RESUMEN
The early migratory phase of pulmonary helminth infections is characterized by tissue injury leading to the release of the alarmin interleukin (IL)-33 and subsequent induction of type 2 immune responses. We recently described a role for IL-17A, through suppression of interferon (IFN)-γ, as an important inducer of type 2 responses during infection with the lung-migrating rodent nematode Nippostrongylus brasiliensis. Here, we aimed to investigate the interaction between IL-17A and IL-33 during the early lung migratory stages of N. brasiliensis infection. In this brief report, we demonstrate that deficiency of IL-17A leads to impaired IL-33 expression and secretion early in infection, independent of IL-17A suppression of IFN-γ. Neutrophil-depletion experiments, which dramatically reduce lung injury, revealed that neutrophils are primarily responsible for the IL-17A-dependent release of IL-33 into the airways. Taken together, our results reveal an IL-17A-neutrophil-axis that can drive IL-33 during helminth infection, highlighting an additional pathway by which IL-17A regulates pulmonary type 2 immunity.
Asunto(s)
Nematodos , Neutrófilos , Animales , Ratones , Interleucina-17/metabolismo , Interleucina-33 , Pulmón , Células Epiteliales/metabolismo , Ratones Endogámicos C57BLRESUMEN
Myelofibrosis is a myeloproliferative neoplasm (MPN) which typically results in reduced length and quality of life due to systemic symptoms and blood count changes arising from fibrotic changes in the bone marrow. While the JAK2 inhibitor ruxolitinib provides some clinical benefit, there remains a substantial unmet need for novel targeted therapies to better modify the disease process or eradicate the cells at the heart of myelofibrosis pathology. Repurposing drugs bypasses many of the hurdles present in drug development, such as toxicity and pharmacodynamic profiling. To this end we undertook a re-analysis of our pre-existing proteomic data sets to identify perturbed biochemical pathways and their associated drugs/inhibitors to potentially target the cells driving myelofibrosis. This approach identified CBL0137 as a candidate for targeting Jak2 mutation-driven malignancies. CBL0137 is a drug derived from curaxin targeting the Facilitates Chromatin Transcription (FACT) complex. It is reported to trap the FACT complex on chromatin thereby activating p53 and inhibiting NF-kB activity. We therefore assessed the activity of CBL0137 in primary patient samples and murine models of Jak2-mutated MPN and found it preferentially targets CD34+ stem and progenitor cells from myelofibrosis patients by comparison with healthy control cells. Further we investigate its mechanism of action in primary haemopoietic progenitor cells and demonstrate its ability to reduce splenomegaly and reticulocyte number in a transgenic murine model of myeloproliferative neoplasms.
Asunto(s)
Trastornos Mieloproliferativos , Mielofibrosis Primaria , Humanos , Ratones , Animales , Mielofibrosis Primaria/tratamiento farmacológico , Mielofibrosis Primaria/genética , Proteómica , Calidad de Vida , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Janus Quinasa 2/metabolismo , Cromatina , MutaciónRESUMEN
Honeycombing is a histological pattern consistent with Usual Interstitial Pneumonia (UIP). Honeycombing refers to cystic airways located at sites of dense fibrosis with marked mucus accumulation. Utilizing laser capture microdissection coupled mass spectrometry (LCM-MS), we interrogated the fibrotic honeycomb airway cells and fibrotic uninvolved airway cells (distant from honeycomb airways and morphologically intact) in specimens from 10 patients with UIP. Non-fibrotic airway cell specimens from 6 patients served as controls. Furthermore, we performed LCM-MS on the mucus plugs found in 6 patients with UIP and 6 patients with mucinous adenocarcinoma. The mass spectrometry data were subject to both qualitative and quantitative analysis and validated by immunohistochemistry. Surprisingly, fibrotic uninvolved airway cells share a similar protein profile to honeycomb airway cells, showing deregulation of the slit and roundabout receptor (Slit and Robo) pathway as the strongest category. We find that (BPI) fold-containing family B member 1 (BPIFB1) is the most significantly increased secretome-associated protein in UIP, whereas Mucin-5AC (MUC5AC) is the most significantly increased in mucinous adenocarcinoma. We conclude that fibrotic uninvolved airway cells share pathological features with fibrotic honeycomb airway cells. In addition, fibrotic honeycomb airway cells are enriched in mucin biogenesis proteins with a marked derangement in proteins essential for ciliogenesis. This unbiased spatial proteomic approach generates novel and testable hypotheses to decipher fibrosis progression.
