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
BACKGROUND: Vascular smooth muscle cell (VSMC) phenotypic switching contributes to cardiovascular diseases. Epigenetic regulation is emerging as a key regulatory mechanism, with the methylcytosine dioxygenase TET2 acting as a master regulator of smooth muscle cell phenotype. The histone acetyl-transferases p300 and CREB-binding protein (CBP) are highly homologous and often considered to be interchangeable, and their roles in smooth muscle cell phenotypic regulation are not known. METHODS: We assessed the roles of p300 and CBP in human VSMC with knockdown, in inducible smooth muscle-specific knockout mice (inducible knockout [iKO]; p300iKO or CBPiKO), and in samples of human intimal hyperplasia. RESULTS: P300, CBP, and histone acetylation were differently regulated in VSMCs undergoing phenotypic switching and in vessel remodeling after vascular injury. Medial p300 expression and activity were repressed by injury, but CBP and histone acetylation were induced in neointima. Knockdown experiments revealed opposing effects of p300 and CBP in the VSMC phenotype: p300 promoted contractile protein expression and inhibited migration, but CBP inhibited contractile genes and enhanced migration. p300iKO mice exhibited severe intimal hyperplasia after arterial injury compared with controls, whereas CBPiKO mice were entirely protected. In normal aorta, p300iKO reduced, but CBPiKO enhanced, contractile protein expression and contractility compared with controls. Mechanistically, we found that these histone acetyl-transferases oppositely regulate histone acetylation, DNA hydroxymethylation, and PolII (RNA polymerase II) binding to promoters of differentiation-specific contractile genes. Our data indicate that p300 and TET2 function together, because p300 was required for TET2-dependent hydroxymethylation of contractile promoters, and TET2 was required for p300-dependent acetylation of these loci. TET2 coimmunoprecipitated with p300, and this interaction was enhanced by rapamycin but repressed by platelet-derived growth factor (PDGF) treatment, with p300 promoting TET2 protein stability. CBP did not associate with TET2, but instead facilitated recruitment of histone deacetylases (HDAC2, HDAC5) to contractile protein promoters. Furthermore, CBP inhibited TET2 mRNA levels. Immunostaining of cardiac allograft vasculopathy samples revealed that p300 expression is repressed but CBP is induced in human intimal hyperplasia. CONCLUSIONS: This work reveals that p300 and CBP serve nonredundant and opposing functions in VSMC phenotypic switching and coordinately regulate chromatin modifications through distinct functional interactions with TET2 or HDACs. Targeting specific histone acetyl-transferases may hold therapeutic promise for cardiovascular diseases.
Asunto(s)
Enfermedades Cardiovasculares , Músculo Liso Vascular , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Enfermedades Cardiovasculares/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Contráctiles/metabolismo , Epigénesis Genética , Histonas/metabolismo , Humanos , Hiperplasia/metabolismo , Ratones , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
The terminal maturation of human erythroblasts requires significant changes in gene expression in the context of dramatic nuclear condensation. Defects in this process are associated with inherited anemias and myelodysplastic syndromes. The progressively dense appearance of the condensing nucleus in maturing erythroblasts led to the assumption that heterochromatin accumulation underlies this process, but despite extensive study, the precise mechanisms underlying this essential biologic process remain elusive. To delineate the epigenetic changes associated with the terminal maturation of human erythroblasts, we performed mass spectrometry of histone posttranslational modifications combined with chromatin immunoprecipitation coupled with high-throughput sequencing, Assay for Transposase Accessible Chromatin, and RNA sequencing. Our studies revealed that the terminal maturation of human erythroblasts is associated with a dramatic decline in histone marks associated with active transcription elongation, without accumulation of heterochromatin. Chromatin structure and gene expression were instead correlated with dynamic changes in occupancy of elongation competent RNA polymerase II, suggesting that terminal erythroid maturation is controlled largely at the level of transcription. We further demonstrate that RNA polymerase II "pausing" is highly correlated with transcriptional repression, with elongation competent RNA polymerase II becoming a scare resource in late-stage erythroblasts, allocated to erythroid-specific genes. Functional studies confirmed an essential role for maturation stage-specific regulation of RNA polymerase II activity during erythroid maturation and demonstrate a critical role for HEXIM1 in the regulation of gene expression and RNA polymerase II activity in maturing erythroblasts. Taken together, our findings reveal important insights into the mechanisms that regulate terminal erythroid maturation and provide a novel paradigm for understanding normal and perturbed erythropoiesis.
