RESUMEN
Dynamic control of interferon-beta (IFN-beta) gene expression requires the regulated assembly and disassembly of the enhanceosome, a higher-order nucleoprotein complex formed in response to virus infection. The enhanceosome activates transcription by recruiting the histone acetyltransferase proteins CREB binding protein (CBP) and p300/CBP-associated factors (PCAF)/GCN5, which, in addition to modifying histones, acetylate HMGI(Y), the architectural component required for enhanceosome assembly. We show that the accurate execution of the IFN-beta transcriptional switch depends on the ordered acetylation of the high-mobility group I protein HMGI(Y) by PCAF/GCN5 and CBP, which acetylate HMGI(Y) at distinct lysine residues on endogenous promoters. Whereas acetylation of HMGI(Y) by CBP at lysine-65 destabilizes the enhanceosome, acetylation of HMGI(Y) by PCAF/GCN5 at lysine-71 potentiates transcription by stabilizing the enhanceosome and preventing acetylation by CBP.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , Interferón beta/genética , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Activación Transcripcional , Acetilación , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Proteína de Unión a CREB , Proteínas de Ciclo Celular , Proteína HMGA1a , Células HeLa , Proteínas del Grupo de Alta Movilidad/química , Histona Acetiltransferasas , Histonas/metabolismo , Humanos , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Recombinantes/metabolismo , Respirovirus/fisiología , Transactivadores/metabolismo , Factores de Transcripción/química , Transfección , Factores de Transcripción p300-CBPRESUMEN
Gene-specific transcriptional regulation in higher eukaryotes is mediated by complex cis-acting control elements that specify the location, timing and magnitude of the response. During the past five years, an argument has been made that in several cases specificity in gene transcription is achieved by the assembly of higher-order three-dimensional transcription factor/enhancer DNA complexes, termed enhanceosomes. The inherent co-operativity in enhanceosome assembly and the embedded synergy in transcription ensure that a specific gene would be selected for activation only if all the enhanceosome components are present in the same nucleus. Enhanceosomes activate transcription by recruiting chromatin-modifying activities and basal transcription factors to the nearby promoters.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Interferón beta/genética , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Humanos , Factores de Transcripción/genéticaRESUMEN
We show that the IRF-2 oncoprotein represses virus-induced IFN-beta gene transcription via a novel mechanism. Virus infection induces recruitment of IRF-2 to some of the endogenous IFN-beta enhancers as part of the enhanceosome. Enhanceosomes bearing IRF-2 cannot activate transcription, due to the presence of a domain in IRF-2 that prevents enhanceosome-dependent recruitment of the CBP-Pol II holoenzyme complex. As a consequence, IRF-2 incorporation into enhanceosomes restricts the number of IFN-beta promoters directing transcription. Remarkably, deletion of the IRF-2 gene increases IFN-beta expression by expanding the number of cells capable of inducing IFN-beta gene transcription in response to virus infection.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Interferón beta/genética , Factores de Transcripción , Transcripción Genética , Virus/inmunología , Animales , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Cromatina/fisiología , Proteínas de Unión al ADN/genética , Genes Reporteros , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Factor 2 Regulador del Interferón , Proteínas Luminiscentes/genética , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , ARN sin Sentido/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , TransfecciónRESUMEN
The human tumor necrosis factor alpha (TNF-alpha) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-alpha enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-alpha gene transcription and selectively associates with the TNF-alpha promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-alpha transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.
Asunto(s)
Proteínas Nucleares , Regiones Promotoras Genéticas/genética , Activación Transcripcional , Factor de Necrosis Tumoral alfa/genética , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos/genética , Humanos , Factores de Transcripción NFATC , Factor de Transcripción Sp1/genética , Factores de Transcripción/genéticaRESUMEN
Transcriptional activation of the virus inducible enhancer of the human interferon-beta (IFN-beta) gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappaB, ATF-2/c-Jun, IRFs and the architectural protein of the mammalian high mobility group I(Y) [HMG I(Y)]. Here, we demonstrate that the first step in enhanceosome assembly, i.e. HMG I(Y)-dependent recruitment of NF-kappaB and ATF-2/c-Jun to the enhancer, is facilitated by discrete regions of HMG I and is mediated by allosteric changes induced in the DNA by HMG I(Y) and not by protein-protein interactions between HMG I(Y) and these proteins. However, we show that completion of the enhanceosome assembly process requires protein-protein interactions between HMG I(Y) and the activators. Finally, we demonstrate that once assembled, the IFN-beta enhanceosome is an unusually stable nucleoprotein structure that can activate transcription at high levels by promoting multiple rounds of reinitiation of transcription.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Interferón beta/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Factor de Transcripción Activador 2 , Regulación Alostérica , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN/genética , ADN/metabolismo , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Dimerización , Proteína HMGA1a , Células HeLa , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Factor 1 Regulador del Interferón , Modelos Genéticos , Mutación , FN-kappa B/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Repetitivas de Aminoácido , Factores de Transcripción/química , Factores de Transcripción/genéticaAsunto(s)
Elementos de Facilitación Genéticos , Interferón beta/genética , