RESUMEN
T-acute lymphoblastic leukemia (T-ALL) is characterized by several genetic alterations and poor prognosis in about 20-25% of patients. Notably, about 60% of T-ALL shows increased Notch1 activity, due to activating NOTCH1 mutations or alterations in the FBW7 gene, which confer to the cell a strong growth advantage. Therapeutic targeting of Notch signaling could be clinically relevant, especially for chemotherapy refractory patients. This study investigated the therapeutic efficacy of a novel anti-Notch1 monoclonal antibody by taking advantage of a collection of pediatric T-ALL engrafted systemically in NOD/SCID mice and genetically characterized with respect to NOTCH1/FBW7 mutations. Anti-Notch1 treatment greatly delayed engraftment of T-ALL cells bearing Notch1 mutations, including samples derived from poor responders or relapsed patients. Notably, the therapeutic efficacy of anti-Notch1 therapy was significantly enhanced in combination with dexamethasone. Anti-Notch1 treatment increased T-ALL cell apoptosis, decreased proliferation and caused strong inhibitory effects on Notch-target genes expression along with complex modulations of gene expression profiles involving cell metabolism. Serial transplantation experiments suggested that anti-Notch1 therapy could compromise leukemia-initiating cell functions. These results show therapeutic efficacy of Notch1 blockade for T-ALL, highlight the potential of combination with dexamethasone and identify surrogate biomarkers of the therapeutic response.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/antagonistas & inhibidores , Adolescente , Animales , Anticuerpos Monoclonales/administración & dosificación , Antineoplásicos/administración & dosificación , Niño , Preescolar , Dexametasona/administración & dosificación , Dexametasona/farmacología , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Terapia Molecular Dirigida , Estadificación de Neoplasias , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/metabolismo , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fusion proteins are commonly used as a source of antigen for producing antibodies and in many cases can be useful for biochemical analyses. This unit describes two widely used expression systems for producing large amounts of proteins in E. coli. One system expresses lacZ fusions using the pUR series of vectors and the other expresses trpE fusions using the pATH vectors. The gene of interest is first subcloned into either a pUR or pATH vector in the correct reading frame. The correct transformant is selected, grown, and then induced with either IPTG or IAA. Sonication of cells in the presence of protease inhibitors is used to prepare extracts containing both types of fusion proteins, as well as other types of proteins overexpressed in E. coli. The extracts are checked for the presence of fusion protein on an SDS-polyacrylamide gel.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , beta-Galactosidasa/metabolismo , Proteínas Bacterianas/genética , Escherichia coli/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , beta-Galactosidasa/genéticaRESUMEN
Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-gamma, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-gamma induction has not been established, experiments were conducted to examine Stat4 activation of IFN-gamma and other genes required for cytokine-induced expression of IFN-gamma. We first examined IL-12 signaling components. Basal expression of IL-12Rss1 and IL-12Rss2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-gamma production are defective in the absence of Stat4. IL-18 induces minimal IFN-gamma production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-kappaB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-gamma production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-gamma.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Interferón gamma/fisiología , Proteínas Proto-Oncogénicas , Transducción de Señal/inmunología , Transactivadores/fisiología , Animales , Células Cultivadas , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Activación Enzimática/genética , Activación Enzimática/inmunología , Interferón gamma/biosíntesis , Interferón gamma/metabolismo , Interleucina-1/fisiología , Interleucina-12/fisiología , Interleucina-18/fisiología , Janus Quinasa 2 , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Factor de Transcripción STAT4 , Transducción de Señal/genética , TYK2 Quinasa , Transactivadores/deficiencia , Transactivadores/genética , Factor de Necrosis Tumoral alfa/fisiologíaAsunto(s)
Citocinas/metabolismo , Proteínas de Unión al ADN/fisiología , Transducción de Señal/genética , Transactivadores/fisiología , Animales , División Celular , Proteínas de Unión al ADN/genética , Ratones , Ratones Transgénicos , Transactivadores/deficiencia , Transactivadores/genética , Activación TranscripcionalRESUMEN
The specificity of the various STAT SH2 domains for different tyrosine-containing peptides enables cytokines to activate different signaling pathways and to induce distinct patterns of gene expression. We show that STAT4 has a unique peptide specificity and binds to the peptide sequence pYLPSNID (where pY represents phosphotyrosine). This motif is found at tyrosine residue 800 in the beta2 subunit of the interleukin-12 receptor and is required for DNA binding and transcriptional activity of STAT4. Our data demonstrate that transfection of interleukin-12 receptor beta1 and beta2 subunits is sufficient for STAT4 activation but not for STAT1 or STAT3 activation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Receptores de Interleucina/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Mutagénesis Sitio-Dirigida , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina-12 , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Factor de Transcripción STAT4 , Tirosina/metabolismoRESUMEN