Asunto(s)
Fibrosis Pulmonar Idiopática , Proteoma , Humanos , Proteómica , Fibrosis Pulmonar Idiopática/patología , Pulmón/patologíaRESUMEN
Despite the big increase in precision medicine targeted therapies developing curative treatments for many cancers is still a major challenge due mainly to the development of drug resistance in cancer stem cells. The cancer stem cells are constantly evolving to survive and targeted drug treatment often increases the selective pressure on these cells from which the disease develops. Chronic myeloid leukaemia is a paradigm of cancer stem cell research. Targeted therapies to the causative oncogene, BCR/ABL, have been developed but drug resistance remains a problem. The introduction of tyrosine kinase inhibitors targeting BCR/ABL were transformative in the management of CML. However, patients are rarely cured as the tyrosine kinase inhibitors fail to eradicate the leukaemic stem cell which often leads to loss of response to therapy as drug resistance develops and progression to more fatal forms of acute leukaemia occurs. New treatment strategies targeting other entities within the leukemic stem cell either alone or in combination with tyrosine kinase are therefore required. Drawing on our previous published work on the development of potential novel targets in CML and other myeloproliferative diseases along with analysis of the facilitates chromatin transcription (FACT) complex in CML we hypothesised that curaxin, a drug that targets the FACT complex and is in clinical trial for the treatment of other cancers, could be of use in the treatment of CML. We therefore assessed the curaxin CBL0137 as a new agent to extinguish CML primitive cells and show its ability to preferentially target CML cells compared to healthy control cells, especially in combination with clinically relevant tyrosine kinase inhibitors.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Células Madre Neoplásicas , Oncogenes , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina QuinasasRESUMEN
Allergic airway inflammation is heterogeneous with variability in immune phenotypes observed across asthmatic patients. Inflammation has been thought to directly contribute to airway remodeling in asthma, but clinical data suggest that neutralizing type 2 cytokines does not necessarily alter disease pathogenesis. Here, we utilized C57BL/6 and BALB/c mice to investigate the development of allergic airway inflammation and remodeling. Exposure to an allergen cocktail for up to 8 weeks led to type 2 and type 17 inflammation, characterized by airway eosinophilia and neutrophilia and increased expression of chitinase-like proteins in both C57BL/6 and BALB/c mice. However, BALB/c mice developed much greater inflammatory responses than C57BL/6 mice, effects possibly explained by a failure to induce pathways that regulate and maintain T-cell activation in C57BL/6 mice, as shown by whole lung RNA transcript analysis. Allergen administration resulted in a similar degree of airway remodeling between mouse strains but with differences in collagen subtype composition. Increased collagen III was observed around the airways of C57BL/6 but not BALB/c mice while allergen-induced loss of basement membrane collagen IV was only observed in BALB/c mice. This study highlights a model of type 2/type 17 airway inflammation in mice whereby development of airway remodeling can occur in both BALB/c and C57BL/6 mice despite differences in immune response dynamics between strains. Importantly, compositional changes in the extracellular matrix between genetic strains of mice may help us better understand the relationships between lung function, remodeling and airway inflammation.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Hipersensibilidad , Alérgenos , Animales , Líquido del Lavado Bronquioalveolar , Modelos Animales de Enfermedad , Humanos , Inflamación , Pulmón , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , OvalbúminaRESUMEN
The transcriptional regulator EVI1 has an essential role in early development and haematopoiesis. However, acute myeloid leukaemia (AML) driven by aberrantly high EVI1 expression has very poor prognosis. To investigate the effects of post-translational modifications on EVI1 function, we carried out a mass spectrometry (MS) analysis of EVI1 in AML and detected dynamic phosphorylation at serine 436 (S436). Wild-type EVI1 (EVI1-WT) with S436 available for phosphorylation, but not non-phosphorylatable EVI1-S436A, conferred haematopoietic progenitor cell self-renewal and was associated with significantly higher organised transcriptional patterns. In silico modelling of EVI1-S436 phosphorylation showed reduced affinity to CtBP1, and CtBP1 showed reduced interaction with EVI1-WT compared with EVI1-S436A. The motif harbouring S436 is a target of CDK2 and CDK3 kinases, which interacted with EVI1-WT. The methyltransferase DNMT3A bound preferentially to EVI1-WT compared with EVI1-S436A, and a hypomethylated cell population associated by EVI1-WT expression in murine haematopoietic progenitors is not maintained with EVI1-S436A. These data point to EVI1-S436 phosphorylation directing functional protein interactions for haematopoietic self-renewal. Targeting EVI1-S436 phosphorylation may be of therapeutic benefit when treating EVI1-driven leukaemia.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Autorrenovación de las Células/fisiología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , Metilación de ADN/fisiología , ADN Metiltransferasa 3A , Metilasas de Modificación del ADN/metabolismo , Humanos , Fosforilación , Pronóstico , Serina/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Aberrantly high expression of EVI1 in acute myeloid leukaemia (AML) is associated with poor prognosis. For targeted treatment of EVI1 overexpressing AML a more detailed understanding of aspects of spatiotemporal interaction dynamics of the EVI1 protein is important. EVI1 overexpressing SB1690CB AML cells were used for quantification and protein interaction studies of EVI1 and ΔEVI1. Cells were cell cycle-synchronised by mimosine and nocodazole treatment and expression of EVI1 and related proteins assessed by western blot, immunoprecipitation and immunofluorescence. EVI1 protein levels oscillate through the cell cycle, and EVI1 is degraded partly by the proteasome complex. Both EVI1 and ΔEVI1 interact with the co-repressor CtBP1 but dissociate from CtBP1 complexes during mitosis. Furthermore, a large fraction of EVI1, but not ΔEVI1 or CtBP1, resides in the nuclear matrix. In conclusion, EVI1- protein levels and EVI1-CtBP1 interaction dynamics vary though the cell cycle and differ between EVI1 and ΔEVI1. These data ad to the functional characterisation of the EVI1 protein in AML and will be important for the development of targeted therapeutic approaches for EVI1-driven AML.
Asunto(s)
Oxidorreductasas de Alcohol/biosíntesis , Relojes Biológicos , Ciclo Celular , Proteínas de Unión al ADN/biosíntesis , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Proteína del Locus del Complejo MDS1 y EV11/biosíntesis , Oxidorreductasas de Alcohol/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Leucemia Mieloide Aguda/genética , Proteína del Locus del Complejo MDS1 y EV11/genéticaRESUMEN
Nippostrongylus brasiliensis is a well-defined model of type-2 immunity but the early lung-migrating phase is dominated by innate IL-17A production. In this study, we confirm previous observations that Il17a-KO mice infected with N. brasiliensis exhibit an impaired type-2 immune response. Transcriptional profiling of the lung on day 2 of N. brasiliensis infection revealed an increased Ifng signature in Il17a-KO mice confirmed by enhanced IFNγ protein production in lung lymphocyte populations. Depletion of early IFNγ rescued type-2 immune responses in the Il17a-KO mice demonstrating that IL-17A-mediated suppression of IFNγ promotes type-2 immunity. Notably, later in infection, once the type-2 response was established, IL-17A limited the magnitude of the type-2 response. IL-17A regulation of type-2 immunity was lung-specific and infection with Trichuris muris revealed that IL-17A promotes a type-2 immune response in the lung even when infection is restricted to the intestine. Together our data reveal IL-17A as a major regulator of pulmonary type-2 immunity such that IL-17A supports early development of a protective type-2 response by suppression of IFNγ but subsequently limits excessive type-2 responses. A failure of this feedback loop may contribute to conditions such as severe asthma, characterised by combined elevation of IL-17 and type-2 cytokines.
