RESUMEN
Reprogramming somatic cells to induced pluripotency by Yamanaka factors is usually slow and inefficient and is thought to be a stochastic process. We identified a privileged somatic cell state, from which acquisition of pluripotency could occur in a nonstochastic manner. Subsets of murine hematopoietic progenitors are privileged whose progeny cells predominantly adopt the pluripotent fate with activation of endogenous Oct4 locus after four to five divisions in reprogramming conditions. Privileged cells display an ultrafast cell cycle of â¼8 hr. In fibroblasts, a subpopulation cycling at a similar ultrafast speed is observed after 6 days of factor expression and is increased by p53 knockdown. This ultrafast cycling population accounts for >99% of the bulk reprogramming activity in wild-type or p53 knockdown fibroblasts. Our data demonstrate that the stochastic nature of reprogramming can be overcome in a privileged somatic cell state and suggest that cell-cycle acceleration toward a critical threshold is an important bottleneck for reprogramming. PAPERCLIP:
Asunto(s)
Reprogramación Celular , Células Progenitoras de Granulocitos y Macrófagos/citología , Células Madre Pluripotentes Inducidas , Animales , Células de la Médula Ósea , Diferenciación Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Genes p53 , Células Progenitoras de Granulocitos y Macrófagos/metabolismo , RatonesRESUMEN
Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
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Azepinas/farmacología , Encéfalo/patología , Proteínas de Ciclo Celular/metabolismo , Interneuronas/patología , Proteína 2 de Unión a Metil-CpG/fisiología , Síndrome de Rett/patología , Factores de Transcripción/metabolismo , Transcriptoma/efectos de los fármacos , Triazoles/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Femenino , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Factores de Transcripción/genéticaRESUMEN
DNA methylation is essential for a wide variety of biological processes, yet the development of a highly efficient and robust technology remains a challenge for routine single-cell analysis. We developed a multiplex scalable single-cell reduced representation bisulfite sequencing (msRRBS) technology. It allows cell-specific barcoded DNA fragments of individual cells to be pooled before bisulfite conversion, free of enzymatic modification or physical capture of the DNA ends, and achieves read mapping rates of 62.5 ± 3.9%, covering 60.0 ± 1.4% of CpG islands and 71.6 ± 1.6% of promoters in K562 cells. Its reproducibility is shown in duplicates of bulk cells with close to perfect correlation (R = 0.97-0.99). At a low 1 Mb of clean reads, msRRBS provides highly consistent coverage of CpG islands and promoters, outperforming the conventional methods with orders of magnitude reduction in cost. Here, we use this method to characterize the distinct methylation patterns and cellular heterogeneity of six cell lines, plus leukemia and hepatocellular carcinoma models. Taking 4 h of hands-on time, msRRBS offers a unique, highly efficient approach for dissecting methylation heterogeneity in a variety of multicellular systems.
Asunto(s)
Metilación de ADN , ADN , Humanos , Islas de CpG/genética , Metilación de ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Células K562 , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodos , Línea Celular TumoralRESUMEN
Single-cell sequencing is widely used in biological and medical studies. However, its application with multiple samples is hindered by inefficient sample processing, high experimental costs, ambiguous identification of true single cells, and technical batch effects. Here, we introduce sample-multiplexing approaches for single-cell sequencing in transcriptomics, epigenomics, genomics, and multiomics. In single-cell transcriptomics, sample multiplexing uses variants of native or artificial features as sample markers, enabling sample pooling and decoding. Such features include: (1) natural genetic variation, (2) nucleotide-barcode anchoring on cellular or nuclear membranes, (3) nucleotide-barcode internalization to the cytoplasm or nucleus, (4) vector-based barcode expression in cells, and (5) nucleotide-barcode incorporation during library construction. Other single-cell omics methods are based on similar concepts, particularly single-cell combinatorial indexing. These methods overcome current challenges, while enabling super-loading of single cells. Finally, selection guidelines are presented that can accelerate technological application.
