RESUMO
Histone deacetylase (HDAC) inhibitors (HDACis) are well-characterized anti-cancer agents with promising results in clinical trials. However, mechanistically little is known regarding their selectivity in killing malignant cells while sparing normal cells. Gene expression-based chemical genomics identified HDACis as being particularly potent against Down syndrome-associated myeloid leukemia (DS-AMKL) blasts. Investigating the antileukemic function of HDACis revealed their transcriptional and post-translational regulation of key autophagic proteins, including ATG7. This leads to suppression of autophagy, a lysosomal degradation process that can protect cells against damaged or unnecessary organelles and protein aggregates. DS-AMKL cells exhibit low baseline autophagy due to mammalian target of rapamycin (mTOR) activation. Consequently, HDAC inhibition repressed autophagy below a critical threshold, which resulted in accumulation of mitochondria, production of reactive oxygen species, DNA damage and apoptosis. Those HDACi-mediated effects could be reverted upon autophagy activation or aggravated upon further pharmacological or genetic inhibition. Our findings were further extended to other major acute myeloid leukemia subgroups with low basal level autophagy. The constitutive suppression of autophagy due to mTOR activation represents an inherent difference between cancer and normal cells. Thus, via autophagy suppression, HDACis deprive cells of an essential pro-survival mechanism, which translates into an attractive strategy to specifically target cancer cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Leucemia Mieloide/patologia , Animais , Humanos , Leucemia Mieloide/imunologia , Leucemia Mieloide/metabolismo , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Transient leukemia (TL) is evident in 5-10% of all neonates with Down syndrome (DS) and associated with N-terminal truncating GATA1 mutations (GATA1s). Here we report that TL-cell clones generate abundant eosinophils in a substantial fraction of patients. Sorted eosinophils from patients with TL and eosinophilia carried the same GATA1s mutations as sorted TL blasts, consistent with their clonal origin. TL blasts exhibited a genetic program characteristic of eosinophils and differentiated along the eosinophil lineage in vitro. Similarly, ectopic expression of Gata1s, but not Gata1, in wild-type CD34(+)-hematopoietic stem and progenitor cells induced hyperproliferation of eosinophil promyelocytes in vitro. Although GATA1s retained the function of GATA1 to induce eosinophil genes by occupying their promoter regions, GATA1s was impaired in its ability to repress oncogenic MYC and the pro-proliferative E2F transcription network. Chromatin Immunoprecipitation Sequencing (ChIP-seq) indicated reduced GATA1s occupancy at the MYC promoter. Knockdown of MYC, or the obligate E2F-cooperation partner DP1, rescued the GATA1s-induced hyperproliferative phenotype. In agreement, terminal eosinophil maturation was blocked in Gata1(Δe2) knockin mice, exclusively expressing Gata1s, leading to accumulation of eosinophil precursors in blood and bone marrow. These data suggest a direct relationship between the N-terminal truncating mutations of GATA1 and clonal eosinophilia in DS patients.