Nuclear factor of activated T cells (NFAT) plays an important role in expression of many cytokine genes including interleukin-2 and interleukin-4. However, its role in interferon-gamma (IFN-gamma) expression is not well understood. In the current studies, two strong NFAT-binding sites in the IFN-gamma promoter were identified by DNase I footprint analysis at positions -280 to -270 and -163 to -155. NFATp bound independently to both sites and was required for the formation of a composite element with AP-1 spanning position -163 to -147. In Jurkat T cells and primary lymphocytes, activation-induced expression of IFN-gamma reporter constructs containing point mutations in either NFAT site or the AP-1 component of the composite site was decreased by approximately 40-65%. Despite elimination of both strong NFAT-binding sites, the IFN-gamma promoter remained completely sensitive to inhibition by cyclosporin. This suggests that other elements in the IFN-gamma promoter, such as the IFN-gamma proximal element, are sufficient for cyclosporin sensitivity of this gene. Ying-Yang 1 (YY1), a potential inhibitor of IFN-gamma expression, binds to sites located between the two NFAT sites. Mutation of the YY1 sites alone had little effect on IFN-gamma promoter activity. However, mutation of both the NFAT and YY1-binding sites abolished activation-induced expression in primary murine splenocytes but not in Jurkat T cells. This suggests that under some conditions, YY1 may play a positive role in activation-induced transcription of IFN-gamma.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interferón gamma/biosíntesis , Proteínas Nucleares , Regiones Promotoras Genéticas , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Ciclosporina/farmacología , Huella de ADN , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Interferón gamma/genética , Células Jurkat , Ratones , Modelos Genéticos , Factores de Transcripción NFATC , Mutación Puntual , Unión Proteica , Proteínas Recombinantes/biosíntesis , Bazo/citología , Factores de Transcripción/genéticaRESUMEN
Nuclear factor of activated T cells (NFAT) is a family of four related transcription factors implicated in cytokine and early response gene expression in activated lymphocytes. Here we report that NFAT4, in contrast to NFATp and NFATc, is preferentially expressed in DP thymocytes. Mice lacking NFAT4 have impaired development of CD4 and CD8 SP thymocytes and peripheral T cells as well as hyperactivation of peripheral T cells. The thymic defect is characterized by increased apoptosis of DP thymocytes. The increased apoptosis and hyperactivation may reflect heightened sensitivity to TcR-mediated signaling. Further, mice lacking NFAT4 have impaired production of Bcl-2 mRNA and protein. NFAT4 thus plays an important role in the successful generation and survival of T cells.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Linfocitos T/citología , Factores de Transcripción/fisiología , Animales , Apoptosis/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Diferenciación Celular/genética , Supervivencia Celular/genética , Proteínas de Unión al ADN/biosíntesis , Expresión Génica/efectos de los fármacos , Marcación de Gen/métodos , Genes bcl-2/genética , Antígenos HLA-DP/análisis , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Recuento de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Factores de Transcripción NFATC , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/fisiología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/fisiología , Bazo/citología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/fisiología , Linfocitos T/inmunología , Linfocitos T/fisiología , Timo/citología , Timo/inmunología , Factores de Transcripción/biosíntesisRESUMEN
The phenotypes of various STAT knockout mice reveal an unexpected specificity in the biological roles of these molecules. The mechanisms involved in generating selectivity and modulating STAT activity have been the focus of intense studies. This work has led to the discovery of novel families of proteins that regulate Jak-STAT signaling. Recently, the structures of a STAT dimer/DNA complex and of the amino-terminal domain have been solved, providing new insights into the function of these versatile proteins.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Tirosina Quinasas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/química , Dimerización , Ratones , Ratones Noqueados , Modelos Biológicos , Familia de Multigenes , Transducción de Señal , Transactivadores/químicaRESUMEN
Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5-17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Válvulas Cardíacas/embriología , Proteínas Nucleares , Factores de Transcripción/fisiología , Animales , Calcineurina/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Endotelio Vascular/embriología , Endotelio Vascular/fisiología , Marcación de Gen , Válvulas Cardíacas/anatomía & histología , Ratones , Morfogénesis , Factores de Transcripción NFATC , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The NF-AT family of transcription factors activates early immune response genes such as cytokines. In the adult, NF-ATc is expressed exclusively in the lymphoid system and is induced upon lymphocyte activation. NF-ATc null mutant mice die in utero of cardiac failure, precluding analysis of the role of NF-ATc in lymphocyte activation. By using RAG-2-deficient blastocyst complementation, we now demonstrate that young, highly chimeric mice lacking NF-ATc have impaired repopulation of both thymus and peripheral lymphoid organs. Furthermore, NF-ATc deficiency impaired T lymphocyte activation and secretion of IL-4. B lymphocytes displayed reduced proliferation and a selective loss of IL-4-driven immunoglobulin isotypes both in vivo and in vitro. Our data demonstrate that NF-ATc is essential for the optimal generation and function of mature T and B lineage cells, with an especially profound effect on IL-4-driven responses.