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1.
J Immunol ; 206(11): 2700-2713, 2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-34021049

RESUMEN

B lymphocyte development is dependent on the interplay between the chromatin landscape and lineage-specific transcription factors. It has been suggested that B lineage commitment is associated with major changes in the nuclear chromatin environment, proposing a critical role for lineage-specific transcription factors in the formation of the epigenetic landscape. In this report, we have used chromosome conformation capture in combination with assay for transposase-accessible chromatin sequencing analysis to enable highly efficient annotation of both proximal and distal transcriptional control elements to genes activated in B lineage specification in mice. A large majority of these genes were annotated to at least one regulatory element with an accessible chromatin configuration in multipotent progenitors. Furthermore, the majority of binding sites for the key regulators of B lineage specification, EBF1 and PAX5, occurred in already accessible regions. EBF1 did, however, cause a dynamic change in assay for transposase-accessible chromatin accessibility and was critical for an increase in distal promoter-enhancer interactions. Our data unravel an extensive epigenetic priming at regulatory elements annotated to lineage-restricted genes and provide insight into the interplay between the epigenetic landscape and transcription factors in cell specification.


Asunto(s)
Linfocitos B/inmunología , Epigénesis Genética/inmunología , Factor de Transcripción PAX5/inmunología , Transactivadores/inmunología , Animales , Epigénesis Genética/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Transactivadores/deficiencia , Transactivadores/genética
2.
J Immunol Methods ; 495: 113070, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-33957108

RESUMEN

The CRISPR/Cas9 system has been used for genome editing of human and mouse cells. In this study, we established a protocol for gene knockout (KO) in mouse hematopoietic stem cells (HSCs). HSCs were highly purified from the bone marrow of tamoxifen-treated Cas9-EGFP/Cre-ER transgenic mice, maintained in serum-free polyvinyl alcohol culture with cytokines, lentivirally transduced with sgRNA-Crimson, and transplanted into lethally irradiated mice with competitor cells. Previous studies of Pax5 KO mice have shown B cell differentiation block. To verify our KO HSC strategy, we deleted Pax5 gene in 600 CD201+CD150+CD48-c-Kit+Sca-1+Lin- cells (HSC1 cells), highly enriched in myeloid-biased HSCs, and CD201+CD150-CD48- c-Kit+Sca-1+Lin- cells (HSC2 cells), highly enriched in lymphoid-biased HSCs. As predicted, both Pax5 KO HSC1 and HSC2 cells showed few B cells in the peripheral blood and the accumulation of pro-B cells in the bone marrow of recipient mice. Our data suggesetd that myeloid-biased and lymphoid-biased HSCs share a common B cell differentiation pathway. This population-specific KO strategy will find its applications for gene editing in a varity of somatic cells, particuarly rare stem and progenitor cells from different tissues.


Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Técnicas de Inactivación de Genes , Células Madre Hematopoyéticas/metabolismo , Factor de Transcripción PAX5/genética , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Hematopoyéticas/inmunología , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción PAX5/deficiencia , Fenotipo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Transducción Genética
3.
Nature ; 542(7642): 479-483, 2017 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-28192788

RESUMEN

B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development, yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL). The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK. Dominant-negative mutants of PAX5 and IKZF1, however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor), TXNIP (encoding a glucose-feedback sensor) and CNR2 (encoding a cannabinoid receptor) as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.


Asunto(s)
Linfocitos B/metabolismo , Metabolismo Energético/genética , Regulación Neoplásica de la Expresión Génica , Glucosa/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Factores de Transcripción/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Carcinogénesis/genética , Proteínas Portadoras/agonistas , Proteínas Portadoras/metabolismo , Muerte Celular , Inmunoprecipitación de Cromatina , Ciclo del Ácido Cítrico , Modelos Animales de Enfermedad , Femenino , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Humanos , Factor de Transcripción Ikaros/metabolismo , Ratones , Ratones Transgénicos , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Ácido Pirúvico/metabolismo , Receptor Cannabinoide CB2/agonistas , Receptor Cannabinoide CB2/metabolismo , Receptores de Glucocorticoides/metabolismo , Análisis de Secuencia de ARN
4.
Leuk Res ; 39(12): 1455-61, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26520622

RESUMEN

Epigenetic dysregulation is a hallmark of cancer executed by a number of complex processes the most important of which converge on DNA methylation and histone protein modifications. Epigenetic marks are potentially reversible and thus promising drug targets. In the setting of acute lymphoblastic leukemia (ALL) they have been associated with clinicopathological features including risk of relapse or molecular subgroups of the disease. Here, using immunocytochemistry of bone marrow smears from diagnosis, we studied global histone H4 acetylation, whose loss was previously linked to treatment failure in adults with ALL, in pediatric patients. We demonstrate that preserved global histone H4 acetylation is significantly associated with favorable outcome (RFS, EFS, OS) in children with B cell progenitor (BCP) ALL, recapitulating the findings from adult populations. Further, for the first time we demonstrate differential histone H4 acetylation in molecular subclasses of BCP-ALL including cases with ETV6-RUNX1 fusion gene or PAX5 deletion or deletions in genes linked to B cell development. We conclude global histone H4 acetylation is a prognostic marker and a potential therapeutic target in ALL.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Epigénesis Genética , Histonas/metabolismo , Proteínas de Fusión Oncogénica/fisiología , Factor de Transcripción PAX5/deficiencia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Acetilación , Adolescente , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Niño , Preescolar , Cromosomas Humanos Par 12/ultraestructura , Cromosomas Humanos Par 21/ultraestructura , Supervivencia sin Enfermedad , Femenino , Humanos , Lactante , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Factor de Transcripción PAX5/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , Procesamiento Proteico-Postraduccional , Inducción de Remisión , Translocación Genética , Resultado del Tratamiento
5.
J Exp Med ; 212(7): 1109-23, 2015 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-26056231

RESUMEN

To investigate how transcription factor levels impact B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19(+) progenitors, these cells displayed an increased T cell potential in vivo and in vitro. The alteration in lineage fate depended on a Notch1-mediated conversion process, whereas no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5(+/-)Ebf1(+/-) pro-B cells were reflected in the transcriptional response. Both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, but only the Pax5(+/-)Ebf1(+/-) pro-B cells down-regulated genes central for the preservation of stable B cell identity. This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling. This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.


Asunto(s)
Linfocitos B/citología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Regulación de la Expresión Génica/inmunología , Factor de Transcripción PAX5/deficiencia , Linfocitos T/citología , Transactivadores/deficiencia , Animales , Antígenos CD19/inmunología , Linfocitos B/inmunología , Secuencia de Bases , Western Blotting , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Citometría de Flujo , Regulación de la Expresión Génica/genética , Pérdida de Heterocigocidad , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación/genética , Factor de Transcripción PAX5/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/metabolismo , Análisis de Secuencia de ARN , Transactivadores/genética
6.
Br J Haematol ; 163(5): 551-64, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24111932

RESUMEN

Multiple myeloma (MM) is a haematological malignancy characterized by the accumulation of clonal plasma cells (PCs) in the bone marrow (BM). Although novel therapeutic strategies have prolonged survival of patients, the disease remains difficult to treat with a high risk of relapse. The failure of therapy is thought to be associated with a persistent population of the so-called MM stem cells or myeloma initiating cells (MIC) that exhibit tumour-initiating potential, self-renewal and resistance to chemotherapy. However, the population responsible for the origin and sustainability of tumour mass has not been clearly characterized so far. This review summarizes current myeloma stem cell concepts and suggests that high phenotypic and intra-clonal heterogeneity, together with plasticity potential of MM might be other contributing factors explaining discrepancies among particular concepts and contributing to the treatment failure.


Asunto(s)
Mieloma Múltiple/patología , Células Madre Neoplásicas/patología , Antígenos de Diferenciación de Linfocitos B/análisis , Antígenos de Neoplasias/análisis , Linfocitos B/patología , Desdiferenciación Celular/genética , Hipoxia de la Célula , Linaje de la Célula , Células Clonales/patología , Regulación Neoplásica de la Expresión Génica , Reordenamiento Génico de Linfocito B , Humanos , Cambio de Clase de Inmunoglobulina , Modelos Biológicos , Terapia Molecular Dirigida , Mieloma Múltiple/terapia , Proteínas de Mieloma/análisis , Proteínas de Mieloma/genética , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Células Plasmáticas/patología
7.
Oncogene ; 31(29): 3419-30, 2012 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-22105368

RESUMEN

Using genome-wide methylation screening, we identified that paired box gene 5 (PAX5) is involved in human cancer development. However, the function of PAX5 in gastric cancer (GC) development is largely unclear. We analyzed its epigenetic inactivation, biological functions and clinical application in GC. PAX5 was silenced in seven out of eight GC cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancerous tissues. The downregulation of PAX5 was closely linked to the promoter hypermethylation status and could be restored with demethylation treatment. Ectopic expression of PAX5 in silenced GC cell lines (AGS and BGC823) inhibited colony formation and cell viability, arrested cell cycle, induced apoptosis, suppressed cell migration and invasion and repressed tumorigenicity in nude mice. Consistent with the induction of apoptosis by PAX5 in vitro, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining showed significantly enhanced apoptotic cells in PAX5-expressed tumors compared with the vector control tumors. On the other hand, knockdown of PAX5 by PAX5-short hairpin RNA increased the cell viability and proliferation. The anti-tumorigenic function of PAX5 was revealed to be mediated by upregulating downstream targets of tumor protein 53 (p53), p21, BCL2-associated X protein, metastasis suppressor 1 and tissue inhibitors of metalloproteinase 1, and downregulating BCL2, cyclin D1, mesenchymal-epithelial transition factor (MET) and matrix metalloproteinase 1. Immunoprecipitation assay demonstrated that PAX5 directly bound to the promoters of p53 and MET. Moreover, PAX5 hypermethylation was detected in 77% (144 of 187) of primary GCs compared with 10.5% (2/19) of normal gastric tissues (P<0.0001). GC patients with PAX5 methylation had a significant poor survival compared with the unmethylated cases as demonstrated by Cox regression model and log-rank test. In conclusion, PAX5 is a novel functional tumor suppressor in gastric carcinogenesis. Detection of methylated PAX5 can be utilized as an independent prognostic factor in GC.


Asunto(s)
Silenciador del Gen , Genes Supresores de Tumor , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Neoplasias Gástricas/diagnóstico , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/genética , Animales , Apoptosis/genética , Biomarcadores de Tumor/deficiencia , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Metilación de ADN/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Persona de Mediana Edad , Invasividad Neoplásica/genética , Factor de Transcripción PAX5/metabolismo , Pronóstico , Regiones Promotoras Genéticas/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Análisis de Supervivencia
8.
Immunity ; 34(2): 175-87, 2011 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-21349430

RESUMEN

V(H)-DJ(H) recombination of the immunoglobulin heavy chain (Igh) locus is temporally and spatially controlled during early B cell development, and yet no regulatory elements other than the V(H) gene promoters have been identified throughout the entire V(H) gene cluster. Here, we discovered regulatory sequences that are interspersed in the distal V(H) gene region. These conserved repeat elements were characterized by the presence of Pax5 transcription factor-dependent active chromatin by binding of the regulators Pax5, E2A, CTCF, and Rad21, as well as by Pax5-dependent antisense transcription in pro-B cells. The Pax5-activated intergenic repeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B cell development. The pro-B cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal V(H)-DJ(H) recombination at the Igh locus.


