RESUMEN
The thrombopoietin receptor (TpoR) plays a central role in myeloproliferative neoplasms (MPNs). Mutations in JAK2, calreticulin, or TpoR itself drive the constitutive activation of TpoR and uncontrolled proliferation and differentiation of hematopoietic stem cells and progenitors. The JAK2 V617F mutation is responsible for most MPNs, and all driver mutants induce pathologic TpoR activation. Existing therapeutic strategies have focused on JAK2 kinase inhibitors that are unable to differentiate between the mutated MPN clone and healthy cells. Surprisingly, the targeting of TpoR itself has remained poorly explored despite its central role in pathology. Here, we performed a comprehensive characterization of human TpoR activation under physiological and pathological conditions, focusing on the JAK2 V617F mutant. Using a system of controlled dimerization of the transmembrane and cytosolic domains of TpoR, we discovered that human TpoR (hTpoR) adopts different dimeric conformations upon Tpo-induced vs JAK2 V617F-mediated activation. We identified the amino acids and specific dimeric conformation of hTpoR responsible for activation in complex with JAK2 V617F and confirmed our findings in the full-length receptor context in hematopoietic cell lines and primary bone marrow cells. Remarkably, we found that the modulation of hTpoR conformations by point mutations allowed for specific inhibition of JAK2 V617F-driven activation without affecting Tpo-induced signaling. Our results demonstrate that modulation of the hTpoR conformation is a viable therapeutic strategy for JAK2 V617F-positive MPNs and set the path for novel drug development by identifying precise residues of hTpoR involved in JAK2 V617F-specific activation.
Asunto(s)
Trastornos Mieloproliferativos , Receptores de Trombopoyetina , Humanos , Receptores de Trombopoyetina/metabolismo , Citocinas/genética , Trastornos Mieloproliferativos/genética , Mutación , Transducción de Señal , Janus Quinasa 2/metabolismoRESUMEN
Embryonic stem cell renewal and differentiation is regulated by metabolites that serve as cofactors for epigenetic enzymes. An increase of α-ketoglutarate (α-KG), a cofactor for histone and DNA demethylases, triggers multilineage differentiation in human embryonic stem cells (hESCs). To gain further insight into how the metabolic fluxes in pluripotent stem cells can be influenced by inactivating mutations in epigenetic enzymes, we generated hESCs deficient for de novo DNA methyltransferases (DNMTs) 3A and 3B. Our data reveal a bidirectional dependence between DNMT3B and α-KG levels: a-KG is significantly upregulated in cells deficient for DNMT3B, while DNMT3B expression is downregulated in hESCs treated with α-KG. In addition, DNMT3B null hESCs exhibit a disturbed mitochondrial fission and fusion balance and a switch from glycolysis to oxidative phosphorylation. Taken together, our data reveal a novel link between DNMT3B and the metabolic flux of hESCs.
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ADN (Citosina-5-)-Metiltransferasas/deficiencia , Células Madre Embrionarias Humanas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocondrias/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/enzimología , Humanos , Mitocondrias/enzimología , Biogénesis de Organelos , ADN Metiltransferasa 3BRESUMEN
Endoplasmic reticulum (ER) stress is thought to activate autophagy via unfolded protein response (UPR)-mediated transcriptional up-regulation of autophagy machinery components and modulation of microtubule-associated protein 1 light chain 3 (LC3). The upstream UPR constituents pancreatic EIF2-α kinase (PERK) and inositol-requiring enzyme 1 (IRE1) have been reported to mediate these effects, suggesting that UPR may stimulate autophagy via PERK and IRE1. However, how the UPR and its components affect autophagic activity has not been thoroughly examined. By analyzing the flux of LC3 through the autophagic pathway, as well as the sequestration and degradation of autophagic cargo, we here conclusively show that the classical ER stressor tunicamycin (TM) enhances autophagic activity in mammalian cells. PERK and its downstream factor, activating transcription factor 4 (ATF4), were crucial for this induction, but surprisingly, IRE1 constitutively suppressed autophagic activity. TM-induced autophagy required autophagy-related 13 (ATG13), Unc-51-like autophagy-activating kinases 1/2 (ULK1/ULK2), and GABA type A receptor-associated proteins (GABARAPs), but interestingly, LC3 proteins appeared to be redundant. Strikingly, ATF4 was activated independently of PERK in both LNCaP and HeLa cells, and our further examination revealed that ATF4 and PERK regulated autophagy through separate mechanisms. Specifically, whereas ATF4 controlled transcription and was essential for autophagosome formation, PERK acted in a transcription-independent manner and was required at a post-sequestration step in the autophagic pathway. In conclusion, our results indicate that TM-induced UPR activates functional autophagy, and whereas IRE1 is a negative regulator, PERK and ATF4 are required at distinct steps in the autophagic pathway.
Asunto(s)
Factor de Transcripción Activador 4/metabolismo , Muerte Celular Autofágica/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Tunicamicina/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 4/genética , Muerte Celular Autofágica/genética , Autofagosomas/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Estrés del Retículo Endoplásmico/genética , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Células PC-3 , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Respuesta de Proteína Desplegada/genética , eIF-2 Quinasa/genéticaRESUMEN
Cytokinesis failure leads to the emergence of tetraploid cells and multiple centrosomes. Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in adults and is characterized by clonal B cell expansion. Here, we show that a significant number of peripheral blood CLL cells are arrested in cytokinesis and that this event occurred after nuclear envelope reformation and before cytoplasmic abscission. mRNA expression data showed that several genes known to be crucial for cell cycle regulation, checkpoint and centromere function, such as ING4, ING5, CDKN1A and CDK4, were significantly dysregulated in CLL samples. Our results demonstrate that CLL cells exhibit difficulties in completing mitosis, which is different from but may, at least in part, explain the previously reported accumulation of CLL cells in G0/1.
Asunto(s)
Puntos de Control del Ciclo Celular , Centrosoma/metabolismo , Citocinesis , Leucemia Linfocítica Crónica de Células B/patología , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Recuento de Células , Proteínas de Ciclo Celular , Línea Celular Tumoral , Citoesqueleto/metabolismo , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His(499) near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His(499) is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His(499) regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain.
Asunto(s)
Proteínas Proto-Oncogénicas/metabolismo , Receptores de Trombopoyetina/metabolismo , Animales , Asparagina/genética , Asparagina/metabolismo , Línea Celular , Histidina/genética , Histidina/metabolismo , Humanos , Ratones , Mutación Missense , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Receptores de Trombopoyetina/genéticaRESUMEN
Ligand binding to the thrombopoietin receptor is thought to stabilize an active receptor dimer that regulates megakaryocyte differentiation and platelet formation, as well as haematopoietic stem cell renewal. By fusing a dimeric coiled coil in all seven possible orientations to the thrombopoietin receptor transmembrane (TM)-cytoplasmic domains, we show that specific biological effects and in vivo phenotypes are imparted by distinct dimeric orientations, which can be visualized by cysteine mutagenesis and crosslinking. Using functional assays and computational searches, we identify one orientation that represents the inactive dimeric state and another similar to a physiologically activated receptor. Several other dimeric orientations are identified that induce proliferation and in vivo myeloproliferative and myelodysplastic disorders, indicating the receptor can signal from several dimeric interfaces. The set of dimeric thrombopoietin receptors with different TM orientations may offer new insights into the activation of distinct signalling pathways by a single receptor and suggests that subtle differences in cytokine receptor dimerization provide a new layer of signalling regulation that is relevant for disease.
Asunto(s)
Multimerización de Proteína/fisiología , Receptores de Trombopoyetina/química , Receptores de Trombopoyetina/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Dominios y Motivos de Interacción de Proteínas/fisiología , Mapas de Interacción de Proteínas , Multimerización de Proteína/genética , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/fisiología , Transducción de Señal/fisiología , EstereoisomerismoRESUMEN
The constitutively active JAK2 V617F mutant is the major determinant of human myeloproliferative neoplasms (MPNs). We show that coexpression of murine JAK2 V617F and the murine thrombopoietin (Tpo) receptor (TpoR, c-MPL) in hematopoietic cell lines or heterozygous knock-in of JAK2 V617F in mice leads to down-modulation of TpoR levels. Enhanced TpoR ubiquitinylation, proteasomal degradation, reduced recycling, and maturation are induced by the constitutive JAK2 V617F activity. These effects can be prevented in cell lines by JAK2 and proteasome inhibitors. Restoration of TpoR levels by inhibitors could be detected in platelets from JAK2 inhibitor-treated myelofibrosis patients that express the JAK2 V617F mutant, and in platelets from JAK2 V617F knock-in mice that were treated in vivo with JAK2 or proteasome inhibitors. In addition, we show that Tpo can induce both proliferative and antiproliferative effects via TpoR at low and high JAK2 activation levels, respectively, or on expression of JAK2 V617F. The antiproliferative signaling and receptor down-modulation by JAK2 V617F were dependent on signaling via TpoR cytosolic tyrosine 626. We propose that selection against TpoR antiproliferative signaling occurs by TpoR down-modulation and that restoration of down-modulated TpoR levels could become a biomarker for the treatment of MPNs.
Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/fisiología , Inhibidores de Proteasoma , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Trombopoyetina/genética , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense/fisiología , Fenilalanina/genética , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Receptores de Trombopoyetina/metabolismo , Transducción de Señal/efectos de los fármacos , Valina/genéticaRESUMEN
Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.
Asunto(s)
Receptores de Trombopoyetina , Transducción de Señal , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/metabolismo , Línea Celular , Mutación , Estructura Secundaria de Proteína , Transducción de Señal/genéticaRESUMEN
Constitutively active JAK2V617F and thrombopoietin receptor (TpoR) W515L/K mutants are major determinants of human myeloproliferative neoplasms (MPNs). We show that a TpoRW515 mutation (W515A), which we detected in 2 myelofibrosis patients, and the Delta5TpoR active mutant, where the juxtamembrane R/KW(515)QFP motif is deleted, induce a myeloproliferative phenotype in mouse bone marrow reconstitution experiments. This phenotype required cytosolic Y112 of the TpoR. Phosphotyrosine immunoprofiling detected phosphorylated cytosolic TpoR Y78 and Y112 in cells expressing TpoRW515A. Mutation of cytosolic Y112 to phenylalanine prevented establishment of the in vivo phenotype and decreased constitutive active signaling by Delta5TpoR and TpoRW515A, especially via the mitogen-activated protein (MAP)-kinase pathway, without decreasing Janus kinase 2 (JAK2) activation. In contrast, mutation of cytosolic Y78 to phenylalanine enhanced the myeloproliferative syndrome induced by the TpoRW515 mutants, by enhancing receptor-induced JAK2 activation. We propose that TpoR cytosolic phosphorylated Y112 and flanking sequences could become targets for pharmacologic inhibition in MPNs.
Asunto(s)
Mutación , Trastornos Mieloproliferativos/genética , Mielofibrosis Primaria/genética , Receptores de Trombopoyetina/genética , Tirosina/genética , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Línea Celular , Proliferación Celular , Humanos , Immunoblotting , Janus Quinasa 2/metabolismo , Ratones , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patología , Fosfoproteínas/metabolismo , Fosforilación , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Mielofibrosis Primaria/metabolismo , Mielofibrosis Primaria/patología , Receptores de Trombopoyetina/metabolismo , Transfección , Tirosina/metabolismoRESUMEN
Ectopic expression of defined transcription factors can reprogram somatic cells to induced pluripotent stem (iPS) cells, but the utility of iPS cells is hampered by the use of viral delivery systems. Small molecules offer an alternative to replace virally transduced transcription factors with chemical signaling cues responsible for reprogramming. In this report we describe a small-molecule screening platform applied to identify compounds that functionally replace the reprogramming factor Klf4. A series of small-molecule scaffolds were identified that activate Nanog expression in mouse fibroblasts transduced with a subset of reprogramming factors lacking Klf4. Application of one such molecule, kenpaullone, in lieu of Klf4 gave rise to iPS cells that are indistinguishable from murine embryonic stem cells. This experimental platform can be used to screen large chemical libraries in search of novel compounds to replace the reprogramming factors that induce pluripotency. Ultimately, such compounds may provide mechanistic insight into the reprogramming process.
Asunto(s)
Benzazepinas/farmacología , Diferenciación Celular , Epigénesis Genética/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Indoles/farmacología , Células Madre Pluripotentes/citología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Fibroblastos/citología , Genes Reporteros , Proteínas de Homeodominio/genética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Luciferasas/genética , Ratones , Proteína Homeótica NanogRESUMEN
Myeloproliferative disorders are clonal haematopoietic stem cell malignancies characterized by independency or hypersensitivity of haematopoietic progenitors to numerous cytokines. The molecular basis of most myeloproliferative disorders is unknown. On the basis of the model of chronic myeloid leukaemia, it is expected that a constitutive tyrosine kinase activity could be at the origin of these diseases. Polycythaemia vera is an acquired myeloproliferative disorder, characterized by the presence of polycythaemia diversely associated with thrombocytosis, leukocytosis and splenomegaly. Polycythaemia vera progenitors are hypersensitive to erythropoietin and other cytokines. Here, we describe a clonal and recurrent mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 (JAK2) gene in most (> 80%) polycythaemia vera patients. The mutation, a valine-to-phenylalanine substitution at amino acid position 617, leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity and induces erythrocytosis in a mouse model. As this mutation is also found in other myeloproliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches.
Asunto(s)
Mutación/genética , Policitemia Vera/metabolismo , Policitemia Vera/patología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Trasplante de Médula Ósea , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Eritropoyetina/farmacología , Exones/genética , Humanos , Interleucina-3/farmacología , Janus Quinasa 2 , Ratones , Policitemia , Policitemia Vera/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Células Cultivadas/fisiología , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Edición Génica/métodos , Reparación del ADN/genética , Reparación del ADN/fisiología , HumanosRESUMEN
Optic atrophy 1 (OPA1), a GTPase at the inner mitochondrial membrane involved in regulating mitochondrial fusion, stability, and energy output, is known to be crucial for neural development: Opa1 heterozygous mice show abnormal brain development, and inactivating mutations in OPA1 are linked to human neurological disorders. Here, we used genetically modified human embryonic and patient-derived induced pluripotent stem cells and reveal that OPA1 haploinsufficiency leads to aberrant nuclear DNA methylation and significantly alters the transcriptional circuitry in neural progenitor cells (NPCs). For instance, expression of the forkhead box G1 transcription factor, which is needed for GABAergic neuronal development, is repressed in OPA1+/- NPCs. Supporting this finding, OPA1+/- NPCs cannot give rise to GABAergic interneurons, whereas formation of glutamatergic neurons is not affected. Taken together, our data reveal that OPA1 controls nuclear DNA methylation and expression of key transcription factors needed for proper neural cell specification.
RESUMEN
The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Inhibidores de Proteínas Quinasas/química , Factores de Transcripción SOXB1/metabolismo , Familia-src Quinasas/antagonistas & inhibidores , Animales , Dasatinib , Doxiciclina/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Factores de Transcripción SOXB1/genética , Tiazoles/química , Tiazoles/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: Hematopoietic stem cell renewal and differentiation are regulated through epigenetic processes. The conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) by ten-eleven-translocation enzymes provides new insights into the epigenetic regulation of gene expression during development. Here, we studied the potential gene regulatory role of 5hmC during human hematopoiesis. RESULTS: We used reduced representation of 5-hydroxymethylcytosine profiling (RRHP) to characterize 5hmC distribution in CD34+ cells, CD4+ T cells, CD19+ B cells, CD14+ monocytes and granulocytes. In all analyzed blood cell types, the presence of 5hmC at gene bodies correlates positively with gene expression, and highest 5hmC levels are found around transcription start sites of highly expressed genes. In CD34+ cells, 5hmC primes for the expression of genes regulating myeloid and lymphoid lineage commitment. Throughout blood cell differentiation, intragenic 5hmC is maintained at genes that are highly expressed and required for acquisition of the mature blood cell phenotype. Moreover, in CD34+ cells, the presence of 5hmC at enhancers associates with increased binding of RUNX1 and FLI1, transcription factors essential for hematopoiesis. CONCLUSIONS: Our study provides a comprehensive genome-wide overview of 5hmC distribution in human hematopoietic cells and new insights into the epigenetic regulation of gene expression during human hematopoiesis.
RESUMEN
Janus kinases (JAKs) are non-receptor tyrosine kinases essential for activation of signaling mediated by cytokine receptors that lack catalytic activity, including receptors for erythropoietin, thrombopoietin, most interleukins and interferon. Upon hormone binding, JAKs phosphorylate tyrosine residues in the receptor cytoplasmic domains and in JAKs themselves leading to recruitment and activation of downstream signaling proteins such as signal transducer and activator of transcription (STAT). The JAK-STAT pathway is important for functional hematopoiesis and several activating mutations in JAK proteins have recently been described as underlying cause of blood disorders. One of the best studied examples is the JAK2 V617F mutant which is found in 95% of polycythemia vera patients and 50% of patients suffering from essential thrombocythemia and primary myelofibrosis. Much effort has been made to understand how the JAK2 V617F affects hematopoietic stem cell (HSC) renewal and lineage differentiation, since convincing evidence has been provided to support the notion that the mutation is acquired at the HSC level. We discuss several in vivo models that support contrary conclusions with respect to the advantage given to HSCs by JAK2 V617F. Moreover, we provide the current knowledge about STAT5 activation and its link to HSC expansion as well as amplification of the erythroid compartment. Evidence for both JAK2 V617F mutated HSCs exhibiting skewed differentiation potential and for amplification occurring after erythroid commitment has been provided, and we will discuss whether this evidence is relevant for the disease.