Asunto(s)
Interleucina-17/metabolismo , Pulmón/inmunología , Nippostrongylus/fisiología , Infecciones por Strongylida/inmunología , Células Th2/inmunología , Animales , Células Cultivadas , Femenino , Tolerancia Inmunológica , Inmunidad Innata , Interferón gamma/metabolismo , Interleucina-17/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasm of early childhood with a poor survival rate, thus there is a requirement for improved treatment strategies. Induced pluripotent stem cells offer the ability to model disease and develop new treatment strategies. JMML is frequently associated with mutations in PTPN11. Children with Noonan syndrome, a development disorder, have an increased incidence of JMML associated with specific germline mutations in PTPN11. We undertook a proteomic assessment of myeloid cells derived from induced pluripotent stem cells obtained from Noonan syndrome patients with PTPN11 mutations, either associated or not associated with an increased incidence of JMML. We report that the proteomic perturbations induced by the leukemia-associated PTPN11 mutations are associated with TP53 and NF-Kκb signaling. We have previously shown that MYC is involved in the differential gene expression observed in Noonan syndrome patients associated with an increased incidence of JMML. Thus, we employed drugs to target these pathways and demonstrate differential effects on clonogenic hematopoietic cells derived from Noonan syndrome patients, who develop JMML and those who do not. Further, we demonstrated these small molecular inhibitors, JQ1 and CBL0137, preferentially extinguish primitive hematopoietic cells from sporadic JMML patients as opposed to cells from healthy individuals.
Asunto(s)
Células Madre Pluripotentes Inducidas , Leucemia Mielomonocítica Juvenil , Síndrome de Noonan , Niño , Preescolar , Humanos , Leucemia Mielomonocítica Juvenil/tratamiento farmacológico , Leucemia Mielomonocítica Juvenil/genética , Mutación , ProteómicaRESUMEN
Myeloproliferative neoplasms (MPN) are clonal stem cell associated disorders inclusive of chronic myeloid leukemia (CML), Polycythaemia vera (PV), myelofibrosis (MF), and essential thrombocythemia (ET). They are characterized by increased production of myeloid cells with minimal effects on terminal differentiation but can undergo transformation to acute leukemias. PV is the most common chronic myeloproliferative neoplasm and in the majority of cases is characterized by a V617F point mutation in JAK2. This JAK2 activating mutation is also found in about half the patients with MF and ET. Such aberrant proteins offer great potential for the treatment of these diseases however inhibitors to JAK2 have had limited success in the clinic in terms of curing the disease. We have previously used advanced proteomic techniques to identify drug targets and thus develop novel treatment strategies to distinguish the leukemic clone in both CML and PV. Here, we build on our proteomic data sets to characterize a new target, the receptor tyrosine kinase AXL. AXL is overexpressed in acute myeloid leukemia and importantly small molecule inhibitors have been developed which are currently in clinical trial hence offer the opportunity to repurpose this drug for the treatment of MPNs. We demonstrate that AXL is upregulated and activated in JAK2 associated MPNs. Further we show that inhibition of AXL preferentially kills early hemopoietic stem cells from PV patients and as such represents a promising therapeutic approach for JAK2 driven MPNs.
