RESUMEN
Human embryonic stem cells (hESCs) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESCs we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single-cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long-term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single-cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm-biased stem cell state.
Asunto(s)
Autorrenovación de las Células , Endodermo/citología , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Biomarcadores , Diferenciación Celular/genética , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Perfilación de la Expresión Génica , Genes Reporteros , Humanos , Inmunofenotipificación , Análisis de la Célula Individual/métodosRESUMEN
In vitro erythroid differentiation systems are used to study the mechanisms underlying normal and abnormal erythropoiesis and to test the effects of various extracellular factors on erythropoiesis. The use of serum or conditioned medium in liquid cultures and the seeding of cultures with heterogeneous peripheral blood mononuclear cells confound the reproducibility of these systems. Newer erythroid differentiation culture systems have overcome some of these limitations by using a fully defined, serum-free medium and initiating cultures using purified CD34+ cells. Although widely used in bulk cultures, these protocols have not been rigorously tested in high-throughput or single-cell assays. Here, we describe a serum-free erythroid differentiation system suitable for small-scale and single-cell experiments. This system generates large numbers of terminally differentiated erythroid cells of very high purity. Here we have adapted this culture system to a 96-well format and have developed a protocol to grow erythroid colonies from single erythroid progenitors in minute culture volumes.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Eritroides/citología , Células Eritroides/metabolismo , Eritropoyesis , Medio de Cultivo Libre de Suero/química , Medio de Cultivo Libre de Suero/farmacología , HumanosRESUMEN
ß-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and ß-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with ß-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with ß-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for ß-thalassemia.ß-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Edición Génica , Células Madre Hematopoyéticas/metabolismo , Globinas alfa/genética , Talasemia beta/genética , Talasemia beta/terapia , Animales , Antígenos CD34/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Femenino , Técnicas de Silenciamiento del Gen , Genoma Humano , Xenoinjertos , Humanos , Ratones , Eliminación de Secuencia/genética , Análisis de la Célula IndividualRESUMEN
ATRX is a chromatin remodelling factor found at a wide range of tandemly repeated sequences including telomeres (TTAGGG)n ATRX mutations are found in nearly all tumours that maintain their telomeres via the alternative lengthening of telomere (ALT) pathway, and ATRX is known to suppress this pathway. Here, we show that recruitment of ATRX to telomeric repeats depends on repeat number, orientation and, critically, on repeat transcription. Importantly, the transcribed telomeric repeats form RNA-DNA hybrids (R-loops) whose abundance correlates with the recruitment of ATRX Here, we show loss of ATRX is also associated with increased R-loop formation. Our data suggest that the presence of ATRX at telomeres may have a central role in suppressing deleterious DNA secondary structures that form at transcribed telomeric repeats, and this may account for the increased DNA damage, stalling of replication and homology-directed repair previously observed upon loss of ATRX function.
Asunto(s)
Ensamble y Desensamble de Cromatina , ADN/genética , ARN/genética , Telómero/genética , Telómero/metabolismo , Proteína Nuclear Ligada al Cromosoma X/metabolismo , Cromatina , ADN/química , Daño del ADN , Replicación del ADN , G-Cuádruplex , Humanos , Homeostasis del Telómero/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína Nuclear Ligada al Cromosoma X/deficiencia , Proteína Nuclear Ligada al Cromosoma X/genéticaAsunto(s)
Inhibidores Enzimáticos/farmacología , Células Eritroides/efectos de los fármacos , Histona Demetilasas/genética , Hidroxiquinolinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Globinas alfa/genética , Antígenos CD34/genética , Antígenos CD34/metabolismo , Ácido Butírico/farmacología , Técnicas de Cultivo de Célula , Células Eritroides/citología , Células Eritroides/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Histona Demetilasas/antagonistas & inhibidores , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Hidroxiurea/farmacología , Anotación de Secuencia Molecular , Piperazinas/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Tranilcipromina/farmacología , Vorinostat , Globinas alfa/antagonistas & inhibidores , Globinas alfa/metabolismo , Talasemia beta/tratamiento farmacológico , Talasemia beta/enzimología , Talasemia beta/genética , Talasemia beta/patologíaRESUMEN
Induced pluripotent stem (iPS) cells offer a unique potential for understanding the molecular basis of disease and development. Here we have generated several human iPS cell lines, and we describe their pluripotent phenotype and ability to differentiate into erythroid cells, monocytes, and endothelial cells. More significantly, however, when these iPS cells were differentiated under conditions that promote lympho-hematopoiesis from human embryonic stem cells, we observed the formation of pre-B cells. These cells were CD45(+)CD19(+)CD10(+) and were positive for transcripts Pax5, IL7αR, λ-like, and VpreB receptor. Although they were negative for surface IgM and CD5 expression, iPS-derived CD45(+)CD19(+) cells also exhibited multiple genomic D-J(H) rearrangements, which supports a pre-B-cell identity. We therefore have been able to demonstrate, for the first time, that human iPS cells are able to undergo hematopoiesis that contributes to the B-cell lymphoid lineage.
Asunto(s)
Linfocitos B/citología , Linfopoyesis/fisiología , Células Madre Pluripotentes/citología , Células Precursoras de Linfocitos B/citología , Adulto , Antígenos CD19/metabolismo , Linfocitos B/fisiología , Línea Celular , Linaje de la Célula/inmunología , Humanos , Inmunoglobulina de Cadenas Ligeras Subrogadas/genética , Inmunofenotipificación , Antígenos Comunes de Leucocito/metabolismo , Neprilisina/metabolismo , Factor de Transcripción PAX5/genética , Células Madre Pluripotentes/fisiología , Células Precursoras de Linfocitos B/fisiología , Receptores de Interleucina-7/genéticaRESUMEN
Precise spatiotemporal control of Gata1 expression is required in both early hematopoietic progenitors to determine erythroid/megakaryocyte versus granulocyte/monocyte lineage output and in the subsequent differentiation of erythroid cells and megakaryocytes. An enhancer element upstream of the mouse Gata1 IE (1st exon erythroid) promoter, mHS-3.5, can direct both erythroid and megakaryocytic expression. However, loss of this element ablates only megakaryocytes, implying that an additional element has erythroid specificity. Here, we identify a double DNaseI hypersensitive site, mHS-25/6, as having erythroid but not megakaryocytic activity in primary cells. It binds an activating transcription factor complex in erythroid cells where it also makes physical contact with the Gata1 promoter. Deletion of mHS-25/6 or mHS-3.5 in embryonic stem cells has only a modest effect on in vitro erythroid differentiation, whereas loss of both elements ablates both primitive and definitive erythropoiesis with an almost complete loss of Gata1 expression. Surprisingly, Gata2 expression was also concomitantly low, suggesting a more complex interaction between these 2 factors than currently envisaged. Thus, whereas mHS-3.5 alone is sufficient for megakaryocytic development, mHS-3.5 and mHS-25/6 collectively regulate erythroid Gata1 expression, demonstrating lineage-specific differences in Gata1 cis-element use important for development of these 2 cell types.
Asunto(s)
Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos/fisiología , Células Eritroides/metabolismo , Eritropoyesis/fisiología , Factor de Transcripción GATA1/biosíntesis , Regulación de la Expresión Génica/fisiología , Megacariocitos/metabolismo , Animales , Células Madre Embrionarias/citología , Células Eritroides/citología , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Megacariocitos/citología , Ratones , Regiones Promotoras Genéticas/fisiología , Eliminación de SecuenciaRESUMEN
It is well established that all of the cis-acting sequences required for fully regulated human alpha-globin expression are contained within a region of approximately 120 kb of conserved synteny. Here, we show that activation of this cluster in erythroid cells dramatically affects expression of apparently unrelated and noncontiguous genes in the 500 kb surrounding this domain, including a gene (NME4) located 300 kb from the alpha-globin cluster. Changes in NME4 expression are mediated by physical cis-interactions between this gene and the alpha-globin regulatory elements. Polymorphic structural variation within the globin cluster, altering the number of alpha-globin genes, affects the pattern of NME4 expression by altering the competition for the shared alpha-globin regulatory elements. These findings challenge the concept that the genome is organized into discrete, insulated regulatory domains. In addition, this work has important implications for our understanding of genome evolution, the interpretation of genome-wide expression, expression-quantitative trait loci, and copy number variant analyses.
Asunto(s)
Expresión Génica , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Cromosomas Humanos Par 16 , Humanos , Secuencias Reguladoras de Ácidos Nucleicos , Telómero , Globinas alfa/genéticaRESUMEN
The transcription factor Runx1 plays a pivotal role in hematopoietic stem cell (HSC) emergence, and studies into its transcriptional regulation should give insight into the critical steps of HSC specification. Recently, we identified the Runx1 +23 enhancer that targets reporter gene expression to the first emerging HSCs of the mouse embryo when linked to the heterologous hsp68 promoter. Endogenous Runx1 is transcribed from 2 alternative promoters, P1 and P2. Here, we examined the in vivo cis-regulatory potential of these alternative promoters and asked whether they act with and contribute to the spatiotemporal specific expression of the Runx1 +23 enhancer. Our results firmly establish that, in contrast to zebrafish runx1, mouse Runx1 promoter sequences do not confer any hematopoietic specificity in transgenic embryos. Yet, both mouse promoters act with the +23 enhancer to drive reporter gene expression to sites of HSC emergence and colonization, in a +23-specific pattern.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Elementos de Facilitación Genéticos , Células Madre Hematopoyéticas/metabolismo , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Embrión de Mamíferos , Células Madre Hematopoyéticas/citología , Ratones , Ratones Transgénicos , Especificidad de la EspecieRESUMEN
Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human alpha- and beta-globin genes are silenced by fundamentally different mechanisms. The alpha-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the beta-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the alpha-cluster by sequences within the CpG islands associated with their promoters; the beta-globin promoters do not lie within such islands. Chromatin associated with the alpha-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The alpha- and beta-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.
Asunto(s)
Silenciador del Gen , Globinas/genética , Proteínas Represoras/metabolismo , Secuencia de Bases , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Proteína Potenciadora del Homólogo Zeste 2 , Células HeLa , Histona Desacetilasas/metabolismo , Humanos , Células Madre Pluripotentes/metabolismo , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Interferencia de ARN , ARN Interferente Pequeño/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The transcription factor Runx1/AML1 is an important regulator of hematopoiesis and is critically required for the generation of the first definitive hematopoietic stem cells (HSCs) in the major vasculature of the mouse embryo. As a pivotal factor in HSC ontogeny, its transcriptional regulation is of high interest but is largely undefined. In this study, we used a combination of comparative genomics and chromatin analysis to identify a highly conserved 531-bp enhancer located at position + 23.5 in the first intron of the 224-kb mouse Runx1 gene. We show that this enhancer contributes to the early hematopoietic expression of Runx1. Transcription factor binding in vivo and analysis of the mutated enhancer in transient transgenic mouse embryos implicate Gata2 and Ets proteins as critical factors for its function. We also show that the SCL/Lmo2/Ldb-1 complex is recruited to the enhancer in vivo. Importantly, transplantation experiments demonstrate that the intronic Runx1 enhancer targets all definitive HSCs in the mouse embryo, suggesting that it functions as a crucial cis-regulatory element that integrates the Gata, Ets, and SCL transcriptional networks to initiate HSC generation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Factor de Transcripción GATA2/fisiología , Células Madre Hematopoyéticas/citología , Proteína Proto-Oncogénica c-ets-1/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Elementos de Facilitación Genéticos/fisiología , Factor de Transcripción GATA2/metabolismo , Proteínas con Dominio LIM , Metaloproteínas/metabolismo , Ratones , Complejos Multiproteicos/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 de la Leucemia Linfocítica T AgudaRESUMEN
We generated five lines of transgenic mice carrying 1-3 copies of the Hb Lepore deltabeta fusion gene, in the context of a Bacterial Artificial Chromosome containing the whole human beta globin gene cluster. Normal developmental regulation of human genes occurred at levels approximating to those of endogenous genes. Deltabeta transgene expression became detectable during fetal life and rose to a mean level of 13.0% in adults, similar to that of humans. Low levels of human gamma chains were detectable as F cells in adult mice, but numbers did not increase after treatment with drugs that raise F cells in human subjects, even on a thalassaemic background.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Globinas/genética , Animales , Secuencia de Bases , Cromosomas Artificiales Bacterianos , Expresión Génica , Hemoglobinas/análisis , Hemoglobinas Anormales/genética , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fragmentos de Péptidos , Recuento de ReticulocitosRESUMEN
During a study of the molecular basis for severe forms of beta thalassemia in Sri Lanka, 2 patients were found to be heterozygous for beta thalassemia mutations. Further analysis revealed that one of them has a previously unreported molecular basis for severe thalassemia intermedia, homozygosity for quadruplicated alpha globin genes in combination with heterozygous beta thalassemia. The other is homozygous for a triplicated alpha globin gene arrangement and heterozygous for beta thalassemia. Their differences in clinical phenotype are explainable by the interaction of other genetic factors and, in particular, their early management. The clinical course of the 2 propositi underlines the importance of full genotyping and a long period of observation before treatment is instituted, particularly in patients with beta thalassemia intermedia associated with extended alpha globin gene arrangements. The hemoglobin (Hb) F levels in these patients with severe beta thalassemia intermedia, compared with other forms of this condition in the Sri Lankan population and elsewhere, are unusually low, a consistent finding in extended alpha globin gene interactions and in dominant beta thalassemia, raising the possibility that increased levels of HbF production in beta thalassemia may require mutations at both beta globin gene loci.