Asunto(s)
Eritroblastos/metabolismo , Células Eritroides/metabolismo , ARN Polimerasa II/metabolismo , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Eritroblastos/citología , Células Eritroides/citología , Eritropoyesis , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , ARN Polimerasa II/genética , Transcripción GenéticaRESUMEN
Histone deacetylases (HDACs) are a group of enzymes that catalyze the removal of acetyl groups from histone and nonhistone proteins. HDACs have been shown to have diverse functions in a wide range of biological processes. However, their roles in mammalian erythropoiesis remain to be fully defined. This study showed that, of the 11 classic HDAC family members, 6 (HDAC1, -2, -3, and HDAC5, -6, -7) are expressed in human erythroid cells, with HDAC5 most significantly upregulated during terminal erythroid differentiation. Knockdown of HDAC5 by either short hairpin RNA or small interfering RNA in human CD34+ cells followed by erythroid cell culture led to increased apoptosis, decreased chromatin condensation, and impaired enucleation of erythroblasts. Biochemical analyses revealed that HDAC5 deficiency resulted in activation of p53 in association with increased acetylation of p53. Furthermore, although acetylation of histone 4 (H4) is decreased during normal terminal erythroid differentiation, HDAC5 deficiency led to increased acetylation of H4 (K12) in late-stage erythroblasts. This increased acetylation was accompanied by decreased chromatin condensation, implying a role for H4 (K12) deacetylation in chromatin condensation. ATAC-seq and RNA sequencing analyses revealed that HDAC5 knockdown leads to increased chromatin accessibility genome-wide and global changes in gene expression. Moreover, pharmacological inhibition of HDAC5 by the inhibitor LMK235 also led to increased H4 acetylation, impaired chromatin condensation, and enucleation. Taken together, our findings have uncovered previously unrecognized roles and molecular mechanisms of action for HDAC5 in human erythropoiesis. These results may provide insights into understanding the anemia associated with HDAC inhibitor treatment.
Asunto(s)
Células Eritroides/citología , Eritropoyesis , Histona Desacetilasas/genética , Apoptosis , Eritroblastos/citología , Eritroblastos/metabolismo , Células Eritroides/metabolismo , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genética , Regulación hacia ArribaRESUMEN
Follicular helper T (Tfh) cells are necessary for germinal center B cell maturation during primary immune responses; however, the T cells that promote humoral recall responses via memory B cells are less well defined. In this article, we characterize a human tonsillar CD4+ T cell subset with this function. These cells are similar to Tfh cells in terms of expression of the chemokine receptor CXCR5 and the inhibitory receptor PD-1, IL-21 secretion, and expression of the transcription factor BCL6; however, unlike Tfh cells that are located within the B cell follicle and germinal center, they reside at the border of the T cell zone and the B cell follicle in proximity to memory B cells, a position dictated by their unique chemokine receptor expression. They promote memory B cells to produce Abs via CD40L, IL-10, and IL-21. Our results reveal a unique extrafollicular CD4+ T cell subset in human tonsils, which specialize in promoting T cell-dependent humoral recall responses.