RESUMEN
The transcriptional regulator EVI1 has an essential role in early hematopoiesis and development. However, aberrantly high expression of EVI1 has potent oncogenic properties and confers poor prognosis and chemo-resistance in leukemia and solid tumors. To investigate to what extent EVI1 function might be regulated by post-translational modifications we carried out mass spectrometry- and antibody-based analyses and uncovered an ATM-mediated double phosphorylation of EVI1 at the carboxy-terminal S858/S860 SQS motif. In the presence of genotoxic stress EVI1-WT (SQS), but not site mutated EVI1-AQA was able to maintain transcriptional patterns and transformation potency, while under standard conditions carboxy-terminal mutation had no effect. Maintenance of hematopoietic progenitor cell clonogenic potential was profoundly impaired with EVI1-AQA compared with EVI1-WT, in particular in the presence of genotoxic stress. Exploring mechanistic events underlying these observations, we showed that after genotoxic stress EVI1-WT, but not EVI1-AQA increased its level of association with its functionally essential interaction partner CtBP1, implying a role for ATM in regulating EVI1 protein interactions via phosphorylation. This aspect of EVI1 regulation is therapeutically relevant, as chemotherapy-induced genotoxicity might detrimentally sustain EVI1 function via stress response mediated phosphorylation, and ATM-inhibition might be of specific targeted benefit in EVI1-overexpressing malignancies.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Autorrenovación de las Células/genética , Proteínas de Unión al ADN/genética , Regulación Leucémica de la Expresión Génica , Proteína del Locus del Complejo MDS1 y EV11/genética , Enfermedad Aguda , Oxidorreductasas de Alcohol/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Proteína del Locus del Complejo MDS1 y EV11/química , Proteína del Locus del Complejo MDS1 y EV11/metabolismo , Mutación , FosforilaciónRESUMEN
The thrombopoietin receptor (MPL) has been shown to be mutated (MPL W515L) in myelofibrosis and thrombocytosis yet new approaches to treat this disorder are still required. We have previously shown that transcriptome and proteomic effects do not correlate well in oncogene-mediated leukemogenesis. We therefore investigated the effects of MPL W515L using proteomics. The consequences of MPL W515L expression on over 3300 nuclear and 3500 cytoplasmic proteins were assessed using relative quantification mass spectrometry. We demonstrate that MPL W515L expression markedly modulates the CXCL12/CXCR4/CD45 pathway associated with stem and progenitor cell chemotactic movement. We also demonstrated that MPL W515L expressing cells displayed increased chemokinesis which required the MPL W515L-mediated dysregulation of MYC expression via phosphorylation of the RNA transport protein THOC5 on tyrosine 225. In addition MPL W515L expression induced TGFß secretion which is linked to sphingosine 1-phosphate production and the increased chemokinesis. These studies identify several pathways which offer potential targets for therapeutic intervention in the treatment of MPL W515L-driven malignancy. We validate our approach by showing that CD34+ cells from MPL W515L positive patients display increased chemokinesis and that treatment with a combination of MYC and sphingosine kinase inhibitors leads to the preferential killing of MPL W515L expressing cells.
Asunto(s)
Proteínas Nucleares/metabolismo , Receptores de Trombopoyetina/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Animales , Estudios de Casos y Controles , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Ratones , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Proteínas Nucleares/genética , Fosforilación , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/metabolismo , Transducción de SeñalRESUMEN
Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQ(TM)) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Hemangioblastos/metabolismo , Fosfoproteínas/metabolismo , Proteómica , Animales , Línea Celular , Linaje de la Célula , Cromatografía Liquida , Bases de Datos de Proteínas , Proteínas Fetales/genética , Proteínas Fetales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Espectrometría de Masas , Ratones , Proteómica/métodos , Transducción de Señal , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Tiempo , Transfección , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The generation of in vivo repopulating hematopoietic cells from in vitro differentiating embryonic stem cells has remained a long-standing challenge. To date, hematopoietic engraftment has mostly been achieved through the enforced expression of ectopic transcription factors. Here, we describe serum-free culture conditions that allow the generation of in vivo repopulating hematopoietic cells in the absence of ectopically expressed factors. We show that repopulating activity arises immediately upon the commitment of mesodermal precursors to the blood program, within the first wave of hematopoietic specification. We establish that the formation of these progenitors is extremely transient and exquisitely sensitive to the cytokine milieu. Our findings define the precise differentiating stage at which hematopoietic repopulating activity first appears in vitro, and suggest that during embryonic stem cell differentiation, all hematopoietic programs are unraveled simultaneously from the mesoderm in the absence of cues that restrict the coordinated emergence of each lineage as is normally observed during embryogenesis.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/citología , Hematopoyesis , Células Madre Hematopoyéticas/citología , Activinas/genética , Activinas/metabolismo , Animales , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Citocinas/farmacología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Factores de Crecimiento de Fibroblastos , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Trasplante de Células Madre , Factores de Crecimiento Endotelial Vascular/genética , Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