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Interleucina-4/fisiología , Tejido Linfoide/fisiología , Proteínas Nucleares , Factores de Transcripción/fisiología , Alelos , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Inmunoglobulina E/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Interleucina-4/biosíntesis , Hígado/embriología , Hígado/fisiología , Activación de Linfocitos/inmunología , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Ratones Endogámicos BALB C , Factores de Transcripción NFATC , Fenotipo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaAsunto(s)
Apoptosis , Proteínas de Unión al ADN/fisiología , Transducción de Señal , Transactivadores/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Caspasa 1 , Línea Celular , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón , Interferón gamma/farmacología , Modelos Genéticos , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Factor de Transcripción STAT1 , Transactivadores/química , Transactivadores/genética , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
STAT proteins (signal transducers and activators of transcription) activate distinct target genes despite having similar DNA binding preferences. The transcriptional specificity of STAT proteins was investigated on natural STAT binding sites near the interferon-gamma gene. These sites are arranged in multiple copies and required cooperative interactions for STAT binding. The conserved amino-terminal domain of STAT proteins was required for cooperative DNA binding, although this domain was not essential for dimerization or binding to a single site. Cooperative binding interactions enabled the STAT proteins to recognize variations of the consensus site. These sites can be specific for the different STAT proteins and may function to direct selective transcriptional activation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Transactivadores/metabolismo , Activación Transcripcional , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/inmunología , Interferón gamma/genética , Intrones , Datos de Secuencia Molecular , Mutación , Oligodesoxirribonucleótidos/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción STAT1 , Factor de Transcripción STAT4 , Eliminación de Secuencia , Transducción de Señal , Transactivadores/química , Transactivadores/inmunologíaRESUMEN
Interactions between cytokine and receptor lead to the activation of multiple signalling molecules, including the family of signal transducer and activator of transcription (STAT) proteins. STAT4 is one member of this family, and is activated only in response to the cytokine interleukin (IL)-12 (refs 5, 6). By gene targeting, we have generated mice deficient in STAT4 to determine whether the function of this transcription factor is redundant with other signalling molecules activated by IL-12. IL-12-induced increases in the production of interferon (IFN)-gamma cellular proliferation and natural killer (NK) cell cytotoxicity are abrogated in lymphocytes from STAT4-deficient mice. The development of Th1 cells in response to either IL-12 of Listeria monocytogenes is also impaired in the absence of Stat4. Furthermore, Stat4-deficient lymphocytes demonstrate a propensity towards the development of Th2 cells. These results demonstrate that Stat4 is essential for mediating responses to IL-12 in lymphocytes, and regulating the differentiation of both Th1 and Th2 cells.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Interleucina-12/fisiología , Células Th2/fisiología , Transactivadores/fisiología , Animales , Diferenciación Celular/fisiología , Línea Celular , Células Cultivadas , Citotoxicidad Inmunológica/inmunología , Proteínas de Unión al ADN/genética , Marcación de Gen , Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Listeria monocytogenes/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Mutación , Factor de Transcripción STAT4 , Transducción de Señal , Bazo/citología , Células TH1/fisiología , Células Th2/inmunología , Transactivadores/genéticaRESUMEN
Interleukin-4 (IL-4) and interleukin-12 (IL-12) control the differentiation of T-helper cells. Here we summarize studies which investigate the mechanism by which these cytokines selectively reprogramme gene expression in T-lymphocytes. Cytokine stimulation leads to the phosphorylation of specific tyrosine residues within the intracellular domain of the corresponding cytokine receptor. These phosphotyrosines serve as docking sites for latent, cytoplasmic transcription factors known as signal transducers and activators of transcription (Stat) proteins. Receptor/Stat interaction is mediated by the src homology 2 (SH2) domain of the corresponding Stat protein. Although Stat binding to the intracellular domain of the cytokine receptor strongly depends on the phosphotyrosine residue, the recruitment of a specific Stat protein is dictated by amino acid residues C-terminal to the phosphotyrosine. Specific docking sites within individual cytokine receptors have been identified for almost all Stat proteins. The direct coupling between cytokine receptor and transcription factor helps to explain how different cytokines elicit distinct patterns of gene expression.
