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1.
Nat Cell Biol ; 25(6): 812-822, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37127714

RESUMEN

Haematopoietic stem cells (HSCs) are multipotent, but individual HSCs can show restricted lineage output in vivo. Currently, the molecular mechanisms and physiological role of HSC fate restriction remain unknown. Here we show that lymphoid fate is epigenetically but not transcriptionally primed in HSCs. In multi-lineage HSCs that produce lymphocytes, lymphoid-specific upstream regulatory elements (LymUREs) but not promoters are preferentially accessible compared with platelet-biased HSCs that do not produce lymphoid cell types, providing transcriptionally silent lymphoid lineage priming. Runx3 is preferentially expressed in multi-lineage HSCs, and reinstating Runx3 expression increases LymURE accessibility and lymphoid-primed multipotent progenitor 4 (MPP4) output in old, platelet-biased HSCs. In contrast, platelet-biased HSCs show elevated levels of epigenetic platelet-lineage priming and give rise to MPP2 progenitors with molecular platelet bias. These MPP2 progenitors generate platelets with faster kinetics and through a more direct cellular pathway compared with MPP2s derived from multi-lineage HSCs. Epigenetic programming therefore predicts both fate restriction and differentiation kinetics in HSCs.


Asunto(s)
Células Madre Hematopoyéticas , Linfocitos , Linaje de la Célula/genética , Células Madre Hematopoyéticas/metabolismo , Diferenciación Celular/genética , Linfocitos/metabolismo , Epigénesis Genética , Células Madre Multipotentes/metabolismo
3.
Nat Commun ; 11(1): 4075, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32796847

RESUMEN

Hematopoietic ageing involves declining erythropoiesis and lymphopoiesis, leading to frequent anaemia and decreased adaptive immunity. How intrinsic changes to the hematopoietic stem cells (HSCs), an altered microenvironment and systemic factors contribute to this process is not fully understood. Here we use bone marrow stromal cells as sensors of age-associated changes to the bone marrow microenvironment, and observe up-regulation of IL-6 and TGFß signalling-induced gene expression in aged bone marrow stroma. Inhibition of TGFß signalling leads to reversal of age-associated HSC platelet lineage bias, increased generation of lymphoid progenitors and rebalanced HSC lineage output in transplantation assays. In contrast, decreased erythropoiesis is not an intrinsic property of aged HSCs, but associated with decreased levels and functionality of erythroid progenitor populations, defects ameliorated by TGFß-receptor and IL-6 inhibition, respectively. These results show that both HSC-intrinsic and -extrinsic mechanisms are involved in age-associated hematopoietic decline, and identify therapeutic targets that promote their reversal.


Asunto(s)
Envejecimiento/metabolismo , Células Madre Hematopoyéticas/metabolismo , Interleucina-6/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Envejecimiento/genética , Animales , Médula Ósea , Ciclo Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Células Precursoras Eritroides , Eritropoyesis/genética , Eritropoyesis/fisiología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Hematopoyesis , Interleucina-6/genética , Linfopoyesis/genética , Linfopoyesis/fisiología , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Células Mieloides , Transducción de Señal , Nicho de Células Madre , Factor de Crecimiento Transformador beta1/genética
4.
Cancer Cell ; 37(5): 690-704.e8, 2020 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-32330454

RESUMEN

Acute erythroid leukemia (AEL) commonly involves both myeloid and erythroid lineage transformation. However, the mutations that cause AEL and the cell(s) that sustain the bilineage leukemia phenotype remain unknown. We here show that combined biallelic Cebpa and Gata2 zinc finger-1 (ZnF1) mutations cooperatively induce bilineage AEL, and that the major leukemia-initiating cell (LIC) population has a neutrophil-monocyte progenitor (NMP) phenotype. In pre-leukemic NMPs Cebpa and Gata2 mutations synergize by increasing erythroid transcription factor (TF) expression and erythroid TF chromatin access, respectively, thereby installing ectopic erythroid potential. This erythroid-permissive chromatin conformation is retained in bilineage LICs. These results demonstrate that synergistic transcriptional and epigenetic reprogramming by leukemia-initiating mutations can generate neomorphic pre-leukemic progenitors, defining the lineage identity of the resulting leukemia.


Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Linaje de la Célula , Transformación Celular Neoplásica/patología , Células Precursoras Eritroides/patología , Factor de Transcripción GATA2/genética , Leucemia Eritroblástica Aguda/patología , Mutación , Neutrófilos/patología , Anciano , Alelos , Animales , Diferenciación Celular , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Células Precursoras Eritroides/metabolismo , Femenino , Factor de Transcripción GATA1/genética , Humanos , Leucemia Eritroblástica Aguda/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neutrófilos/metabolismo , Dedos de Zinc
5.
Nat Commun ; 10(1): 5455, 2019 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-31784538

RESUMEN

Acute Myeloid Leukemia (AML) develops due to the acquisition of mutations from multiple functional classes. Here, we demonstrate that activating mutations in the granulocyte colony stimulating factor receptor (CSF3R), cooperate with loss of function mutations in the transcription factor CEBPA to promote acute leukemia development. The interaction between these distinct classes of mutations occurs at the level of myeloid lineage enhancers where mutant CEBPA prevents activation of a subset of differentiation associated enhancers. To confirm this enhancer-dependent mechanism, we demonstrate that CEBPA mutations must occur as the initial event in AML initiation. This improved mechanistic understanding will facilitate therapeutic development targeting the intersection of oncogene cooperativity.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Leucemia Mieloide Aguda/genética , Receptores del Factor Estimulante de Colonias/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Humanos , Células K562 , Mutación con Pérdida de Función , Ratones , Mutación
6.
Sci Immunol ; 4(35)2019 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-31126997

RESUMEN

Human myelopoiesis has been proposed to occur through oligopotent common myeloid progenitor (CMP) and lymphoid-primed multipotent progenitor (LMPP) populations. However, other studies have proposed direct commitment of multipotent cells to unilineage fates, without specific intermediary lineage cosegregation patterns. We here show that distinct human myeloid progenitor populations generate the neutrophil/monocyte and mast cell/basophil/eosinophil lineages as previously shown in mouse. Moreover, we find that neutrophil/monocyte potential selectively cosegregates with lymphoid lineage and mast cell/basophil/eosinophil potentials with megakaryocyte/erythroid potential early during lineage commitment. Furthermore, after this initial commitment step, mast cell/basophil/eosinophil and megakaryocyte/erythroid potentials colocalize at the single-cell level in restricted oligopotent progenitors. These results show that human myeloid lineages are generated through two distinct cellular pathways defined by complementary oligopotent cell populations.


Asunto(s)
Células Progenitoras Linfoides/metabolismo , Células Progenitoras Mieloides/metabolismo , Mielopoyesis/fisiología , Adulto , Antígenos de Superficie/genética , Diferenciación Celular/fisiología , Linaje de la Célula/fisiología , Eritrocitos/metabolismo , Expresión Génica , Voluntarios Sanos , Humanos , Masculino , Megacariocitos/metabolismo , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Adulto Joven
7.
Haematologica ; 104(11): 2215-2224, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-30975913

RESUMEN

Somatic mutations in acute myeloid leukemia are acquired sequentially and hierarchically. First, pre-leukemic mutations, such as t(8;21) that encodes AML1-ETO, are acquired within the hematopoietic stem cell (HSC) compartment, while signaling pathway mutations, including KRAS activating mutations, are late events acquired during transformation of leukemic progenitor cells and are rarely detectable in HSC. This raises the possibility that signaling pathway mutations are detrimental to clonal expansion of pre-leukemic HSC. To address this hypothesis, we used conditional genetics to introduce Aml1-ETO and K-RasG12D into murine HSC, either individually or in combination. In the absence of activated Ras, Aml1-ETO-expressing HSC conferred a competitive advantage. However, activated K-Ras had a marked detrimental effect on Aml1-ETO-expressing HSC, leading to loss of both phenotypic and functional HSC. Cell cycle analysis revealed a loss of quiescence in HSC co-expressing Aml1-ETO and K-RasG12D, accompanied by an enrichment in E2F and Myc target gene expression and depletion of HSC self-renewal-associated gene expression. These findings provide a mechanistic basis for the observed absence of KRAS signaling mutations in the pre-malignant HSC compartment.


Asunto(s)
Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Mutación , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteína 1 Compañera de Translocación de RUNX1/genética , Proteína 1 Compañera de Translocación de RUNX1/metabolismo , Animales , Proliferación Celular/genética , Expresión Génica , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/patología , Humanos , Ratones , Ratones Transgénicos , Modelos Animales , Modelos Biológicos , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo
8.
Cell Stem Cell ; 19(2): 148-150, 2016 08 04.
Artículo en Inglés | MEDLINE | ID: mdl-27494670

RESUMEN

Hematopoiesis is a complex process that requires a high degree of transcriptional diversification during lineage commitment and differentiation. de Graaf et al. (2016) have now generated a comprehensive gene expression dataset that allows cell-type-specific genes as well as associated transcription factor expression patterns to be readily identified.