Asunto(s)
Genómica , Análisis de la Célula Individual , Epigenómica , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nucleótidos , Análisis de la Célula Individual/métodosRESUMEN
Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by a unique BCR-ABL fusion gene. Tyrosine kinase inhibitors (TKIs) were developed to target the BCR-ABL oncoprotein, inhibiting its abnormal kinase activity. TKI treatments have significantly improved CML patient outcomes. However, the patients can develop drug resistance and relapse after therapy discontinues largely due to intratumor heterogeneity. It is critical to understand the differences in therapeutic responses among subpopulations of cells. Single-cell RNA sequencing measures the transcriptome of individual cells, allowing us to differentiate and analyze individual cell populations. Here, we integrated a single-cell RNA sequencing profile of CML stem cells and network analysis to decipher the mechanisms of distinct TKI responses. Compared to normal hematopoietic stem cells, a set of genes that were concordantly differentially expressed in various types of stem cells of CML patients was revealed. Further transcription regulatory network analysis found that most of these genes were directly controlled by one or more transcript factors and the genes have more regulators in the cells of the patients who responded to the treatment. The molecular markers including a known drug-resistance gene and novel gene signatures for treatment response were also identified. Moreover, we combined protein-protein interaction network construction with a cancer drug database and uncovered the drugs that target the marker genes directly or indirectly via the protein interactions. The gene signatures and their interacted proteins identified by this work can be used for treatment response prediction and lead to new strategies for drug resistance monitoring and prevention. Our single-cell-based findings offered novel insights into the mechanisms underlying the therapeutic response of CML.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Transcriptoma , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Resistencia a Antineoplásicos/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de Fusión bcr-ablRESUMEN
With the advent of next generation high-throughput DNA sequencing technologies, omics experiments have become the mainstay for studying diverse biological effects on a genome wide scale. Chromatin immunoprecipitation (ChIP-seq) is the omics technique that enables genome wide localization of transcription factor (TF) binding or epigenetic modification events. Since the inception of ChIP-seq in 2007, many methods have been developed to infer ChIP-target binding loci from the resultant reads after mapping them to a reference genome. However, interpreting these data has proven challenging, and as such these algorithms have several shortcomings, including susceptibility to false positives due to artifactual peaks, poor localization of binding sites and the requirement for a total DNA input control which increases the cost of performing these experiments. We present Ritornello, a new approach for finding TF-binding sites in ChIP-seq, with roots in digital signal processing that addresses all of these problems. We show that Ritornello generally performs equally or better than the peak callers tested and recommended by the ENCODE consortium, but in contrast, Ritornello does not require a matched total DNA input control to avoid false positives, effectively decreasing the sequencing cost to perform ChIP-seq. Ritornello is freely available at https://github.com/KlugerLab/Ritornello.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Factores de Transcripción/metabolismo , Algoritmos , Artefactos , Sitios de Unión , ADN/química , ADN/metabolismo , Motivos de NucleótidosRESUMEN
Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population.
Asunto(s)
Islas de CpG , Metilación de ADN , Epigénesis Genética , Regiones Promotoras Genéticas , Análisis de la Célula Individual/métodos , Línea Celular , Línea Celular Tumoral , Mapeo Cromosómico , Enzimas de Restricción del ADN/química , Fibroblastos/citología , Fibroblastos/metabolismo , Variación Genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células K562 , Linfocitos/citología , Linfocitos/metabolismoRESUMEN
The HLA-F adjacent transcript 10 (FAT10) is a member of the ubiquitin-like gene family that alters protein function/stability through covalent ligation. Although FAT10 is induced by inflammatory mediators and implicated in immunity, the physiological functions of FAT10 are poorly defined. We report the discovery that FAT10 regulates lifespan through pleiotropic actions on metabolism and inflammation. Median and overall lifespan are increased 20% in FAT10ko mice, coincident with elevated metabolic rate, preferential use of fat as fuel, and dramatically reduced adiposity. This phenotype is associated with metabolic reprogramming of skeletal muscle (i.e., increased AMP kinase activity, ß-oxidation and -uncoupling, and decreased triglyceride content). Moreover, knockout mice have reduced circulating glucose and insulin levels and enhanced insulin sensitivity in metabolic tissues, consistent with elevated IL-10 in skeletal muscle and serum. These observations suggest novel roles of FAT10 in immune metabolic regulation that impact aging and chronic disease.