Asunto(s)
Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Citocinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-12/farmacología , Interleucina-4/farmacología , Fosfotirosina/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal , Linfocitos T Colaboradores-Inductores/inmunología , Activación TranscripcionalRESUMEN
NF-ATp is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In this study, we show that mice with a null mutation in the NF-ATp gene have splenomegaly with hyperproliferation of both B and T cells. They also display early defects in the transcription of multiple genes encoding cytokines and cell surface receptors, including CD40L and FasL. A striking defect in early IL-4 production was observed after ligation of the TCR complex by treatment with anti-CD3 in vivo. The transcription of other cytokines including IL-13, GM-CSF, and TNF alpha was also affected, though to a lesser degree. Interestingly, the cytokines IL-2 and IFN gamma were minimally affected. Despite this early defect in IL-4 transcription, Th2 development was actually enhanced at later timepoints as evidenced by increased IL-4 production and IgE levels in situations that favor the formation of Th2 cells both in vitro and in vivo. These data suggest that NF-ATp may be involved in cell growth, and that it is important for the balanced transcription of the IL-4 gene during the course of an immune response.
Asunto(s)
Interleucina-4/biosíntesis , Proteínas Nucleares , Factores de Transcripción/deficiencia , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Linfocitos B/patología , Secuencia de Bases , Ligando de CD40 , División Celular , Cartilla de ADN/genética , Proteínas de Unión al ADN , Proteína Ligando Fas , Expresión Génica , Marcación de Gen , Interleucina-4/genética , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Esplenomegalia/genética , Esplenomegalia/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patología , Células Th2/inmunología , Células Th2/metabolismo , Células Th2/patologíaRESUMEN
The transcription factors NFAT and AP-1 have been shown to be essential for inducible interleukin-2 (IL-2) expression in activated T cells. NFAT has been previously reported to bind to two sites in the IL-2 promoter: in association with AP-1 at the distal antigen response element at -280 and at -135. On the basis of DNase I footprinting with recombinant NFAT and AP-1 proteins, gel shift assays, and transfection experiments, we have identified three additional NFAT sites in the IL-2 promoter. Strikingly, all five NFAT sites are essential for the full induction of promoter activity in response to T-cell receptor stimulation. Four of the five NFAT sites are part of composite elements able to bind AP-1 in association with NFAT. These sites display a diverse range of cooperativity and interdependency on NFAT and AP-1 proteins for binding. One of the NFAT sites directly overlaps the CD28-responsive element. We present evidence that CD28 inducibility is conferred by the AP-1 component in NFAT-AP-1 composite elements. These findings provide further insight into the mechanisms involved in the regulation of the IL-2 promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Interleucina-2/genética , Proteínas Nucleares , Regiones Promotoras Genéticas , Receptores de Antígenos de Linfocitos T/fisiología , Células TH1/fisiología , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Antígenos CD28/fisiología , Línea Celular , Núcleo Celular/metabolismo , Huella de ADN , Activación de Linfocitos , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Oligodesoxirribonucleótidos/química , Transducción de SeñalRESUMEN
