RESUMEN
Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.
Asunto(s)
Neoplasias Encefálicas/inmunología , Epigénesis Genética , Glioblastoma/inmunología , Evasión Inmune/inmunología , Células Mieloides/inmunología , Células Madre Neoplásicas/inmunología , Microambiente Tumoral/inmunología , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proliferación Celular , Metilación de ADN , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Mieloides/metabolismo , Células Mieloides/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state.
Asunto(s)
Células Madre Embrionarias/citología , Genes myc , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Blastocisto/metabolismo , Proliferación Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , Técnicas de Inactivación de Genes , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Técnicas Citológicas , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Humanos , Factor 4 Similar a Kruppel , Ratones , Mitocondrias/metabolismo , Proteína Homeótica Nanog , Células Madre Pluripotentes/citología , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Chromatin remodeling complexes play essential roles in metazoan development through widespread control of gene expression, but the precise molecular mechanisms by which they do this in vivo remain ill defined. Using an inducible system with fine temporal resolution, we show that the nucleosome remodeling and deacetylation (NuRD) complex controls chromatin architecture and the protein binding repertoire at regulatory regions during cell state transitions. This is primarily exerted through its nucleosome remodeling activity while deacetylation at H3K27 follows changes in gene expression. Additionally, NuRD activity influences association of RNA polymerase II at transcription start sites and subsequent nascent transcript production, thereby guiding the establishment of lineage-appropriate transcriptional programs. These findings provide a detailed molecular picture of genome-wide modulation of lineage-specific transcription by an essential chromatin remodeling complex as well as insight into the orchestration of molecular events involved in transcriptional transitions in vivo. VIDEO ABSTRACT.
Asunto(s)
Regulación de la Expresión Génica , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Acetilación , Animales , Línea Celular , Histonas/genética , Histonas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Ratones , Células Madre Embrionarias de Ratones/citología , Nucleosomas/genética , ARN Polimerasa II/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Glioblastoma multiforme (GBM) is an aggressive brain tumor driven by cells with hallmarks of neural stem (NS) cells. GBM stem cells frequently express high levels of the transcription factors FOXG1 and SOX2. Here we show that increased expression of these factors restricts astrocyte differentiation and can trigger dedifferentiation to a proliferative NS cell state. Transcriptional targets include cell cycle and epigenetic regulators (e.g., Foxo3, Plk1, Mycn, Dnmt1, Dnmt3b, and Tet3). Foxo3 is a critical repressed downstream effector that is controlled via a conserved FOXG1/SOX2-bound cis-regulatory element. Foxo3 loss, combined with exposure to the DNA methylation inhibitor 5-azacytidine, enforces astrocyte dedifferentiation. DNA methylation profiling in differentiating astrocytes identifies changes at multiple polycomb targets, including the promoter of Foxo3 In patient-derived GBM stem cells, CRISPR/Cas9 deletion of FOXG1 does not impact proliferation in vitro; however, upon transplantation in vivo, FOXG1-null cells display increased astrocyte differentiation and up-regulate FOXO3. In contrast, SOX2 ablation attenuates proliferation, and mutant cells cannot be expanded in vitro. Thus, FOXG1 and SOX2 operate in complementary but distinct roles to fuel unconstrained self-renewal in GBM stem cells via transcriptional control of core cell cycle and epigenetic regulators.
Asunto(s)
Neoplasias Encefálicas/fisiopatología , Epigenómica , Factores de Transcripción Forkhead/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/fisiopatología , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/citología , Factores de Transcripción SOXB1/genética , Secuencias de Aminoácidos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Azacitidina/farmacología , Neoplasias Encefálicas/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Cromatina/metabolismo , Metilación de ADN , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/genética , Humanos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Factores de Transcripción SOXB1/metabolismo , Células Tumorales CultivadasRESUMEN
DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals.
Asunto(s)
Núcleo Celular/metabolismo , Momento de Replicación del ADN , Proteínas de Unión a Telómeros/metabolismo , Animales , Proliferación Celular , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Islas de CpG/genética , Fase G1 , Eliminación de Gen , Regulación de la Expresión Génica , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Unión a Telómeros/química , Sitio de Iniciación de la TranscripciónRESUMEN
Dosage compensation, mediated by the MSL complex, regulates X-chromosomal gene expression in Drosophila. Here we report that the histone H4 lysine 16 (H4K16) specific histone acetyltransferase MOF displays differential binding behavior depending on whether the target gene is located on the X chromosome versus the autosomes. More specifically, on the male X chromosome, where MSL1 and MSL3 are preferentially associated with the 3' end of dosage compensated genes, MOF displays a bimodal distribution binding to promoters and the 3' ends of genes. In contrast, on MSL1/MSL3 independent X-linked genes and autosomal genes in males and females, MOF binds primarily to promoters. Binding of MOF to autosomes is functional, as H4K16 acetylation and the transcription levels of a number of genes are affected upon MOF depletion. Therefore, MOF is not only involved in the onset of dosage compensation, but also acts as a regulator of gene expression in the Drosophila genome.