Asunto(s)
Adiposidad/genética , Longevidad/genética , Ubiquitinas/genética , Adipocitos/metabolismo , Animales , Biomarcadores/metabolismo , Metabolismo Energético , Femenino , Masculino , Ratones , Ratones Noqueados , Oxidación-Reducción , Triglicéridos/metabolismoRESUMEN
Measurement of telomere length currently requires a large population of cells, which masks telomere length heterogeneity in single cells, or requires FISH in metaphase arrested cells, posing technical challenges. A practical method for measuring telomere length in single cells has been lacking. We established a simple and robust approach for single-cell telomere length measurement (SCT-pqPCR). We first optimized a multiplex preamplification specific for telomeres and reference genes from individual cells, such that the amplicon provides a consistent ratio (T/R) of telomeres (T) to the reference genes (R) by quantitative PCR (qPCR). The average T/R ratio of multiple single cells corresponded closely to that of a given cell population measured by regular qPCR, and correlated with those of telomere restriction fragments (TRF) and quantitative FISH measurements. Furthermore, SCT-pqPCR detected the telomere length for quiescent cells that are inaccessible by quantitative FISH. The reliability of SCT-pqPCR also was confirmed using sister cells from two cell embryos. Telomere length heterogeneity was identified by SCT-pqPCR among cells of various human and mouse cell types. We found that the T/R values of human fibroblasts at later passages and from old donors were lower and more heterogeneous than those of early passages and from young donors, that cancer cell lines show heterogeneous telomere lengths, that human oocytes and polar bodies have nearly identical telomere lengths, and that the telomere lengths progressively increase from the zygote, two-cell to four-cell embryo. This method will facilitate understanding of telomere heterogeneity and its role in tumorigenesis, aging, and associated diseases.
Asunto(s)
Blastocisto/metabolismo , Cuerpos Polares/metabolismo , Telómero/metabolismo , Animales , Blastocisto/citología , Células HeLa , Humanos , Ratones , Cuerpos Polares/citología , Reacción en Cadena de la Polimerasa/métodos , Telómero/genéticaRESUMEN
Human embryonic stem cells (hESCs) can be induced and differentiated to form a relatively homogeneous population of neuronal precursors in vitro. We have used this system to screen for genes necessary for neural lineage development by using a pooled human short hairpin RNA (shRNA) library screen and massively parallel sequencing. We confirmed known genes and identified several unpredicted genes with interrelated functions that were specifically required for the formation or survival of neuronal progenitor cells without interfering with the self-renewal capacity of undifferentiated hESCs. Among these are several genes that have been implicated in various neurodevelopmental disorders (i.e., brain malformations, mental retardation, and autism). Unexpectedly, a set of genes mutated in late-onset neurodegenerative disorders and with roles in the formation of RNA granules were also found to interfere with neuronal progenitor cell formation, suggesting their functional relevance in early neurogenesis. This study advances the feasibility and utility of using pooled shRNA libraries in combination with next-generation sequencing for a high-throughput, unbiased functional genomic screen. Our approach can also be used with patient-specific human-induced pluripotent stem cell-derived neural models to obtain unparalleled insights into developmental and degenerative processes in neurological or neuropsychiatric disorders with monogenic or complex inheritance.