During embryonic development, the emergence of hematopoiesis and vasculogenesis is tightly associated, with many transcription factors implicated in both developmental processes. Among those factors, ETV2 acts at the top of the hierarchy and controls the formation of both lineages. However, it is not known at which stage of mesoderm development ETV2 is acting and whether ETV2 activity is further required once mesodermal precursors have been specified to the hematopoietic and endothelial fates. In this study, we characterize the developmental window during which ETV2 expression is required for hematopoietic and endothelial development. Using cre-mediated deletion of ETV2, we demonstrate that ETV2 is acting prior to or at the time of FLK1 expression in mesodermal precursors to initiate the hematopoietic and endothelial program. Using the in vitro differentiation of embryonic stem cells as a model system, we further show that ETV2 re-expression in Etv2(-/-) Flk1-negative precursors drives hematopoiesis specification and switches on the expression of most genes known to be implicated in hematopoietic and endothelial development. Among the downstream targets of ETV2, we identify the transcription factors SCL, GATA2, and FLI1 known to operate a recursive loop controlling hematopoietic development. Surprisingly, SCL re-expression in Etv2(-/-) cells fully rescues hematopoiesis, while the re-expression of FLI1 or GATA2 promotes only a very limited rescue. Altogether, our data establish that ETV2 is required very transiently to specify mesodermal precursors to hematopoiesis and vasculogenesis and that SCL is one of the key downstream targets of ETV2 in controlling hematopoietic specification.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Factores de Transcripción/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Inmunoprecipitación de Cromatina , Citometría de Flujo , Hematopoyesis/genética , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Transcripción/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
At early stages of vertebrate ontogeny, blood and endothelial cells develop from a common mesodermal progenitor, the haemangioblast. Upon haematopoietic commitment, the haemangioblast generates blood precursors through populations of endothelial cells with haemogenic properties. Although several transcription factors have been implicated in haemangioblast differentiation, the precise mechanisms governing cell fate decisions towards the generation of haemogenic endothelium precursors remain largely unknown. Under defined conditions, embryonic stem (ES) cells can be differentiated into haemangioblast-like progenitors that faithfully recapitulate early embryonic haematopoiesis. Here, we made use of mouse ES cells as a model system to understand the role of SOX7, a member of a large family of transcription factors involved in a wide range of developmental processes. During haemangioblast differentiation, SOX7 is expressed in haemogenic endothelium cells and is downregulated in nascent blood precursors. Gain-of-function assays revealed that the enforced expression of Sox7 in haemangioblast-derived blast colonies blocks further differentiation and sustains the expression of endothelial markers. Thus, to explore the transcriptional activity of SOX7, we focused on the endothelial-specific adhesion molecule VE-cadherin. Similar to SOX7, VE-cadherin is expressed in haemogenic endothelium and is downregulated during blood cell formation. We show that SOX7 binds and activates the promoter of VE-cadherin, demonstrating that this gene is a novel downstream transcriptional target of SOX7. Altogether, our findings suggest that SOX7 is involved in the transcriptional regulation of genes expressed in the haemogenic endothelium and provide new clues to decipher the molecular pathways that drive early embryonic haematopoiesis.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/fisiología , Hemangioblastos/metabolismo , Hematopoyesis/fisiología , Factores de Transcripción SOXF/metabolismo , Animales , Antígenos CD/genética , Western Blotting , Cadherinas/genética , Inmunoprecipitación de Cromatina , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Hemangioblastos/citología , Hematopoyesis/genética , Luciferasas , Ratones , Regiones Promotoras Genéticas/genética , Factores de Transcripción SOXF/genéticaRESUMEN
Many lineage-specific developmental regulator genes are transcriptionally primed in embryonic stem (ES) cells; RNA Pol(II) is bound at their promoters but is prevented from productive elongation by the activity of polycomb repressive complexes (PRC) 1 and 2. This epigenetically poised state is thought to enable ES cells to rapidly execute multiple differentiation programs and is recognized by a simultaneous enrichment for trimethylation of lysine 4 and trimethylation of lysine 27 of histone H3 (bivalent chromatin) across promoter regions. Here we show that the chromatin profile of this important cohort of genes is progressively modified as ES cells differentiate toward blood-forming precursors. Surprisingly however, neural specifying genes, such as Nkx2-2, Nkx2-9, and Sox1, remain bivalent and primed even in committed hemangioblasts, as conditional deletion of PRC1 results in overt and inappropriate expression of neural genes in hemangioblasts. These data reinforce the importance of PRC1 for normal hematopoietic differentiation and reveal an unexpected epigenetic plasticity of mesoderm-committed hemangioblasts.
