprinting control regions (ICRs) that are constitutively marked by DNA methylation imprints on the maternal or paternal allele. Mono-allelic DNA methylation is strictly required for the process of imprinting and has to be faithfully maintained during the entire life-span. While the regulation of DNA methylation itself is well understood, the mechanisms whereby the opposite allele remains unmethylated are unclear. Here, we show that in the mouse, at maternally methylated ICRs, the paternal allele, which is constitutively associated with H3K4me2/3, is marked by default by H3K27me3 when these ICRs are transcriptionally inactive, leading to the formation of a bivalent chromatin signature. Our data suggest that at ICRs, chromatin bivalency has a protective role by ensuring that DNA on the paternal allele remains unmethylated and protected against spurious and unscheduled gene expression. Moreover, they provide the proof of concept that, beside pluripotent cells, chromatin bivalency is the default state of transcriptionally inactive CpG island promoters, regardless of the developmental stage, thereby contributing to protect cell identity.
Asunto(s)
Alelos , Cromatina/metabolismo , Impresión Genómica , Animales , Células Cultivadas , Cromatina/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Células Madre Embrionarias/fisiología , Femenino , Regulación de la Expresión Génica , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Especificidad de Órganos , Regiones Promotoras GenéticasRESUMEN
Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Células Madre Multipotentes/trasplante , Infarto del Miocardio/terapia , Miocitos Cardíacos/citología , Trasplante de Células Madre , Animales , Proteína Morfogenética Ósea 2/farmacología , Diferenciación Celular , Células Cultivadas , Células Madre Embrionarias/citología , Humanos , Antígeno Lewis X/análisis , Macaca mulatta , MicroARNs/análisis , Células Madre Multipotentes/citología , Factor 3 de Transcripción de Unión a Octámeros/análisis , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisisRESUMEN
Oct4 exerts a dose-dependent dual action, as both a gatekeeper for stem cell pluripotency and in driving cells toward specific lineages. Here, we identify the molecular mechanism underlying this dual function. BMP2- or transgene-induced Oct4 up-regulation drives human embryonic and induced pluripotent stem cells to become cardiac progenitors. When embryonic stem cell pluripotency is achieved, Oct4 switches from the Sox2 to the Sox17 promoter. This switch allows the cells to turn off the pluripotency Oct4-Sox2 loop and to turn on the Sox17 promoter. This powerful process generates a subset of endoderm-expressing Sox17 and Hex, both regulators of paracrine signals for cardiogenesis (i.e., Wnt, BMP2) released into the medium surrounding colonies of embryonic stem cells. Our data thus reveal a novel molecular Oct4- and Sox17-mediated mechanism that disrupts the stem cell microenvironment favoring pluripotency to provide a novel paracrine endodermal environment in which cell lineage is determined and commits the cells to a cardiogenic fate.
Asunto(s)
Corazón/embriología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/fisiología , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXF/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Humanos , Factor 3 de Transcripción de Unión a Octámeros/genética , Comunicación Paracrina , Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXF/genéticaRESUMEN
Human embryonic stem (HES) cells are pluripotent and give rise to any cell lineage. More specifically, how the first embryonic lineage (i.e., cardiac lineage) is acquired remains in many aspects questionable. Herein, we summarize the protocols that have been used to direct the fate of HES cells toward the cardiomyocytic lineage. We further discuss the regulation of transcriptional pathways underlying this process of differentiation. Finally, we propose perspectives of this research in the near future.
Asunto(s)
Linaje de la Célula , Células Madre Embrionarias/citología , Miocardio/citología , Humanos , MicroARNs/metabolismoRESUMEN
Muscle wasting, characterized by the loss of protein mass in myofibers, is in most cases largely due to the activation of intracellular protein degradation by the ubiquitin proteasome system (UPS). During the last decade, mechanisms contributing to this activation have been unraveled and key mediators of this process identified. Even though much remains to be understood, the available information already suggests screens for new compounds inhibiting these mechanisms and highlights the potential for pharmaceutical drugs able to treat muscle wasting when it becomes deleterious. This review presents an overview of the main pathways contributing to UPS activation in muscle and describes the present state of efforts made to develop new strategies aimed at blocking or slowing muscle wasting. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Atrofia Muscular/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Animales , Sistemas de Liberación de Medicamentos/tendencias , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Atrofia Muscular/tratamiento farmacológico , Inhibidores de Proteasoma , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidoresRESUMEN
In eukaryotic cells, mRNA localization can provide local protein synthesis. Metallothionein-1 (MT-1) mRNA is associated with the perinuclear cytoskeleton, and this is essential for subsequent nuclear import of the protein. The present study defines the cis-acting localization signal and a trans-acting binding protein. Gel retardation and UV cross-linking assays using MT-1 3'UTR transcripts and CHO cell extracts revealed formation of a complex containing a approximately 50-kDa protein. Only localization-positive mutant transcripts competed for binding of this protein. Using an RNA affinity technique, Western blotting, mass spectrometry, and a supershift assay, the protein was identified as Elongation factor 1alpha (eEF1alpha). Mutation and deletion analysis showed that two regions, nucleotides 21-36 and 66-76, were required for both binding and localization. RNA-folding prediction combined with chemical and enzymatic probing experiments suggest that these regions are in juxtaposition within a stem/internal loop structure. Mutations that are predicted to alter this structure abrogate protein binding. Our hypothesis is that the cis-acting signal in MT-1 3'UTR is formed by this stem/internal loop, that it binds eEF1alpha, and that eEF1alpha-cytoskeleton interactions play a role in perinuclear mRNA localization.
Asunto(s)
Núcleo Celular/metabolismo , Metalotioneína/genética , Factor 1 de Elongación Peptídica/metabolismo , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/química , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Células CHO , Cricetinae , Cartilla de ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Factor 1 de Elongación Peptídica/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/química , ARN Mensajero/genética , Ratas , Transcripción GenéticaRESUMEN
Localization of mRNAs provides a novel mechanism for synthesis of proteins close to their site of function. MT1 (metallothionein-1) is a small, metal-binding protein that is largely cytoplasmic but which can be found in the nucleus. The localization of rat MT1 requires the perinuclear localization of its mRNA by a mechanism dependent on the 3'-UTR (3'-untranslated region). The present study investigates the nature of this mRNA localization signal using Chinese-hamster ovary cells transfected with gene constructs in which either MT1 or the globin coding region is linked to different sequences from the MT1 3'-UTR. Deletion, mutagenesis and antisense oligonucleotide approaches indicate that nt 45-76 of the 3'-UTR, in particular nt 66-76, are required for the localization of either MT1 mRNA or chimaeric transcripts in which a beta-globin coding region is linked to sequences from the MT1 3'-UTR. This section of the 3'-UTR contains a CACC repeat. Two mutations that are predicted to alter the secondary structure of this region also impair localization. Our hypothesis is that the perinuclear localization signal in MT1 mRNA is formed by a combination of the CACC repeat and its structural context.