Asunto(s)
Formación de Anticuerpos , Linfocitos B/inmunología , Inmunoglobulina G/inmunología , Memoria Inmunológica , Linfocitos T Colaboradores-Inductores/inmunología , Adolescente , Linfocitos B/citología , Niño , Preescolar , Citocinas/inmunología , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Masculino , Receptor de Muerte Celular Programada 1/inmunología , Proteínas Proto-Oncogénicas c-bcl-6/inmunología , Receptores CXCR5/inmunología , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
Tactile-foraging ducks are specialist birds known for their touch-dependent feeding behavior. They use dabbling, straining, and filtering to find edible matter in murky water, relying on the sense of touch in their bill. Here, we present the molecular characterization of embryonic duck bill, which we show contains a high density of mechanosensory corpuscles innervated by functional rapidly adapting trigeminal afferents. In contrast to chicken, a visually foraging bird, the majority of duck trigeminal neurons are mechanoreceptors that express the Piezo2 ion channel and produce slowly inactivating mechano-current before hatching. Furthermore, duck neurons have a significantly reduced mechano-activation threshold and elevated mechano-current amplitude. Cloning and electrophysiological characterization of duck Piezo2 in a heterologous expression system shows that duck Piezo2 is functionally similar to the mouse ortholog but with prolonged inactivation kinetics, particularly at positive potentials. Knockdown of Piezo2 in duck trigeminal neurons attenuates mechano current with intermediate and slow inactivation kinetics. This suggests that Piezo2 is capable of contributing to a larger range of mechano-activated currents in duck trigeminal ganglia than in mouse trigeminal ganglia. Our results provide insights into the molecular basis of mechanotransduction in a tactile-specialist vertebrate.
Asunto(s)
Proteínas Aviares/genética , Pico/fisiología , Patos/fisiología , Mecanorreceptores/metabolismo , Percepción del Tacto/fisiología , Tacto/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Aviares/antagonistas & inhibidores , Proteínas Aviares/metabolismo , Pico/citología , Pico/inervación , Pollos , Clonación Molecular , Embrión no Mamífero , Expresión Génica , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/genética , Canales Iónicos/metabolismo , Cinética , Mecanorreceptores/citología , Mecanotransducción Celular , Ratones , Técnicas de Placa-Clamp , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Ganglio del Trigémino/citología , Ganglio del Trigémino/metabolismoRESUMEN
Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.
Asunto(s)
Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales Iónicos/genética , Adulto , Anemia Hemolítica Congénita/metabolismo , Niño , Estudios de Cohortes , Análisis Mutacional de ADN , Deshidratación/genética , Deshidratación/metabolismo , Eritrocitos/metabolismo , Familia , Femenino , Células HEK293 , Humanos , Hidropesía Fetal/metabolismo , Mutación INDEL , Recién Nacido , Canales Iónicos/metabolismo , Cinética , Masculino , Mutación Missense , Presión Osmótica/fisiologíaRESUMEN
The ten-eleven translocation (TET) family of proteins plays important roles in a wide range of biological processes by oxidizing 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine. However, their function in erythropoiesis has remained unclear. We show here that TET2 and TET3 but not TET1 are expressed in human erythroid cells, and we explore the role of these proteins in erythropoiesis. Knockdown experiments revealed that TET2 and TET3 have different functions. Suppression of TET3 expression in human CD34+ cells markedly impaired terminal erythroid differentiation, as reflected by increased apoptosis, the generation of bi/multinucleated polychromatic/orthochromatic erythroblasts, and impaired enucleation, although without effect on erythroid progenitors. In marked contrast, TET2 knockdown led to hyper-proliferation and impaired differentiation of erythroid progenitors. Surprisingly, knockdown of neither TET2 nor TET3 affected global levels of 5mC. Thus, our findings have identified distinct roles for TET2 and TET3 in human erythropoiesis, and provide new insights into their role in regulating human erythroid differentiation at distinct stages of development. Moreover, because knockdown of TET2 recapitulates certain features of erythroid development defects characteristic of myelodysplastic syndromes (MDSs), and the TET2 gene mutation is one of the most common mutations in MDS, our findings may be relevant for improved understanding of dyserythropoiesis of MDS.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Dioxigenasas/metabolismo , Eritropoyesis/fisiología , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Antígenos CD34/genética , Antígenos CD34/metabolismo , Proteínas de Unión al ADN/genética , Dioxigenasas/genética , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/citología , Humanos , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Proteínas Proto-Oncogénicas/genéticaRESUMEN
The modulation of chromatin structure is a key step in transcription regulation in mammalian cells and eventually determines lineage commitment and differentiation. USF1/2, Setd1a and NURF complexes interact to regulate chromatin architecture in erythropoiesis, but the mechanistic basis for this regulation is hitherto unknown. Here we showed that Setd1a and NURF complexes bind to promoters to control chromatin structural alterations and gene activation in a cell context dependent manner. In human primary erythroid cells USF1/2, H3K4me3 and the NURF complex were significantly co-enriched at transcription start sites of erythroid genes, and their binding was associated with promoter/enhancer accessibility that resulted from nucleosome repositioning. Mice deficient for Setd1a, an H3K4 trimethylase, in the erythroid compartment exhibited reduced Ter119/CD71 positive erythroblasts, peripheral blood RBCs and hemoglobin levels. Loss of Setd1a led to a reduction of promoter-associated H3K4 methylation, inhibition of gene transcription and blockade of erythroid differentiation. This was associated with alterations in NURF complex occupancy at erythroid gene promoters and reduced chromatin accessibility. Setd1a deficiency caused decreased associations between enhancer and promoter looped interactions as well as reduced expression of erythroid genes such as the adult ß-globin gene. These data indicate that Setd1a and NURF complexes are specifically targeted to and coordinately regulate erythroid promoter chromatin dynamics during erythroid lineage differentiation.