Asunto(s)
Cromatina/genética , ADN Intergénico/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Factor de Transcripción PAX5/fisiología , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Sitios de Unión , Factor de Unión a CCCTC , Inmunoprecipitación de Cromatina , Secuencia Conservada , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Células Precursoras de Linfocitos B/metabolismo , ARN sin Sentido/biosíntesis , ARN sin Sentido/genética , Proteínas Represoras/fisiología , Transcripción Genética
9.
Cell Stem Cell ; 6(3): 279-86, 2010 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-20207230

RESUMEN

Polycomb group (PcG) proteins are essential regulators of stem cells. PcG and trithorax group proteins mark developmental regulator gene promoters with bivalent domains consisting of overlapping repressive and activating histone modifications to keep them poised for activation in embryonic stem cells. Bmi1, a component of PcG complexes, maintains the self-renewal capacity of adult stem cells, but its role in multipotency remains obscure. Here we show that Bmi1 is critical for multipotency of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). B cell lineage developmental regulator genes, Ebf1 and Pax5, appeared to be transcriptionally repressed by bivalent domains before lineage commitment. Loss of Bmi1 resulted in a resolution of bivalent domains at the Ebf1 and Pax5 loci, leading to their premature expression in HSC/MPPs accompanied by accelerated lymphoid specification and a marked reduction in HSC/MPPs. Thus, Bmi1 is required to reinforce bivalent domains at key developmental regulator gene loci to maintain lineage specification poised for activation in adult stem cells.


Asunto(s)
Linaje de la Célula , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Diferenciación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/deficiencia , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/metabolismo , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Represoras/deficiencia , Transactivadores/deficiencia , Transactivadores/metabolismo
10.
Eur J Immunol ; 38(12): 3520-9, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18991270

RESUMEN

Between 10 and 20% of the peripheral gammadelta T cells express cytoplasmic TCR-beta proteins, but whether such TCR-beta chains can partake in alphabeta T-cell development has never been systematically investigated. Therefore, we reconstituted the T-cell compartment of CD3epsilon-deficient mice with Pax5-TCR-beta deficient proB cells expressing, via a retroviral vector, TCR-beta chains from either peripheral gammadelta or alphabeta T cells. Recipient thymi reconstituted with proB cells containing empty vector were small (<15x10(6) cells), contained few gammadelta T but no alphabeta T cells. In contrast, thymi from mice receiving proB cells containing gammadelta or alphabeta T-cell-derived TCR-beta chains contained 80-130x10(6) cells, and showed a normal CD4, CD8 and alphabeta TCR expression pattern. However, regardless of the source of TCR-beta chain, reconstituted mice rapidly showed signs of autoimmunity dying 5-15 wk following reconstitution. Autoimmune disease induction could be prevented by co-transfer of Treg cells thereby allowing the functionality of the generated T cells to be assessed. Results obtained show that TCR-beta chains from gammadelta T cells can efficiently take part in alphabeta T-cell development. The implications of these findings for gammadelta T-cell development will be discussed.


Asunto(s)
Diferenciación Celular/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/deficiencia , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Timo/inmunología
11.
Blood ; 112(5): 1673-82, 2008 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-18552207

RESUMEN

Low-level expression of multiple lineage-specific genes is a hallmark of hematopoietic stem cells (HSCs). HSCs predominantly express genes specific for the myeloid or megakaryocytic-erythroid lineages, whereas the transcription of lymphoid specific genes appears to begin after lymphoid specification. It has been demonstrated for a number of genes that epigenetic priming occurs before gene expression and lineage specification; however, little is known about how epigenetic priming of lymphoid genes is regulated. To address the question of how B cell-restricted expression is established, we studied activation of the Cd19 gene during hematopoietic development. We identified a B cell-specific upstream enhancer and showed that the developmental regulation of Cd19 expression involves precisely coordinated alterations in transcription factor binding and chromatin remodeling at Cd19 cis-regulatory elements. In multipotent progenitor cells, Cd19 chromatin is first remodeled at the upstream enhancer, and this remodeling is associated with binding of E2A. This is followed by the binding of EBF and PAX5 during B-cell differentiation. The Cd19 promoter is transcriptionally activated only after PAX5 binding. Our experiments give important mechanistic insights into how widely expressed and B lineage-specific transcription factors cooperate to mediate the developmental regulation of lymphoid genes during hematopoiesis.