RESUMEN
Activating mutations in JAK1 have been reported in acute lymphoblastic leukemias, but little is known about the mechanisms involved in their constitutive activation. Here, we studied the ability of JAK1 V658F and A634D to activate the Janus kinase (JAK)/STAT pathway upon ectopic expression in HEK293 cells alone or together with the other components of the interleukin-9 receptor complex (IL-9Ralpha, gammac, and JAK3). Expression of JAK1 mutants alone failed to trigger STAT activation, but co-expression of the IL-9Ralpha chain promoted JAK1 mutant phosphorylation and STAT activation. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Ralpha involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with a functional IL-9Ralpha to activate STAT factors. Several lines of evidence indicated that IL-9Ralpha homodimerization was involved in this process. IL-9Ralpha variants with mutations of the JAK-interacting BOX1 region not only failed to promote JAK1 activation but also acted as dominant negative forms reverting the effect of wild-type IL-9Ralpha. Coimmunoprecipitation experiments also showed the formation of IL-9Ralpha homodimers. Interestingly, STAT activation was partially inhibited by expression of gammac, suggesting that overlapping residues are involved in IL-9Ralpha homodimerization and IL-9Ralpha/gammac heterodimerization. Co-expression of wild-type JAK3 partially reverted the inhibition by gammac, indicating that JAK3 cooperates with JAK1 mutants within the IL-9 receptor complex. Similar results were observed with IL-2Rbeta. Taken together, our results show that IL-9Ralpha and IL-2Rbeta homodimers efficiently mediate constitutive activation of ALL-associated JAK1 mutants.
Asunto(s)
Janus Quinasa 1/metabolismo , Mutación Missense , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Interleucina-9/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Sustitución de Aminoácidos , Animales , Línea Celular Tumoral , Dimerización , Activación Enzimática/genética , Humanos , Subunidad beta del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/metabolismo , Janus Quinasa 1/genética , Janus Quinasa 3/genética , Janus Quinasa 3/metabolismo , Ratones , Fosforilación/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Estructura Terciaria de Proteína/genética , Receptores de Interleucina-9/genética , Factores de Transcripción STAT/genéticaRESUMEN
Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the postimplantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the nonobese diabetic (NOD) mouse strain, which prior to this study was considered "nonpermissive" for ESC derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ESCs and iPSCs appear to be "metastable," as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.
Asunto(s)
Hemostasis , Células Madre Pluripotentes/citología , Animales , Desdiferenciación Celular , Células Madre Embrionarias/citología , Estratos Germinativos/citología , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
Explaining the uniqueness of the acquired somatic JAK2 V617F mutation, which is present in more than 95% of polycythemia vera patients, has been a challenge. The V617F mutation in the pseudokinase domain of JAK2 renders the unmutated kinase domain constitutively active. We have performed random mutagenesis at position 617 of JAK2 and tested each of the 20 possible amino acids for ability to induce constitutive signaling in Ba/F3 cells expressing the erythropoietin receptor. Four JAK2 mutants, V617W, V617M, V617I, and V617L, were able to induce cytokine independence and constitutive downstream signaling. Only V617W induced a level of constitutive activation comparable with V617F. Also, only V617W stabilized tyrosine-phosphorylated suppressor of cytokine signaling 3 (SOCS3), a mechanism by which JAK2 V617F overcomes inhibition by SOCS3. The V617W mutant induced a myeloproliferative disease in mice, mainly characterized by erythrocytosis and megakaryocytic proliferation. Although JAK2 V617W would predictably be pathogenic in humans, the substitution of the Val codon, GTC, by TTG, the codon for Trp, would require three base pair changes, and thus it is unlikely to occur. We discuss how the predicted conformations of the activated JAK2 mutants can lead to better screening assays for novel small molecule inhibitors.