Asunto(s)
Linaje de la Célula/genética , Células Madre Hematopoyéticas , Diferenciación Celular , Hematopoyesis/genética , Humanos , Factores de Transcripción/genética
9.
Nat Immunol ; 17(6): 666-676, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-27043410

RESUMEN

According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)Flt3(hi)) and common myeloid progenitors (CMPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)CD41(hi)) establish an early branch point for separate lineage-commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre-granulocyte-macrophage progenitor (pre-GM) (Lin(-)Sca-1(-)c-Kit(+)CD41(-)FcγRII/III(-)CD150(-)CD105(-)). By single-cell transcriptome profiling of pre-GMs, we identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential.


Asunto(s)
Diferenciación Celular , Linaje de la Célula , Linfocitos/fisiología , Células Mieloides/fisiología , Células Progenitoras Mieloides/fisiología , Animales , Antígenos CD/metabolismo , Células Cultivadas , Biología Computacional , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Hematopoyesis , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Análisis de Matrices Tisulares , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismo
10.
Blood ; 115(17): 3463-71, 2010 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-20154211

RESUMEN

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 Secuencia
11.
Exp Hematol ; 33(10): 1083-91, 2005 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-16219530

RESUMEN

OBJECTIVE: To design a culture method allowing the quantitative and qualitative analysis of terminal erythroid differentiation. METHODS: Primary erythroid progenitors derived either from mouse tissues or from human umbilical cord blood were differentiated using hanging drop cultures and compared to methylcellulose cultures. Cultured cells were analyzed by FACS to assess differentiation. RESULTS: We describe a practical culture method by adapting the previously described hanging drop culture system to conditions allowing terminal differentiation of primary erythroid progenitors. Using minimal volumes of media and small numbers of cells, we obtained quantitative terminal erythroid differentiation within two days of culture in the case of murine cells and 4 days in the case of human cells. CONCLUSIONS: The established methods for ex vivo culture of primary erythroid progenitors, such as methylcellulose-based burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) assays, allow the detection of committed erythroid progenitors but are of limited value to study terminal erythroid differentiation. We show that the application of hanging drop cultures is a practical alternative that, in combination with clonogenic assays, enables a comprehensive assessment of the behavior of primary erythroid cells ex vivo in the context of genetic and drug-induced perturbations.


Asunto(s)
Diferenciación Celular/fisiología , Células Precursoras Eritroides/fisiología , Eritropoyesis/fisiología , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Células Precursoras Eritroides/citología , Citometría de Flujo/métodos , Ratones , Ratones Transgénicos
12.
Mol Cell Biol ; 25(12): 5205-14, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15923635

RESUMEN

Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Kruppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in beta-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.


Asunto(s)
Membrana Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Eritrocitos/fisiología , Eritropoyesis/fisiología , Regulación de la Expresión Génica , Hemoglobinas/metabolismo , Fenotipo , Factores de Transcripción/metabolismo , Animales , Antineoplásicos Hormonales/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Diferenciación Celular/fisiología , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Eritrocitos/citología , Perfilación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel , Ratones , Ratones Noqueados , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tamoxifeno/metabolismo , Factores de Transcripción/genética
13.
Genes Dev ; 18(20): 2485-90, 2004 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-15489291

RESUMEN

Three-dimensional organization of a gene locus is important for its regulation, as recently demonstrated for the beta-globin locus. When actively expressed, the cis-regulatory elements of the beta-globin locus are in proximity in the nuclear space, forming a compartment termed the Active Chromatin Hub (ACH). However, it is unknown which proteins are involved in ACH formation. Here, we show that EKLF, an erythroid transcription factor required for adult beta-globin gene transcription, is also required for ACH formation. We conclude that transcription factors can play an essential role in the three-dimensional organization of gene loci.


Asunto(s)
Cromatina/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Globinas/genética , Factores de Transcripción/genética , Animales , Reactivos de Enlaces Cruzados , ADN/metabolismo , Cartilla de ADN , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , Formaldehído , Globinas/química , Globinas/metabolismo , Factores de Transcripción de Tipo Kruppel , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Mutantes , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo
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