Asunto(s)
Linaje de la Célula , Ensamble y Desensamble de Cromatina , Eritrocitos/citología , Eritropoyesis , Regulación de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Antígenos Nucleares/metabolismo , Linaje de la Célula/genética , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Eritroblastos/citología , Eritroblastos/metabolismo , Recuento de Eritrocitos , Eritrocitos/metabolismo , Eritropoyesis/genética , Femenino , Hemoglobinas/metabolismo , N-Metiltransferasa de Histona-Lisina/deficiencia , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Metilación , Ratones , Ratones Noqueados , Nucleasa Microcócica/metabolismo , Complejos Multiproteicos/química , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas/genética , Bazo/citología , Factores de Transcripción/metabolismo , Factores Estimuladores hacia 5'/metabolismoRESUMEN
Hereditary xerocytosis (HX; MIM 194380) is an autosomal-dominant hemolytic anemia characterized by primary erythrocyte dehydration. In many patients, heterozygous mutations associated with delayed channel inactivation have been identified in PIEZO1. This report describes patients from 2 well-phenotyped HX kindreds, including from one of the first HX kindreds described, who lack predicted heterozygous PIEZO1-linked variants. Whole-exome sequencing identified novel, heterozygous mutations affecting the Gardos channel, encoded by the KCNN4 gene, in both kindreds. Segregation analyses confirmed transmission of the Gardos channel mutations with disease phenotype in affected individuals. The KCNN4 variants were different mutations in the same residue, which is highly conserved across species and within members of the small-intermediate family of calcium-activated potassium channel proteins. Both mutations were predicted to be deleterious by mutation effect algorithms. In sickle erythrocytes, the Gardos channel is activated under deoxy conditions, leading to cellular dehydration due to salt and water loss. The identification of KCNN4 mutations in HX patients supports recent studies that indicate it plays a critical role in normal erythrocyte deformation in the microcirculation and participates in maintenance of erythrocyte volume homeostasis.
Asunto(s)
Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Mutación Missense , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Análisis Mutacional de ADN , Exoma/genética , Femenino , Genes Dominantes , Estudios de Asociación Genética , Heterocigoto , Humanos , Canales Iónicos/genética , Masculino , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de AminoácidoRESUMEN
A 17-year-old male presented with acute hemolysis with stomatocytosis, elevated mean corpuscular hemoglobin concentration (MCHC), and osmotic gradient ektacytometry consistent with marked erythrocyte dehydration. Erythrocytes from both parents also demonstrated evidence of dehydration with elevated MCHC and abnormal ektacytometry, but neither to the degree of the patient. Genetic studies revealed the patient had hereditary xerocytosis (HX) due to a novel PIEZO1 mutation inherited from his mother and hemoglobin C (HbC) trait inherited from his father. HbC trait accentuated the erythrocyte dehydration of HX. Coinheritance of interrelated disorders and/or modifier alleles should be considered whenever severe erythrocyte dehydration is observed.