Asunto(s)
Antígenos CD19/genética , Linfocitos B/inmunología , Células Madre Hematopoyéticas/inmunología , Animales , Linfocitos B/citología , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Islas de CpG , ADN/genética , ADN/metabolismo , Metilación de ADN , Elementos de Facilitación Genéticos , Epigénesis Genética , Hematopoyesis/genética , Ratones , Datos de Secuencia Molecular , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal/genética , Transducción de Señal/inmunología , Transactivadores/metabolismo , Activación Transcripcional
13.
Nature ; 449(7161): 473-7, 2007 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-17851532

RESUMEN

Lineage commitment and differentiation to a mature cell type are considered to be unidirectional and irreversible processes under physiological conditions. The commitment of haematopoietic progenitors to the B-cell lineage and their development to mature B lymphocytes critically depend on the transcription factor encoded by the paired box gene 5 (Pax5). Here we show that conditional Pax5 deletion in mice allowed mature B cells from peripheral lymphoid organs to dedifferentiate in vivo back to early uncommitted progenitors in the bone marrow, which rescued T lymphopoiesis in the thymus of T-cell-deficient mice. These B-cell-derived T lymphocytes carried not only immunoglobulin heavy- and light-chain gene rearrangements but also participated as functional T cells in immune reactions. Mice lacking Pax5 in mature B cells also developed aggressive lymphomas, which were identified by their gene expression profile as progenitor cell tumours. Hence, the complete loss of Pax5 in late B cells could initiate lymphoma development and uncovered an extraordinary plasticity of mature peripheral B cells despite their advanced differentiation stage.


Asunto(s)
Linfocitos B/citología , Diferenciación Celular , Células Madre/citología , Linfocitos T/citología , Animales , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Eliminación de Gen , Reordenamiento Génico de Linfocito B/genética , Inmunoglobulinas/genética , Inmunoglobulinas/inmunología , Linfoma/genética , Linfoma/patología , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Linfocitos T/inmunología , Timo/citología , Timo/metabolismo
15.
Genes Cells ; 12(3): 359-73, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17352740

RESUMEN

We previously reported that histone deacetylase-2 (HDAC2) controls the amount of IgM H-chain at the steps of transcription of its gene and alternative processing of its pre-mRNA in DT40 cells. Here, we showed not only that the HDAC2-deficiency caused repressions of gene expressions for HDAC7, EBF1, Pax5, Aiolos and Ikaros, and elevations of gene expressions for HDAC4, HDAC5, PCAF and E2A, but also that it caused altered acetylation levels of several Lys residues of core histones. Using gene targeting techniques, we generated three homozygous DT40 mutants: EBF1(-/-), Aiolos(-/-) and E2A(-/-), devoid of EBF1, Aiolos and E2A genes, respectively. Semiquantitative RT-PCR analysis of the resultant mutants revealed not only that EBF1 and Aiolos down-regulate expressions of IgM H- and L-chain genes, but also that E2A up-regulates expressions of these two genes. These results, together with others, indicate that HDAC2 controls indirectly expressions of IgM H- and L-chain genes through opposite transcriptional regulations of EBF1, Pax5, Aiolos plus Ikaros and E2A genes.


