RESUMEN
Transcription factors can function as DNA-binding-specific activators or as coactivators. c-Jun drives gene expression via binding to AP-1 sequences or as a cofactor for PU.1 in macrophages. c-Jun heterodimers bind AP-1 sequences with higher affinity than homodimers, but how c-Jun works as a coactivator is unknown. Here, we provide in vitro and in vivo evidence that c-Jun homodimers are recruited to the interleukin-1beta (IL-1beta) promoter in the absence of direct DNA binding via protein-protein interactions with DNA-anchored PU.1 and CCAAT/enhancer-binding protein beta (C/EBPbeta). Unexpectedly, the interaction interface with PU.1 and C/EBPbeta involves four of the residues within the basic domain of c-Jun that contact DNA, indicating that the capacities of c-Jun to function as a coactivator or as a DNA-bound transcription factor are mutually exclusive. Our observations indicate that the IL-1beta locus is occupied by PU.1 and C/EBPbeta and poised for expression and that c-Jun enhances transcription by facilitating a rate-limiting step, the assembly of the RNA polymerase II preinitiation complex, with minimal effect on the local chromatin status. We propose that the basic domain of other transcription factors may also be redirected from a DNA interaction mode to a protein-protein interaction mode and that this switch represents a novel mechanism regulating gene expression profiles.
Asunto(s)
Proteínas Proto-Oncogénicas c-jun/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células COS , Chlorocebus aethiops , ADN/metabolismo , Dimerización , Expresión Génica , Interleucina-1beta/genética , Activación de Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-jun/química , ARN Polimerasa II/metabolismo , Moldes Genéticos , Regulación hacia Arriba/genéticaRESUMEN
TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser(213) and Ser(257). TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser(213) and Ser(257) functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Represoras/metabolismo , Animales , Diferenciación Celular/fisiología , Línea Celular , Proteínas de Unión al ADN/genética , Células Eritroides/citología , Células Eritroides/metabolismo , Humanos , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-ets , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Serina/metabolismo , Proteína ETS de Variante de Translocación 6RESUMEN
TEL belongs to a member of the ETS family transcription factors that represses transcription of target genes such as FLI-1. Although TEL is essential for establishing hematopoiesis in neonatal bone marrow, its role in erythroid lineage is not understood. To investigate a role for TEL in erythroid differentiation, we introduced TEL into mouse erythroleukemia (MEL) cells. Overexpressing wild-type-TEL in MEL cells enhanced differentiation induced by hexamethylene bisacetamide or dimethylsulfoxide, as judged by the increased levels of erythroid-specific delta-aminolevulinate synthase and beta-globin mRNAs. TEL bound to a corepressor mSin3A through the helix-loop-helix domain. A TEL mutant lacking this domain still bound to the ETS binding site, but lost its transrepressional effect. This mutant completely blocked erythroid differentiation in MEL cells. Moreover, it showed dominant-negative effects over TEL-mediated transcriptional repression and acceleration of erythroid differentiation. Endogenous TEL mRNA was found to increase during the first 3 days in differentiating MEL cells and drastically decrease thereafter. All these data suggest that TEL might play some role in erythroid cell differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/fisiología , Virus de la Leucemia Murina de Friend/aislamiento & purificación , Leucemia Eritroblástica Aguda/patología , Proteínas Represoras/fisiología , 5-Aminolevulinato Sintetasa/genética , Animales , Secuencia de Bases , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Globinas/genética , Células HeLa , Humanos , Leucemia Eritroblástica Aguda/genética , Leucemia Eritroblástica Aguda/virología , Ratones , Proteínas Proto-Oncogénicas c-ets , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína ETS de Variante de Translocación 6Asunto(s)
Leucemia/diagnóstico , Leucocitos/enzimología , Peroxidasa/sangre , Enfermedad Aguda , Reacción de Fase Aguda/diagnóstico , Biomarcadores/sangre , Pruebas Enzimáticas Clínicas/métodos , Pruebas Hematológicas/métodos , Humanos , Leucemia/clasificación , Leucemia Mielógena Crónica BCR-ABL Positiva/clasificación , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Peroxidasa/deficiencia , Valores de Referencia , Manejo de EspecímenesAsunto(s)
Fosfatasa Alcalina/sangre , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Neutrofílica Crónica/diagnóstico , Neutrófilos/enzimología , Enfermedad Aguda , Biomarcadores/sangre , Pruebas Enzimáticas Clínicas/métodos , Pruebas Hematológicas/métodos , Hemoglobinuria Paroxística/diagnóstico , Humanos , Leucemia/diagnóstico , Policitemia Vera/diagnóstico , Mielofibrosis Primaria/diagnóstico , Valores de Referencia , Manejo de EspecímenesRESUMEN
TEL is an ETS family transcription factor that is critical for maintaining hematopoietic stem cells in adult bone marrow. To investigate the roles of TEL in myeloid proliferation and differentiation, we introduced TEL cDNA into mouse myeloid 32Dcl3 cells. Overexpression of TEL repressed interleukin-3-dependent proliferation through blocking cell cycle progression. Also, the presence of TEL triggered apoptosis through the mitochondrial intrinsic pathway on exposure to granulocyte colony-stimulating factor. We found an increase in p53 protein and its DNA binding in the TEL-overexpressing cells. Forced expression of TEL stimulated transcription via the p53-responsive element and increased the expression of cellular target genes for p53 such as cell cycle regulator p21 and apoptosis inducer Puma. Consistently, induction of apoptosis was delayed by pifithrin-alpha treatment and completely blocked by increased expression of Bcl-2 in the TEL-overexpressing cells. These data collectively suggest that TEL exerts a tumor suppressive function through augmenting the p53 pathway and facilitates normal development of myelopoiesis.