Asunto(s)
Diferenciación Celular , Genoma Humano , Neuronas/citología , Células Madre/citología , Trastorno Autístico/genética , Silenciador del Gen , Marcación de Gen , Humanos , Discapacidad Intelectual/genética , Neuronas/metabolismo , ARN/metabolismo , Células Madre/metabolismoRESUMEN
The ability to determine the gene expression pattern in low quantities of cells or single cells is important for resolving a variety of problems in many biological disciplines. A robust description of the expression signature of a single cell requires determination of the full-length sequence of the expressed mRNAs in the cell, yet existing methods have either 3' biased or variable transcript representation. Here, we report our protocols for the amplification and high-throughput sequencing of very small amounts of RNA for sequencing using procedures of either semirandom primed PCR or phi29 DNA polymerase-based DNA amplification, for the cDNA generated with oligo-dT and/or random oligonucleotide primers. Unlike existing methods, these protocols produce relatively uniformly distributed sequences covering the full length of almost all transcripts independent of their sizes, from 1,000 to 10 cells, and even with single cells. Both protocols produced satisfactory detection/coverage of the abundant mRNAs from a single K562 erythroleukemic cell or a single dorsal root ganglion neuron. The phi29-based method produces long products with less noise, uses an isothermal reaction, and is simple to practice. The semirandom primed PCR procedure is more sensitive and reproducible at low transcript levels or with low quantities of cells. These methods provide tools for mRNA sequencing or RNA sequencing when only low quantities of cells, a single cell, or even degraded RNA are available for profiling.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Mensajero/genética , Análisis de la Célula Individual/métodos , Cartilla de ADN/genética , Humanos , Células K562 , Reacción en Cadena de la Polimerasa/métodosRESUMEN
PPARGC1A is a transcriptional coactivator that binds to and coactivates a variety of transcription factors (TFs) to regulate the expression of target genes. PPARGC1A plays a pivotal role in regulating energy metabolism and has been implicated in several human diseases, most notably type II diabetes. Previous studies have focused on the interplay between PPARGC1A and individual TFs, but little is known about how PPARGC1A combines with all of its partners across the genome to regulate transcriptional dynamics. In this study, we describe a core PPARGC1A transcriptional regulatory network operating in HepG2 cells treated with forskolin. We first mapped the genome-wide binding sites of PPARGC1A using chromatin-IP followed by high-throughput sequencing (ChIP-seq) and uncovered overrepresented DNA sequence motifs corresponding to known and novel PPARGC1A network partners. We then profiled six of these site-specific TF partners using ChIP-seq and examined their network connectivity and combinatorial binding patterns with PPARGC1A. Our analysis revealed extensive overlap of targets including a novel link between PPARGC1A and HSF1, a TF regulating the conserved heat shock response pathway that is misregulated in diabetes. Importantly, we found that different combinations of TFs bound to distinct functional sets of genes, thereby helping to reveal the combinatorial regulatory code for metabolic and other cellular processes. In addition, the different TFs often bound near the promoters and coding regions of each other's genes suggesting an intricate network of interdependent regulation. Overall, our study provides an important framework for understanding the systems-level control of metabolic gene expression in humans.
Asunto(s)
Redes Reguladoras de Genes , Proteínas de Choque Térmico/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión/genética , Proteínas Portadoras/metabolismo , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Regulación de la Expresión Génica , Células Hep G2 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Motivos de Nucleótidos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Unión Proteica/genética , Transporte de Proteínas , Transcripción GenéticaRESUMEN
Genetic variation between individuals has been extensively investigated, but differences between tissues within individuals are far less understood. It is commonly assumed that all healthy cells that arise from the same zygote possess the same genomic content, with a few known exceptions in the immune system and germ line. However, a growing body of evidence shows that genomic variation exists between differentiated tissues. We investigated the scope of somatic genomic variation between tissues within humans. Analysis of copy number variation by high-resolution array-comparative genomic hybridization in diverse tissues from six unrelated subjects reveals a significant number of intraindividual genomic changes between tissues. Many (79%) of these events affect genes. Our results have important consequences for understanding normal genetic and phenotypic variation within individuals, and they have significant implications for both the etiology of genetic diseases such as cancer and for immortalized cell lines that might be used in research and therapeutics.
Asunto(s)
Variación Genética , Hibridación Genómica Comparativa , Dosificación de Gen , HumanosRESUMEN
Two mechanisms that play important roles in cell fate decisions are control of a "core transcriptional network" and repression of alternative transcriptional programs by antagonizing transcription factors. Whether these two mechanisms operate together is not known. Here we report that GATA-1, SCL, and Klf1 form an erythroid core transcriptional network by co-occupying >300 genes. Importantly, we find that PU.1, a negative regulator of terminal erythroid differentiation, is a highly integrated component of this network. GATA-1, SCL, and Klf1 act to promote, whereas PU.1 represses expression of many of the core network genes. PU.1 also represses the genes encoding GATA-1, SCL, Klf1, and important GATA-1 cofactors. Conversely, in addition to repressing PU.1 expression, GATA-1 also binds to and represses >100 PU.1 myelo-lymphoid gene targets in erythroid progenitors. Mathematical modeling further supports that this dual mechanism of repressing both the opposing upstream activator and its downstream targets provides a synergistic, robust mechanism for lineage specification. Taken together, these results amalgamate two key developmental principles, namely, regulation of a core transcriptional network and repression of an alternative transcriptional program, thereby enhancing our understanding of the mechanisms that establish cellular identity.