Assuntos
Proliferação de Células , Síndrome de Down/patologia , Eosinofilia/patologia , Fator de Transcrição GATA1/genética , Leucemia Mieloide Aguda/patologia , Mutação/genética , Animais , Apoptose , Diferenciação Celular , Síndrome de Down/complicações , Síndrome de Down/genética , Eosinofilia/etiologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/complicações , Leucemia Mieloide Aguda/genética , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Tumors are thought to be sustained by a reservoir of self-renewing cells, termed tumor-initiating cells or cancer stem cells. Osteosarcomas are high-grade sarcomas derived from osteoblast progenitor cells and are the most common pediatric bone malignancy. In this report we show that the stem cell transcription factor Sox2 is highly expressed in human and murine osteosarcoma (mOS) cell lines as well as in the tumor samples. Osteosarcoma cells have increased ability to grow in suspension as osteospheres, that are greatly enriched in expression of Sox2 and the stem cell marker, Sca-1. Depletion of Sox2 by short-hairpin RNAs in independent mOS-derived cells drastically reduces their transformed properties in vitro and their ability to form tumors. Sox2-depleted osteosarcoma cells can no longer form osteospheres and differentiate into mature osteoblasts. Concomitantly, they exhibit decreased Sca-1 expression and upregulation of the Wnt signaling pathway. Thus, despite other mutations, these cells maintain a requirement for Sox2 for tumorigenicity. Our data indicate that Sox2 is required for osteosarcoma cell self renewal, and that Sox2 antagonizes the pro-differentiation Wnt pathway that can in turn reduce Sox2 expression. These studies define Sox2 as a survival factor and a novel biomarker of self renewal in osteosarcomas, and support a tumor suppressive role for the Wnt pathway in tumors of mesenchymal origin. Our findings could provide the basis for novel therapeutic strategies based on inhibiting Sox2 or enhancing Wnt signaling for the treatment of osteosarcomas.
Assuntos
Neoplasias Ósseas/genética , Proliferação de Células , Células-Tronco Neoplásicas , Osteossarcoma/genética , Fatores de Transcrição SOXB1/genética , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Neoplasias Ósseas/patologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Osteossarcoma/patologia , Transdução de Sinais , Via de Sinalização WntRESUMO
Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.
Assuntos
Epigênese Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Reprogramação Celular/genética , Metilação de DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genoma/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Técnicas de Transferência Nuclear , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Although the physiological role of tissue-specific translational control of gene expression in mammals has long been suspected on the basis of biochemical studies, direct evidence has been lacking. Here, we report on the targeted disruption of the gene encoding the heme-regulated eIF2alpha kinase (HRI) in mice. We establish that HRI, which is expressed predominantly in erythroid cells, regulates the synthesis of both alpha- and beta-globins in red blood cell (RBC) precursors by inhibiting the general translation initiation factor eIF2. This inhibition occurs when the intracellular concentration of heme declines, thereby preventing the synthesis of globin peptides in excess of heme. In iron-deficient HRI(-/-) mice, globins devoid of heme aggregated within the RBC and its precursors, resulting in a hyperchromic, normocytic anemia with decreased RBC counts, compensatory erythroid hyperplasia and accelerated apoptosis in bone marrow and spleen. Thus, HRI is a physiological regulator of gene expression and cell survival in the erythroid lineage.
Assuntos
Eritrócitos/citologia , Eritrócitos/enzimologia , Regulação Enzimológica da Expressão Gênica , Deficiências de Ferro , Biossíntese de Proteínas , eIF-2 Quinase/metabolismo , eIF-2 Quinase/fisiologia , Animais , Apoptose , Northern Blotting , Western Blotting , Linhagem da Célula , Separação Celular , Sobrevivência Celular , Clonagem Molecular , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Fator de Iniciação 2 em Eucariotos/metabolismo , Citometria de Fluxo , Biblioteca Gênica , Genótipo , Heme/biossíntese , Ferro/metabolismo , Camundongos , Microscopia Eletrônica , Modelos Biológicos , Fosforilação , Polirribossomos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Protoporfirinas/biossíntese , Reticulócitos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico , Fatores de TempoRESUMO