Asunto(s)
Compensación de Dosificación (Genética) , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica , Histona Acetiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Región de Flanqueo 3' , Acetilación , Animales , Línea Celular , Femenino , Genoma de los Insectos , Histonas/genética , Histonas/metabolismo , Masculino , Regiones Promotoras Genéticas , Cromosoma XRESUMEN
Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies have provided transcriptome data from human pre-implantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors, such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here, we address these issues to assemble a complete gene expression time course spanning human pre-implantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post-hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naïve state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.
Asunto(s)
Blastocisto/citología , Blastocisto/metabolismo , Animales , Masa Celular Interna del Blastocisto/citología , Masa Celular Interna del Blastocisto/metabolismo , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Mapeo Cromosómico , Técnicas de Cultivo de Embriones , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Perfilación de la Expresión Génica , Marcadores Genéticos , Estratos Germinativos/citología , Estratos Germinativos/embriología , Estratos Germinativos/metabolismo , Humanos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Primates , Análisis de la Célula IndividualRESUMEN
The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single cell RNA-seq. Zygotic genome activation correlates with the presence of polycomb repressive complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species, stage and lineage specific. The pluripotency network in the primate epiblast lacks certain regulators that are operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and non-human primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development, and underscores the molecular adaptability of early mammalian embryogenesis.
Asunto(s)
Callithrix/genética , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Análisis de la Célula Individual , Transcriptoma/genética , Animales , Secuencia Conservada/genética , Endodermo/metabolismo , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Estratos Germinativos/metabolismo , Humanos , Ratones , Factores de Transcripción Otx , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Transcripción GenéticaRESUMEN
Epigenetic changes are frequently observed in cancer. However, their role in establishing or sustaining the malignant state has been difficult to determine due to the lack of experimental tools that enable resetting of epigenetic abnormalities. To address this, we applied induced pluripotent stem cell (iPSC) reprogramming techniques to invoke widespread epigenetic resetting of glioblastoma (GBM)-derived neural stem (GNS) cells. GBM iPSCs (GiPSCs) were subsequently redifferentiated to the neural lineage to assess the impact of cancer-specific epigenetic abnormalities on tumorigenicity. GiPSCs and their differentiating derivatives display widespread resetting of common GBM-associated changes, such as DNA hypermethylation of promoter regions of the cell motility regulator TES (testis-derived transcript), the tumor suppressor cyclin-dependent kinase inhibitor 1C (CDKN1C; p57KIP2), and many polycomb-repressive complex 2 (PRC2) target genes (e.g., SFRP2). Surprisingly, despite such global epigenetic reconfiguration, GiPSC-derived neural progenitors remained highly malignant upon xenotransplantation. Only when GiPSCs were directed to nonneural cell types did we observe sustained expression of reactivated tumor suppressors and reduced infiltrative behavior. These data suggest that imposing an epigenome associated with an alternative developmental lineage can suppress malignant behavior. However, in the context of the neural lineage, widespread resetting of GBM-associated epigenetic abnormalities is not sufficient to override the cancer genome.
Asunto(s)
Reprogramación Celular/genética , Metilación de ADN , Epigénesis Genética , Glioblastoma/patología , Células-Madre Neurales/citología , Animales , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Transformación Celular Neoplásica/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Humanos , Ratones , Ratones Endogámicos NOD , Células Madre Pluripotentes/citología , Trasplante HeterólogoRESUMEN
Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24â h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.
Asunto(s)
Elementos Transponibles de ADN/genética , Epigénesis Genética/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Metilación de ADN/genética , Metilación de ADN/fisiología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Genes Ligados a X/genética , Humanos , Hibridación Fluorescente in Situ , Ratones , Reacción en Cadena en Tiempo Real de la Polimerasa , Inactivación del Cromosoma X/genéticaRESUMEN
Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.