Asunto(s)
Anemia Hemolítica Congénita/complicaciones , Anemia Hemolítica Congénita/genética , Eritrocitos/patología , Enfermedad de la Hemoglobina C/complicaciones , Enfermedad de la Hemoglobina C/genética , Hidropesía Fetal/genética , Adolescente , Anemia Hemolítica Congénita/sangre , Índices de Eritrocitos , Enfermedad de la Hemoglobina C/sangre , Humanos , Hidropesía Fetal/sangre , Canales Iónicos/genética , Masculino , MutaciónRESUMEN
Relying almost exclusively on their acute sense of touch, tactile-foraging birds can feed in murky water, but the cellular mechanism is unknown. Mechanical stimuli activate specialized cutaneous end organs in the bill, innervated by trigeminal afferents. We report that trigeminal ganglia (TG) of domestic and wild tactile-foraging ducks exhibit numerical expansion of large-diameter mechanoreceptive neurons expressing the mechano-gated ion channel Piezo2. These features are not found in visually foraging birds. Moreover, in the duck, the expansion of mechanoreceptors occurs at the expense of thermosensors. Direct mechanical stimulation of duck TG neurons evokes high-amplitude depolarizing current with a low threshold of activation, high signal amplification gain, and slow kinetics of inactivation. Together, these factors contribute to efficient conversion of light mechanical stimuli into neuronal excitation. Our results reveal an evolutionary strategy to hone tactile perception in vertebrates at the level of primary afferents.
Asunto(s)
Patos/fisiología , Conducta Alimentaria , Mecanotransducción Celular , Neuronas/fisiología , Tacto/fisiología , Animales , Regulación hacia Abajo , Activación del Canal Iónico , Canales Iónicos/metabolismo , Umbral Sensorial , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPV/metabolismo , Termorreceptores/metabolismo , Ganglio del Trigémino/fisiología , Regulación hacia ArribaRESUMEN
T follicular helper (Tfh) cells are a subset of CD4(+) T helper cells that migrate into germinal centers and promote B-cell maturation into memory B and plasma cells. Tfh cells are necessary for promotion of protective humoral immunity following pathogen challenge, but when aberrantly regulated, drive pathogenic antibody formation in autoimmunity and undergo neoplastic transformation in angioimmunoblastic T-cell lymphoma and other primary cutaneous T-cell lymphomas. Limited information is available on the expression and regulation of genes in human Tfh cells. Using a fluorescence-activated cell sorting-based strategy, we obtained primary Tfh and non-Tfh T effector cells from tonsils and prepared genome-wide maps of active, intermediate, and poised enhancers determined by chromatin immunoprecipitation-sequencing, with parallel transcriptome analyses determined by RNA sequencing. Tfh cell enhancers were enriched near genes highly expressed in lymphoid cells or involved in lymphoid cell function, with many mapping to sites previously associated with autoimmune disease in genome-wide association studies. A group of active enhancers unique to Tfh cells associated with differentially expressed genes was identified. Fragments from these regions directed expression in reporter gene assays. These data provide a significant resource for studies of T lymphocyte development and differentiation and normal and perturbed Tfh cell function.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Transcriptoma/genética , Células Cultivadas , Citometría de Flujo , Genoma Humano/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Tonsila Palatina/citología , Cultivo Primario de Células , Análisis de Secuencia de ARN , Subgrupos de Linfocitos T/citología , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
We recently developed fluorescence-activated cell sorting (FACS)-based methods to purify morphologically and functionally discrete populations of cells, each representing specific stages of terminal erythroid differentiation. We used these techniques to obtain pure populations of both human and murine erythroblasts at distinct developmental stages. RNA was prepared from these cells and subjected to RNA sequencing analyses, creating unbiased, stage-specific transcriptomes. Tight clustering of transcriptomes from differing stages, even between biologically different replicates, validated the utility of the FACS-based assays. Bioinformatic analyses revealed that there were marked differences between differentiation stages, with both shared and dissimilar gene expression profiles defining each stage within transcriptional space. There were vast temporal changes in gene expression across the differentiation stages, with each stage exhibiting unique transcriptomes. Clustering and network analyses revealed that varying stage-specific patterns of expression observed across differentiation were enriched for genes of differing function. Numerous differences were present between human and murine transcriptomes, with significant variation in the global patterns of gene expression. These data provide a significant resource for studies of normal and perturbed erythropoiesis, allowing a deeper understanding of mechanisms of erythroid development in various inherited and acquired erythroid disorders.