Asunto(s)
Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Linfocitos/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Inmunoprecipitación de Cromatina , Eritrocitos , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Modelos Teóricos , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/citología , Proteína 1 de la Leucemia Linfocítica T Aguda , Transactivadores/metabolismo , Transcripción GenéticaRESUMEN
Since the creation of Dolly via somatic cell nuclear transfer (SCNT), more than a dozen species of mammals have been cloned using this technology. One hypothesis for the limited success of cloning via SCNT (1%-5%) is that the clones are likely to be derived from adult stem cells. Support for this hypothesis comes from the findings that the reproductive cloning efficiency for embryonic stem cells is five to ten times higher than that for somatic cells as donors and that cloned pups cannot be produced directly from cloned embryos derived from differentiated B and T cells or neuronal cells. The question remains as to whether SCNT-derived animal clones can be derived from truly differentiated somatic cells. We tested this hypothesis with mouse hematopoietic cells at different differentiation stages: hematopoietic stem cells, progenitor cells and granulocytes. We found that cloning efficiency increases over the differentiation hierarchy, and terminally differentiated postmitotic granulocytes yield cloned pups with the greatest cloning efficiency.
Asunto(s)
Células Madre Adultas/fisiología , Diferenciación Celular/fisiología , Clonación de Organismos/métodos , Células Madre Hematopoyéticas/citología , Técnicas de Transferencia Nuclear , Células Madre Adultas/citología , Animales , Embrión de Mamíferos/citología , Femenino , Perfilación de la Expresión Génica , Granulocitos/citología , Granulocitos/fisiología , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Modelos Biológicos , Embarazo , Células Madre/citología , Células Madre/fisiologíaRESUMEN
DNA replication, repair, transcription and chromatin structure are intricately associated nuclear processes, but the molecular links between these events are often obscure. In this study, we have surveyed the protein complexes that bind at ß-globin locus control region, and purified and characterized the function of one such multiprotein complex from human erythroleukemic K562 cells. We further validated the existence of this complex in human CD34+ cell-derived normal erythroid cells. This complex contains ILF2/ILF3 transcription factors, p300 acetyltransferase and proteins associated with DNA replication, transcription and repair. RNAi knockdown of ILF2, a DNA-binding component of this complex, abrogates the recruitment of the complex to its cognate DNA sequence and inhibits transcription, histone acetylation and usage of the origin of DNA replication at the ß-globin locus. These results imply a direct link between mammalian DNA replication, transcription and histone acetylation mediated by a single multiprotein complex.
Asunto(s)
Reparación del ADN/fisiología , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Transcripción Genética/fisiología , Globinas beta/metabolismo , Línea Celular Tumoral , Reparación del ADN/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Humanos , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/fisiología , Proteína del Factor Nuclear 45/genética , Proteína del Factor Nuclear 45/metabolismo , Proteínas del Factor Nuclear 90/genética , Proteínas del Factor Nuclear 90/metabolismo , Oligonucleótidos/genética , Interferencia de ARN , Transcripción Genética/genética , Globinas beta/genética , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
HOTAIRM1 is a long intergenic non-coding RNA encoded in the human HOXA gene cluster, with gene expression highly specific for maturing myeloid cells. Knockdown of HOTAIRM1 in the NB4 acute promyelocytic leukemia cell line retarded all-trans retinoid acid (ATRA)-induced granulocytic differentiation, resulting in a significantly larger population of immature and proliferating cells that maintained cell cycle progression from G1 to S phases. Correspondingly, HOTAIRM1 knockdown resulted in retained expression of many otherwise ATRA-suppressed cell cycle and DNA replication genes, and abated ATRA induction of cell surface leukocyte activation, defense response, and other maturation-related genes. Resistance to ATRA-induced cell cycle arrest at the G1/S phase transition in knockdown cells was accompanied by retained expression of ITGA4 (CD49d) and decreased induction of ITGAX (CD11c). The coupling of cell cycle progression with temporal dynamics in the expression patterns of these integrin genes suggests a regulated switch to control the transit from the proliferative phase to granulocytic maturation. Furthermore, ITGAX was among a small number of genes showing perturbation in transcript levels upon HOTAIRM1 knockdown even without ATRA treatment, suggesting a direct pathway of regulation. These results indicate that HOTAIRM1 provides a regulatory link in myeloid maturation by modulating integrin-controlled cell cycle progression at the gene expression level.