The retinoblastoma tumour suppressor (Rb) pathway is believed to have a critical role in the control of cellular proliferation by regulating E2F activities. E2F1, E2F2 and E2F3 belong to a subclass of E2F factors thought to act as transcriptional activators important for progression through the G1/S transition. Here we show, by taking a conditional gene targeting approach, that the combined loss of these three E2F factors severely affects E2F target expression and completely abolishes the ability of mouse embryonic fibroblasts to enter S phase, progress through mitosis and proliferate. Loss of E2F function results in an elevation of p21Cip1 protein, leading to a decrease in cyclin-dependent kinase activity and Rb phosphorylation. These findings suggest a function for this subclass of E2F transcriptional activators in a positive feedback loop, through down-modulation of p21Cip1, that leads to the inactivation of Rb-dependent repression and S phase entry. By targeting the entire subclass of E2F transcriptional activators we provide direct genetic evidence for their essential role in cell cycle progression, proliferation and development.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Proteínas de Ligação a DNA , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Regulação para Baixo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Fator de Transcrição E2F3 , Fibroblastos/citologia , Marcação de Genes , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína do Retinoblastoma/metabolismo , Fase S/genética , Fase S/fisiologia , Fatores de Transcrição/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
E2F activity is critical for the control of the G(1) to S phase transition. We show that the combined loss of E2F1 and E2F2 results in profound effects on hematopoietic cell proliferation and differentiation, as well as increased tumorigenesis and decreased lymphocyte tolerance. The loss of E2F1 and E2F2 impedes B-cell differentiation, and hematopoietic progenitor cells in the bone marrow of mice lacking E2F1 and E2F2 exhibit increased cell cycling. Importantly, we show that E2F1 and E2F2 double-knockout T cells exhibit more rapid entry into S phase following antigenic stimulation. Furthermore, T cells lacking E2F1 and E2F2 proliferate much more extensively in response to subthreshold antigenic stimulation. Consistent with these observations, E2F1/E2F2 mutant mice are highly predisposed to the development of tumors, and some mice exhibit signs of autoimmunity.
Assuntos
Antígenos/metabolismo , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Neoplasias/metabolismo , Linfócitos T/citologia , Linfócitos T/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Fatores Etários , Animais , Western Blotting , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Divisão Celular , Células Cultivadas , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Feminino , Citometria de Fluxo , Genótipo , Linfonodos/citologia , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Ribonucleases/metabolismo , Baço/citologia , Fatores de TempoRESUMO
The development of naive CD4+ T cells into a T helper (Th) 2 subset capable of producing interleukin (IL)-4, IL-5, and IL-13 involves a signal transducer and activator of transcription (Stat)6-dependent induction of GATA-3 expression, followed by Stat6-independent GATA-3 autoactivation. The friend of GATA (FOG)-1 protein regulates GATA transcription factor activity in several stages of hematopoietic development including erythrocyte and megakaryocyte differentiation, but whether FOG-1 regulates GATA-3 in T cells is uncertain. We show that FOG-1 can repress GATA-3-dependent activation of the IL-5 promoter in T cells. Also, FOG-1 overexpression during primary activation of naive T cells inhibited Th2 development in CD4+ T cells. FOG-1 fully repressed GATA-3-dependent Th2 development and GATA-3 autoactivation, but not Stat6-dependent induction of GATA-3. FOG-1 overexpression repressed development of Th2 cells from naive T cells, but did not reverse the phenotype of fully committed Th2 cells. Thus, FOG-1 may be one factor capable of regulating the Th2 development.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Animais , Fator de Transcrição GATA3 , Interleucina-4/farmacologia , Camundongos , Camundongos Transgênicos , Fator de Transcrição STAT6 , Células Th2/fisiologia , Fatores de Transcrição , Transcrição GênicaRESUMO
Over the past year, significant new insights have been gained in our understanding of the lineage determination of red blood cells. In particular, evidence has emerged demonstrating that cross-antagonism of lineage-specific transcription factors plays an important role in determining cell phenotype by actively repressing alternate lineage gene programs.