The activation of cytokine genes in response to antigenic stimulation of T cells is mediated by NF-AT proteins. Previous studies have identified two NF-AT proteins, NF-ATp and NF-ATc, that are homologous within a 290 aa domain distantly related to the Rel domain. We have isolated two additional members of this gene family, NF-AT3 and NF-AT4, which encode proteins 65% identical to the other NF-AT proteins within the Rel domain. The four NF-AT genes are transcribed in different sets of tissues that included many sites of expression outside the immune system. The Rel homology domain is sufficient for DNA recognition and cooperative binding interactions with AP-1. Although other members of the Rel family bind DNA as dimers, NF-AT proteins are monomers in solution or bound to DNA. Transfection assays indicate that each of the four NF-AT proteins can activate the IL-2 promoter in T cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas Nucleares , Factores de Transcripción/genética , Secuencia de Aminoácidos , Cartilla de ADN/química , Proteínas de Unión al ADN/química , Expresión Génica , Genes , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Factores de Transcripción NFATC , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The transcription factor NF-AT plays an essential role in the inducible transcription of several cytokine genes during T cell activation. The distal NF-AT site of the murine IL-2 promoter binds both NF-AT and AP-1 proteins, and thus represents a composite regulatory site that integrates Ca(2+)- and PKC-dependent signaling pathways in T cell activation. However, the individual contributions of the NF-AT and AP-1 components to promoter activity via this composite site have not been resolved, owing to the absence of a clearly defined AP-1 binding site, which, when mutated abolishes AP-1 binding. We describe here an apparently analogous NF-AT/AP-1 composite site in the murine IL-4 promoter, which can be mutated to selectively block the recruitment of each component. We show that the cooperative and coordinate involvement of both NF-AT and AP-1 is necessary for full activity of the NF-AT/AP-1 composite site, and, ultimately, the entire IL-4 promoter.
Asunto(s)
Proteínas de Unión al ADN/genética , Interleucina-4/genética , Proteínas Nucleares , Regiones Promotoras Genéticas , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/química , Antígeno 2 Relacionado con Fos , Genes , Interleucina-2/genética , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Oligodesoxirribonucleótidos/química , Proteínas Proto-Oncogénicas c-fos/química , Células Th2 , Factor de Transcripción AP-1/química , Factores de Transcripción/químicaRESUMEN
Enhancement of RNA polymerase II transcription by the viral transactivator VP16 requires the TFIID complex, which consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs). Here we report the molecular cloning, expression, and biochemical characterization of Drosophila TAFII40 (dTAFII40), a subunit of TFIID. In vitro protein-protein interaction assays revealed direct binding between dTAFII40 and a 39 amino acid VP16 activation domain. In addition, affinity chromatography indicated a direct binding of the basal factor TFIIB to immobilized dTAFII40. Since VP16 also binds TFIIB, our results suggest a ternary interaction among an activator, a coactivator, and a basal transcription factor. Antibodies directed against dTAFII40 inhibited activation by GAL4-VP16 without affecting basal transcription. These results, taken together with previous studies of Sp1 and dTAFII110, establish that different activators interact with distinct TAFs in the TFIID complex and that TAFs can contact both activators and basal factors.
Asunto(s)
Proteínas de Drosophila , Drosophila/genética , Factores Asociados con la Proteína de Unión a TATA , Transactivadores/metabolismo , Factor de Transcripción TFIID , Factores de Transcripción/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Proteína de Unión a TATA-Box , Transactivadores/biosíntesis , Transactivadores/genética , Factor de Transcripción TFIIBRESUMEN
The TATA-binding protein TBP is necessary for the transcription of eukaryotic genes. Multi-protein complexes formed by TBP and different TBP-associated factors are involved in the initiation of transcription by polymerases I and II, and probably III as well. During the formation of an active initiation complex, TBP makes specific contacts with other proteins, for example TFIIB and RNA polymerase II (refs 2-4). Here we describe the cloning and characterization of a Drosophila gene product with considerable sequence similarity to TBP and a highly restricted expression pattern in the embryo. This TBP-related factor is a DNA-binding protein but is not likely to be a basal transcription factor. Our results suggest that TBP-related factor is a sequence-specific transcription factor that shares the DNA-binding properties of TBP.