Asunto(s)
Ciclo Celular/genética , Diferenciación Celular/genética , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , ARN Largo no Codificante/genética , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Integrina alfa4/genética , Integrina alfa4/metabolismo , Leucemia Promielocítica Aguda/patología , Tretinoina/farmacologíaRESUMEN
Erythroid myeloid lymphoid (EML) cells are an established multipotent hematopoietic precursor cell line that can be maintained in medium including stem cell factor (SCF). EML cultures contain a heterogeneous mixture of cells, including a lineage-negative, CD34+ subset of cells that propagate rapidly in SCF and can clonally regenerate the mixed population. A second major subset of EML cells consists of lineage-negative. CD34- cells that can be propagated in IL-3 but grow slowly, if at all, in SCF, although they express the SCF receptor (c-kit). The response of these cells to IL-3 is stimulated synergistically by SCF, and we present evidence that both the synergy and the inhibition of c-kit responses may be mediated by direct interaction with IL-3 receptor. Further, the relative level of tyrosine phosphorylation of various substrates by either cytokine alone differs from that produced by the combination of the two cytokines, suggesting that cell signaling by the combination of the two cytokines differs from that produced by either alone.
Asunto(s)
Antígenos CD34 , Células Madre Hematopoyéticas/citología , Interleucina-3/farmacología , Factor de Células Madre/farmacología , Animales , Línea Celular , Células Madre Hematopoyéticas/metabolismo , Humanos , Interleucina-3/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de Interleucina-3/metabolismo , Factor de Células Madre/metabolismoRESUMEN
The DNA methylation status of human X chromosomes from male and female neutrophils was identified by high-throughput sequencing of HpaII and MspI digested fragments. In the intergenic and intragenic regions on the X chromosome, the sites outside CpG islands were heavily hypermethylated to the same degree in both genders. Nearly half of X chromosome promoters were either hypomethylated or hypermethylated in both females and males. Nearly one third of X chromosome promoters were a mixture of hypomethylated and heterogeneously methylated sites in females and were hypomethylated in males. Thus, a large fraction of genes that are silenced on the inactive X chromosome are hypomethylated in their promoter regions. These genes frequently belong to the evolutionarily younger strata of the X chromosome. The promoters that were hypomethylated at more than two sites contained most of the genes that escaped silencing on the inactive X chromosome. The overall levels of expression of X-linked genes were indistinguishable in females and males, regardless of the methylation state of the inactive X chromosome. Thus, in addition to DNA methylation, other factors are involved in the fine tuning of gene dosage compensation in neutrophils.
Asunto(s)
Cromosomas Humanos X/genética , Metilación de ADN , Regulación de la Expresión Génica , Expresión Génica , Genes Ligados a X , Femenino , Humanos , Masculino , Neutrófilos/metabolismo , Regiones Promotoras Genéticas , Factores SexualesRESUMEN
Epstein-Barr virus (EBV) is associated with several types of lymphomas and epithelial tumors including Burkitt's lymphoma (BL), HIV-associated lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. EBV nuclear antigen 1 (EBNA1) is expressed in all EBV associated tumors and is required for latency and transformation. EBNA1 initiates latent viral replication in B cells, maintains the viral genome copy number, and regulates transcription of other EBV-encoded latent genes. These activities are mediated through the ability of EBNA1 to bind viral-DNA. To further elucidate the role of EBNA1 in the host cell, we have examined the effect of EBNA1 on cellular gene expression by microarray analysis using the B cell BJAB and the epithelial 293 cell lines transfected with EBNA1. Analysis of the data revealed distinct profiles of cellular gene changes in BJAB and 293 cell lines. Subsequently, chromatin immune-precipitation revealed a direct binding of EBNA1 to cellular promoters. We have correlated EBNA1 bound promoters with changes in gene expression. Sequence analysis of the 100 promoters most enriched revealed a DNA motif that differs from the EBNA1 binding site in the EBV genome.