Assuntos
Linhagem da Célula , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/metabolismo , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Humanos , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismoRESUMO
Patients with paroxysmal nocturnal hemoglobinuria (PNH) have blood cells deficient in glycosyl phosphatidylinositol (GPI)-linked proteins owing to a somatic mutation in the X-linked PIGA gene. To target Piga recombination to the erythroid/megakaryocytic lineage in mice, the Cre/loxP system was used, and Cre was expressed under the transcriptional regulatory sequences of GATA-1. Breeding of GATA1-cre (G) transgenic mice with mice carrying a floxed Piga (L) allele was associated with high embryonic lethality. However, double-transgenic (GL) mice that escaped early recombination looked healthy and were observed for 16 months. Flow cytometric analysis of peripheral blood cells showed that GL mice had up to 100% of red cells deficient in GPI-linked proteins. The loss of GPI-linked proteins on the cell surface occurred late in erythroid differentiation, causing a proportion of red cells to express low residual levels of GPI-linked proteins. Red cells with residual expression of GPI-linked proteins showed an intermediate sensitivity toward complement and thus resemble PNH type II cells in patients with PNH. Recombination of the floxed Piga allele was also detected in cultured megakaryocytes, mast cells, and eosinophils, but not in neutrophils, lymphocytes, or nonhematopoietic tissues. In summary, GATA1-Cre causes high-efficiency Piga gene inactivation in a GATA-1-specific pattern. For the first time, mice were generated that have almost 100% of red cells deficient in GPI-linked proteins. These animals will be valuable to further investigate the consequences of GPI-anchor deficiency on erythroid/megakaryocytic cells.
Assuntos
Proteínas de Ligação a DNA/farmacologia , Eritropoese/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Glicosilfosfatidilinositóis/deficiência , Integrases/farmacologia , Proteínas de Membrana/genética , Fatores de Transcrição/farmacologia , Proteínas Virais/farmacologia , Animais , Células da Medula Óssea/metabolismo , Linhagem da Célula , Proteínas de Ligação a DNA/fisiologia , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Hemoglobinúria Paroxística/patologia , Integrases/fisiologia , Fígado/citologia , Fígado/embriologia , Fígado/metabolismo , Mastócitos/citologia , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Camundongos , Camundongos Transgênicos , Recombinação Genética , Fatores de Transcrição/fisiologia , Proteínas Virais/fisiologiaRESUMO
Hematopoietic stem cells are derived from ventral mesoderm during vertebrate development. Gene targeting experiments in the mouse have demonstrated key roles for the basic helix-loop-helix transcription factor SCL and the GATA-binding protein GATA-1 in hematopoiesis. When overexpressed in Xenopus animal cap explants, SCL and GATA-1 are each capable of specifying mesoderm to become blood. Forced expression of either factor in whole embryos, however, does not lead to ectopic blood formation. This apparent paradox between animal cap assays and whole embryo phenotype has led to the hypothesis that additional factors are involved in specifying hematopoietic mesoderm. SCL and GATA-1 interact in a transcriptional complex with the LIM domain protein LMO-2. We have cloned the Xenopus homolog of LMO-2 and show that it is expressed in a similar pattern to SCL during development. LMO-2 can specify hematopoietic mesoderm in animal cap assays. SCL and LMO-2 act synergistically to expand the blood island when overexpressed in whole embryos. Furthermore, co-expression of GATA-1 with SCL and LMO-2 leads to embryos that are ventralized and have blood throughout the dorsal-ventral axis. The synergistic effect of SCL, LMO-2 and GATA-1, taken together with the findings that these factors can form a complex in vitro, suggests that this complex specifies mesoderm to become blood during embryogenesis.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Eritropoese/fisiologia , Sequências Hélice-Alça-Hélice , Metaloproteínas/fisiologia , Proteínas Proto-Oncogênicas , Fatores de Transcrição/fisiologia , Proteínas de Xenopus , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/genética , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA1 , Expressão Gênica , Globinas/genética , Humanos , Proteínas com Domínio LIM , Metaloproteínas/genética , Camundongos , Dados de Sequência Molecular , Proteínas Smad , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transativadores/genética , Fatores de Transcrição/genética , Xenopus laevis/embriologiaRESUMO
Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fase S/fisiologia , Fatores de Transcrição/metabolismo , Adenoviridae/genética , Animais , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F3 , Fator de Transcrição E2F4 , Fibroblastos/fisiologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The Drosophila FOG homologue U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homologue Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. In this report, we show that Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of ush function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Hemócitos/citologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Embrião não Mamífero/fisiologia , Olho/embriologia , Olho/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Coração/crescimento & desenvolvimento , Hemócitos/fisiologia , Larva , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Dedos de ZincoRESUMO
Reporter mouse strains are important tools for monitoring Cre recombinase-mediated excision in vivo. In practice, excision may be incomplete in a given population due to threshold level or variegated expression of Cre. Hence, it is desirable in many experimental contexts to isolate cells that have undergone excision to assess the consequences of gene ablation. To generate alternative reporter mice, an enhanced green fluorescent protein (EGFP) gene was targeted to the retroviral-trapped ROSA26 locus. Upon Cre-mediated excision of "Stop" sequences, EGFP was expressed ubiquitously during embryogenesis and in adult tissues (including T cells, B cells, and myeloid cells). Using this new reporter strain, separation of excised from nonexcised cells in vitro was achieved in thymocytes in a noninvasive manner based on activated EGFP expression. This new EGFP reporter strain should facilitate a variety of conditional gene-targeting experiments, including the functional studies of hematopoietic cells in lineage-specific knockout mice.
Assuntos
Genes Reporter , Integrases/metabolismo , Proteínas Luminescentes/biossíntese , Proteínas/genética , Proteínas Virais , Animais , Células Sanguíneas , Embrião de Mamíferos , Regulação da Expressão Gênica , Marcação de Genes/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde , Indicadores e Reagentes , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , RNA não Traduzido , Timo/citologiaRESUMO
E2Fs are important regulators of proliferation, differentiation, and apoptosis. Here we characterize the phenotype of mice deficient in E2F2. We show that E2F2 is required for immunologic self-tolerance. E2F2(-/-) mice develop late-onset autoimmune features, characterized by widespread inflammatory infiltrates, glomerular immunocomplex deposition, and anti-nuclear antibodies. E2F2-deficient T lymphocytes exhibit enhanced TCR-stimulated proliferation and a lower activation threshold, leading to the accumulation of a population of autoreactive effector/memory T lymphocytes, which appear to be responsible for causing autoimmunity in E2F2-deficient mice. Finally, we provide support for a model to explain E2F2's unexpected role as a suppressor of T lymphocyte proliferation. Rather than functioning as a transcriptional activator, E2F2 appears to function as a transcriptional repressor of genes required for normal S phase entry, particularly E2F1.
Assuntos
Doenças Autoimunes/genética , Autoimunidade/imunologia , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , Regulação da Expressão Gênica/imunologia , Proteínas Repressoras/fisiologia , Tolerância a Antígenos Próprios/imunologia , Linfócitos T/citologia , Fatores de Transcrição/fisiologia , Animais , Apoptose , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Autoimunidade/genética , Divisão Celular , Quimera , Deleção Clonal , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F2 , Glomerulonefrite Membranoproliferativa/genética , Glomerulonefrite Membranoproliferativa/imunologia , Antígeno H-Y/genética , Antígeno H-Y/imunologia , Humanos , Memória Imunológica , Inflamação , Células Jurkat , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/imunologia , Proteínas Repressoras/genética , Fase S/genética , Tolerância a Antígenos Próprios/genética , Esplenomegalia/genética , Esplenomegalia/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Timo/imunologia , Timo/patologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , TransfecçãoRESUMO
Malignant rhabdoid tumor (MRT) is an aggressive, highly lethal cancer of young children. Tumors occur in various locations, including kidney, brain, and soft tissues. Despite intensive therapy, 80% of affected children die, often within 1 year of diagnosis. The majority of MRT samples and cell lines have sustained biallelic inactivating mutations of the hSNF5 (integrase interactor 1) gene, suggesting that hSNF5 may act as a tumor suppressor. We sought to examine the role of Snf5 in development and cancer in a murine model. Here we report that Snf5 is widely expressed during embryogenesis with focal areas of high-level expression in the mandibular portion of the first branchial arch and central nervous system. Homozygous knockout of Snf5 results in embryonic lethality by embryonic day 7, whereas heterozygous mice are born at the expected frequency and appear normal. However, beginning as early as 5 weeks of age, heterozygous mice develop tumors consistent with MRT. The majority of tumors arise in soft tissues derived from the first branchial arch. Our findings constitute persuasive genetic evidence that Snf5, a core member of the Swi/Snf chromatin-remodeling complex, functions as a tumor suppressor gene, and, moreover, Snf5 heterozygotes provide a murine model of this lethal pediatric cancer.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Predisposição Genética para Doença , Tumor Rabdoide/genética , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Cromossômicas não Histona , Clonagem Molecular , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteína SMARCB1 , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Megakaryocytes lacking transcription factor GATA-1 fail to complete maturation in vivo and hyperproliferate. To define how GATA-1 regulates megakaryocyte cell growth we searched for mRNA transcripts expressed in primary wild-type, but not GATA-1(-), megakaryocytes. One differentially expressed transcript encodes inositol polyphosphate 4-phosphatase type I (4-Ptase I). This enzyme hydrolyses phosphatidylinositol 3,4-bisphosphate and also has lesser activity against soluble analogues of this lipid, inositol 3, 4-bisphosphate and inositol 1,3,4-triphosphate. Reintroduction of 4-Ptase I into both primary GATA-1(-) and wild-type megakaryocytes significantly retards cell growth, suggesting that absence of 4-Ptase I may contribute to the hyperproliferative phenotype of GATA-1(-) megakaryocytes. Overexpression of 4-Ptase I also markedly reduces growth of NIH 3T3 fibroblasts. Taken together, these data indicate that 4-Ptase I is a regulator of cell proliferation.
Assuntos
Divisão Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Fatores de Transcrição/fisiologia , Células 3T3 , Animais , Sequência de Bases , Primers do DNA , Fatores de Ligação de DNA Eritroide Específicos , Fibroblastos/citologia , Fator de Transcrição GATA1 , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Dados de Sequência MolecularRESUMO
The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1-estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1-mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein-protein interactions. These findings contribute to understanding how protein-protein interactions participate in hematopoietic differentiation and leukemogenesis. (Blood. 2000;96:2641-2648)
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , DNA/metabolismo , Células Precursoras Eritroides/metabolismo , Eritropoese/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Transporte Biológico , Diferenciação Celular/fisiologia , Linhagem da Célula , Núcleo Celular/metabolismo , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Estradiol/farmacologia , Fator de Transcrição GATA1 , Regulação Leucêmica da Expressão Gênica/fisiologia , Genes Sintéticos , Humanos , Células K562/metabolismo , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/análise , RNA Neoplásico/análise , Receptores de Estrogênio/genética , Proteínas Recombinantes de Fusão/fisiologia , Transativadores/química , Transativadores/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
The receptor tyrosine kinase Flk-1 is essential for embryonic blood vessel development and for tumor angiogenesis. To identify upstream transcriptional regulators of Flk-1, the gene regulatory elements that mediate endothelium-specific expression in mouse embryos were characterized. By mutational analysis, binding sites for SCL/Tal-1, GATA, and Ets transcription factors located in the Flk-1 enhancer were identified as critical elements for the endothelium-specific Flk-1 gene expression in transgenic mice. c-Ets1, a transcription factor that is coexpressed with Flk-1 during embryonic development and tumor angiogenesis, activated the Flk-1 promoter via 2 binding sites. One of these sites was required for Flk-1 promoter function in the embryonic vasculature. These results provide the first evidence that SCL/Tal-1, GATA, and Ets transcription factors act upstream of Flk-1 in a combinatorial fashion to determine embryonic blood vessel formation and are key regulators not only of the hematopoietic program, but also of vascular development.