Asunto(s)
Células Madre Embrionarias/metabolismo , Epigénesis Genética , Hemangioblastos/fisiología , Proteínas de Homeodominio/genética , Proteínas Represoras/fisiología , Factores de Transcripción SOXB1/genética , Factores de Transcripción/genética , Animales , Western Blotting , Diferenciación Celular , Células Cultivadas , Cromatina/genética , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/citología , Proteínas Fetales/genética , Proteínas Fluorescentes Verdes/genética , Histonas/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/metabolismo , Integrasas/metabolismo , Ratones , Ratones Noqueados , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1/antagonistas & inhibidores , Factores de Transcripción SOXB1/metabolismo , Proteínas de Dominio T Box/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Proteínas de Pez CebraRESUMEN
During embryogenesis, the hematopoietic program is specified from the mesodermal germ layer through the formation of hemangioblast. This precursor gives rise to a hemogenic endothelium that later on matures to generate primitive and definitive hematopoietic precursors. A lack of specific cell surface markers to identify cells with discrete developmental potential is a major hurdle in the quest to further understand the cellular and molecular program governing blood formation. In the present study, we identify CD40 and Icam2, two markers typically associated with the adult immunological compartment, as expressed at the earliest stages of blood specification both in vitro and in vivo. Using in vitro serum-free culture conditions that support the efficient and directed differentiation of embryonic stem cells, we show that the sequential expression of CD40 and Icam2 delineate a transition in the acquisition of the blood potential from hemangioblast to hemogenic endothelium leading to the formation of primitive and definitive hematopoietic progenitors. CD40 is transiently expressed at the onset of blood development and marks first the hemangioblast then the hemogenic endothelium but is no longer expressed on fully committed hematopoietic precursors within the fetal liver. In contrast, Icam2 is first expressed on the hemogenic endothelium and its expression persists on fetal liver hematopoietic progenitors. Taken together, our data identify novel cell surface markers allowing us to further refine our understanding of the events marking progressive hematopoietic commitment from the mesoderm germ layer.
Asunto(s)
Antígenos CD40/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Cultivadas/citología , Hematopoyesis , Mesodermo/metabolismo , Animales , Antígenos CD , Linaje de la Célula , Ratones , Ratones Endogámicos ICRRESUMEN
We have previously shown that Sox7 was transiently expressed at the onset of blood specification and was implicated in the regulation of cell survival, proliferation, and maturation of hematopoietic precursors. Here, we assessed, using embryonic stem cell differentiation as a model system, whether Sox17 and Sox18, 2 close homologs of Sox7, may act similarly to Sox7 at the onset of hematopoietic development. Sox18-enforced expression led to the enhanced proliferation of early hematopoietic precursors while blocking their maturation, phenotype highly reminiscent of Sox7-enforced expression. In striking contrast, Sox17-enforced expression dramatically increased the apoptosis of these early precursors. Similarly to Sox7, Sox18 was transiently expressed during early hematopoiesis, but its expression was predominantly observed in CD41(+) cells, contrasting with Sox7, mostly expressed in Flk1(+) cells. Conversely, Sox17 remained marginally expressed during blood specification. Overall, our data uncover contrasting effect and expression pattern for Sox18 and Sox17 at the onset of hematopoiesis specification.