Asunto(s)
Apoptosis/fisiología , Proteínas Proto-Oncogénicas c-ets/fisiología , Proteínas Represoras/fisiología , Transducción de Señal , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/efectos de los fármacos , Benzotiazoles , Northern Blotting , Western Blotting , Línea Celular , Proliferación Celular/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Interleucina-3/farmacología , Potenciales de la Membrana/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , Mutación , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Tiazoles/farmacología , Tolueno/análogos & derivados , Tolueno/farmacología , Transcripción Genética/efectos de los fármacos , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína ETS de Variante de Translocación 6RESUMEN
The TEL/AML1 chimeric gene is generated by the t(12;21) translocation in pre-B cell acute lymphoblastic leukemia. TEL/AML1 consists of the helix-loop-helix (HLH) dimerization domain from TEL and almost the entire of AML1, but loses the ETS DNA-binding domain from TEL. Dominant-negative effects of TEL/AML1 over wild-type-AML1 are believed to trigger the development of this type of leukemia. However, it could also be possible that TEL/AML1 affects wild-type-TEL's molecular and tumor suppressive functions through the HLH domain. To test this possibility, we first confirmed that TEL/AML1 associates with wild-type-TEL. TEL/AML1 neither bound to the ETS-binding consensus site nor repressed transcription through it. Regardless, this prevented wild-type-TEL-induced transcriptional repression. Moreover, TEL/AML1 concomitantly inhibited wild-type-TEL-induced growth suppression and wild-type-AML1-mediated transforming activity in NIH3T3 cells. All these data indicate that TEL/AML1 exerts dominant-interfering effects on both AML1 and TEL, and that expression of TEL/AML1 could result in inactivation of TEL's tumor suppressive functions in t(12;21)-carrying leukemia.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Epistasis Genética , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Células COS , Chlorocebus aethiops , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Leucemia de Células B/etiología , Leucemia de Células B/genética , Leucemia de Células B/metabolismo , Ratones , Células 3T3 NIH , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Proteínas Represoras/genética , Factores de Transcripción/genética , Translocación Genética , Proteína ETS de Variante de Translocación 6RESUMEN
A transcriptional repressor TEL belongs to the ETS family transcription factors and acts as a tumor suppressor. We identified five alternatively spliced TEL isoforms generated possibly through exon skipping mechanisms, by using reverse transcriptase-polymerase chain reaction analysis. Among them, we examined molecular and biological functions of a DeltaETS-TEL isoform (TEL-f). This isoform abrogated specific DNA-binding capacity to and trans-repressional ability through the ETS-binding site. Regardless, it showed dominant-negative effects over wild-type-TEL (TEL-a)-mediated transcriptional repression partly through sequestration of TEL-a from nucleus to cytoplasm. Moreover, TEL-f dominantly interfered with TEL-a-mediated erythroid differentiation in MEL cells and growth suppression in NIH3T3 cells. Interestingly, TEL isoforms without the entire (Delta exons 6+7-TEL) or a part (Delta exon 7-TEL) of ETS domain were expressed more frequently in myelodysplastic syndrome-derived leukemia than in myelodysplastic syndrome before transformation. This observation suggests that accumulation of the dominant-negative DeltaETS-TEL molecules could be a related phenomenon to leukemic progression of myelodysplastic syndrome.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Represoras/fisiología , Empalme Alternativo , Animales , Células COS , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Eritrocitos/citología , Eritrocitos/metabolismo , Genes Dominantes , Células HeLa , Humanos , Leucocitos Mononucleares/metabolismo , Ratones , Células 3T3 NIH , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-ets , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/biosíntesis , Proteínas Represoras/química , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/fisiología , Proteína ETS de Variante de Translocación 6RESUMEN
TEL is a nuclear phosphoprotein that belongs to a member of the ETS family transcription factors. TEL acts as a tumor suppressor and is essential for establishing hematopoiesis in neonatal bone marrow. Because TEL possesses multiple putative mitogen-activated protein (MAP) kinase phosphorylation sites, we here investigated functional regulation of TEL via stress signaling pathways. We showed that TEL becomes phosphorylated in vivo by activated p38 but not by JNK1. The constitutive and inducible phosphorylation sites were found to be Ser(22) and Ser(257), respectively. TEL bound to p38 and was directly phosphorylated in vitro by p38. In vivo p38-dependent phosphorylation reduced trans-repressional abilities of TEL through ETS-binding consensus site. These data indicate that TEL's functions are potentially regulated by p38 which is activated by various kinds of stresses. TEL could be a constituent downstream of the specific MAP kinase in the signal transduction system.