Asunto(s)
Diferenciación Celular/genética , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/metabolismo , Eritropoyesis/genética , Transcriptoma , Animales , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Ratones , Análisis de Secuencia de ARN , Factores de Transcripción/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
We present a patient with a compound heterozygosity codon 39 (C > T) (ß0) [or ß39(C5)GlnâStop (G39X); CAG > TAG; HBB: c.118C > T] and -87 (C > T) (ß+) (HBB: c.-137C > T) ß-globin mutations, a non transfusion-dependent thalassemia phenotype and 97.0% fetal hemoglobin. A novel heterozygous mutation was identified in a highly conserved residue in the COOH-terminus of the Krüppel-like factor 1, R360H, that likely altered DNA-binding and impaired transactivation.
Asunto(s)
Hemoglobina Fetal/análisis , Factores de Transcripción de Tipo Kruppel/genética , Fenotipo , Talasemia beta/genética , Secuencia Conservada , Heterocigoto , Humanos , Mutación , Globinas beta/genéticaRESUMEN
PURPOSE OF REVIEW: Research into the fundamental mechanisms of erythropoiesis has provided critical insights into inherited and acquired disorders of the erythrocyte. Studies of human erythropoiesis have primarily utilized in-vitro systems, whereas murine models have provided insights from in-vivo studies. This report reviews recent insights into human and murine erythropoiesis gained from transcriptome-based analyses. RECENT FINDINGS: The availability of high-throughput genomic methodologies has allowed attainment of detailed gene expression data from cells at varying developmental and differentiation stages of erythropoiesis. Transcriptome analyses of human and murine reveal both stage and species-specific similarities and differences across terminal erythroid differentiation. Erythroid-specific long noncoding RNAs exhibit poor sequence conservation between human and mouse. Genome-wide analyses of alternative splicing reveal that complex, dynamic, stage-specific programs of alternative splicing program are utilized during terminal erythroid differentiation. Transcriptome data provide a significant resource for understanding mechanisms of normal and perturbed erythropoiesis. Understanding these processes will provide innovative strategies to detect, diagnose, prevent, and treat hematologic disease. SUMMARY: Understanding the shared and different mechanisms controlling human and murine erythropoiesis will allow investigators to leverage the best model system to provide insights in normal and perturbed erythropoiesis.
Asunto(s)
Eritropoyesis/genética , Animales , Diferenciación Celular , Humanos , RatonesRESUMEN
In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.
Asunto(s)
Linaje de la Célula , Proteínas de Unión al ADN/metabolismo , Proteínas Musculares/metabolismo , Factores de Transcripción/metabolismo , Animales , Blastocisto/citología , Blastocisto/metabolismo , Masa Celular Interna del Blastocisto/citología , Masa Celular Interna del Blastocisto/metabolismo , Blastómeros/citología , Blastómeros/metabolismo , Western Blotting , Factor de Transcripción CDX2 , Bovinos , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Macaca mulatta , Ratones , Ratones Transgénicos , Proteínas Musculares/genética , Interferencia de ARN , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de Dominio TEA , Factores de Transcripción/genéticaRESUMEN
A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34- cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34- cells, using RNA-Seq. We subsequently identified the target genes bound by TCF7, using ChIP-Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34- state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Células Madre Hematopoyéticas/metabolismo , Factor 1 de Transcripción de Linfocitos T/genética , Factor 1 de Transcripción de Linfocitos T/metabolismo , Animales , Antígenos CD34/metabolismo , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/citología , Factor Nuclear 1-alfa del Hepatocito , Humanos , Ratones , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño , Análisis de Secuencia de ARN , Factor 1 de Transcripción de Linfocitos T/antagonistas & inhibidores , Factores de Transcripción/clasificación , Factores de Transcripción/metabolismoRESUMEN
Identification of cell type-specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses, and candidate enhancers were identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the transcriptional start site (TSS) of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or phenotype. Candidate enhancers exhibited moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a set of erythroid-associated, biologically relevant, SNPs from the genome-wide association studies (GWAS) catalogue of NHGRI, National Institutes of Health. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function and provide insights into inherited and acquired hematologic disease.