Asunto(s)
Rastreo Celular , Células Madre Embrionarias/citología , Células Madre Pluripotentes/citología , Animales , Linaje de la Célula , Autorrenovación de las Células , Metilación de ADN/genética , Regulación hacia Abajo , Embrión de Mamíferos/citología , Células Madre Embrionarias/metabolismo , Genes Reporteros , Estratos Germinativos/citología , Cinética , Ratones , Células Madre Pluripotentes/metabolismo , Trasplante de Células Madre , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Citrulina/metabolismo , Histonas/química , Histonas/metabolismo , Células Madre Pluripotentes/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Arginina/química , Arginina/metabolismo , Sitios de Unión , Reprogramación Celular/genética , Cromatina/química , ADN/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica , Hidrolasas/metabolismo , Ratones , Células Madre Pluripotentes/citología , Unión Proteica , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica , Proteómica , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Sall4 is an essential transcription factor for early mammalian development and is frequently overexpressed in cancer. Although it is reported to play an important role in embryonic stem cell (ESC) self-renewal, whether it is an essential pluripotency factor has been disputed. Here, we show that Sall4 is dispensable for mouse ESC pluripotency. Sall4 is an enhancer-binding protein that prevents precocious activation of the neural gene expression programme in ESCs but is not required for maintenance of the pluripotency gene regulatory network. Although a proportion of Sall4 protein physically associates with the Nucleosome Remodelling and Deacetylase (NuRD) complex, Sall4 neither recruits NuRD to chromatin nor influences transcription via NuRD; rather, free Sall4 protein regulates transcription independently of NuRD. We propose a model whereby enhancer binding by Sall4 and other pluripotency-associated transcription factors is responsible for maintaining the balance between transcriptional programmes in pluripotent cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Inmunoprecipitación de Cromatina , Biología Computacional , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Espectrometría de Masas , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones , Nucleosomas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
Digital production, transmission and storage have revolutionized how we access and use information but have also made archiving an increasingly complex task that requires active, continuing maintenance of digital media. This challenge has focused some interest on DNA as an attractive target for information storage because of its capacity for high-density information encoding, longevity under easily achieved conditions and proven track record as an information bearer. Previous DNA-based information storage approaches have encoded only trivial amounts of information or were not amenable to scaling-up, and used no robust error-correction and lacked examination of their cost-efficiency for large-scale information archival. Here we describe a scalable method that can reliably store more information than has been handled before. We encoded computer files totalling 739 kilobytes of hard-disk storage and with an estimated Shannon information of 5.2 × 10(6) bits into a DNA code, synthesized this DNA, sequenced it and reconstructed the original files with 100% accuracy. Theoretical analysis indicates that our DNA-based storage scheme could be scaled far beyond current global information volumes and offers a realistic technology for large-scale, long-term and infrequently accessed digital archiving. In fact, current trends in technological advances are reducing DNA synthesis costs at a pace that should make our scheme cost-effective for sub-50-year archiving within a decade.
Asunto(s)
Archivos , ADN/química , ADN/síntesis química , Gestión de la Información/métodos , Secuencia de Bases , Computadores , ADN/economía , Gestión de la Información/economía , Datos de Secuencia Molecular , Análisis de Secuencia de ADN/economía , Biología Sintética/economía , Biología Sintética/métodosRESUMEN
Self-renewal of pluripotent mouse embryonic stem (ES) cells is sustained by the cytokine leukaemia inhibitory factor (LIF) acting through the transcription factor Stat3. Several targets of Stat3 have previously been identified, most notably the reprogramming factor Klf4. However, such factors are neither required nor sufficient for the potent effect of LIF. We took advantage of Stat3 null ES cells to confirm that Stat3 mediates the self-renewal response to LIF. Through comparative transcriptome analysis intersected with genome location data, we arrived at a set of candidate transcription factor effectors. Among these, Tfcp2l1 (also known as Crtr-1) was most abundant. Constitutive expression of Tfcp2l1 at levels similar to those induced by LIF effectively substituted for LIF or Stat3 in sustaining clonal self-renewal and pluripotency. Conversely, knockdown of Tfcp2l1 profoundly compromised responsiveness to LIF. We further found that Tfcp2l1 is both necessary and sufficient to direct molecular reprogramming of post-implantation epiblast stem cells to naïve pluripotency. These results establish Tfcp2l1 as the principal bridge between LIF/Stat3 input and the transcription factor core of naïve pluripotency.
Asunto(s)
Células Madre Embrionarias/metabolismo , Factor Inhibidor de Leucemia/metabolismo , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Células Cultivadas , Células Madre Embrionarias/citología , Factor 4 Similar a Kruppel , Ratones , Células Madre Pluripotentes/citología , Factor de Transcripción STAT3/genéticaRESUMEN
Pluripotent cells possess the ability to differentiate into any cell type. Commitment to differentiate into specific lineages requires strict control of gene expression to coordinate the downregulation of lineage inappropriate genes while enabling the expression of lineage-specific genes. The nucleosome remodelling and deacetylation complex (NuRD) is required for lineage commitment of pluripotent cells; however, the mechanism through which it exerts this effect has not been defined. Here, we show that histone deacetylation by NuRD specifies recruitment for Polycomb Repressive Complex 2 (PRC2) in embryonic stem (ES) cells. NuRD-mediated deacetylation of histone H3K27 enables PRC2 recruitment and subsequent H3K27 trimethylation at NuRD target promoters. We propose a gene-specific mechanism for modulating expression of transcriptionally poised genes whereby NuRD controls the balance between acetylation and methylation of histones, thereby precisely directing the expression of genes critical for embryonic development.