Asunto(s)
Genes de Inmunoglobulinas , Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Acetilación , Animales , Secuencia de Bases , Línea Celular , Pollos/genética , Pollos/inmunología , Cartilla de ADN/genética , Regulación de la Expresión Génica , Marcación de Gen , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Genes de las Cadenas Ligeras de las Inmunoglobulinas , Histona Acetiltransferasas/metabolismo , Histona Desacetilasa 2 , Histona Desacetilasas/deficiencia , Histona Desacetilasas/genética , Histonas/química , Histonas/metabolismo , Factor de Transcripción Ikaros/deficiencia , Factor de Transcripción Ikaros/genética , Inmunoglobulina M/genética , Inmunoglobulina M/metabolismo , Lisina/química , Modelos Biológicos , Mutación , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/genética , Proteínas Represoras/genética , Factores de Transcripción/deficiencia
16.
Eur J Immunol ; 36(12): 3294-304, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17111353

RESUMEN

Unlike early B/T cell development, NK cell lineage commitment is not well understood, with a major limitation being the lack of a robust culture system to assay NK cell progenitors. Here we have exploited the multi-lineage potential of Pax5(-/-) pro-B cells to establish an effective system to direct differentiation of progenitors into the NK cell lineage. Cultivation of Pax5(-/-) pro-B cells on OP9 cells expressing the Notch ligand Delta-Like1 (OP9-DL1) in the presence of IL-7 efficiently induced T and NK cell potential. For NK cells, Notch was only transiently required, as prolonged signaling decreased NK and increased T cell development. Pure NK cell populations could be obtained by the culture of these Notch signal-experienced cells onto OP9 stroma and IL-15. A similar transient exposure to Notch was also compatible with the differentiation of NK cells from hematopoietic progenitors, while sustained Notch signaling impaired NK cell generation. Pax5(-/-) pro-B cell-derived NK cells were cytotoxic, secreted cytokines and expressed all the expected NK cell-specific surface markers examined except the Ly49 family, a phenotype similar to fetal NK cells. These data indicate that Notch signaling induces T/NK cell differentiation in Pax5(-/-) pro-B cells that is strikingly similar to early thymopoiesis.


Asunto(s)
Linfocitos B/citología , Células Asesinas Naturales/citología , Factor de Transcripción PAX5/deficiencia , Receptores Notch/fisiología , Transducción de Señal/inmunología , Células Madre/citología , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Línea Celular Tumoral , Linaje de la Célula/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción PAX5/genética , Transducción de Señal/genética , Células Madre/inmunología , Células Madre/metabolismo
17.
Eur J Immunol ; 36(3): 526-32, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16482516

RESUMEN

The precise roles played by the transmembrane receptor tyrosine kinase c-kit and its ligand stem cell factor in early T cell development are difficult to study. Using cloned Pax5-deficient progenitor B cells, we show that following Notch signaling, which induces their commitment to the T cell developmental pathway, c-kit expression is rapidly up-regulated at both the transcriptional and cell surface level. Using either an anti-c-kit monoclonal antibody or Gleevec, a pharmacological inhibitor of c-kit signaling, we show that the Notch-induced T cell differentiation of either Pax5-deficient progenitor B cells, or the equivalent cell from the bone marrow of normal mice, is strictly dependent on c-kit signaling, whereas the differentiation of normal progenitors into the B cell lineage is not. Moreover, we show that the Notch and IL-7 signaling-induced proliferation and differentiation of CD44+CD25-c-kit(high) and CD44+CD25+c-kithigh thymocytes along the T cell, but not natural killer cell or macrophage, pathway also requires c-kit signaling, whereas the Notch-induced proliferation and differentiation of CD44-CD25+c-kitint cells along the T cell pathway is independent of c-kit. These results further highlight the complex inter-relationships existing between c-kit, Notch and IL-7 receptor signaling that control the proliferation and differentiation of early T cell progenitors.


Asunto(s)
Diferenciación Celular/inmunología , Proteínas Proto-Oncogénicas c-kit/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Timo/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Linfocitos B/inmunología , Benzamidas , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Femenino , Receptores de Hialuranos/inmunología , Mesilato de Imatinib , Interleucina-7/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ratones , Factor de Transcripción PAX5/deficiencia , Factor de Transcripción PAX5/inmunología , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Receptores de Interleucina-2/inmunología , Receptores Notch/inmunología , Transducción de Señal/genética , Células Madre/inmunología , Linfocitos T/citología , Timo/citología
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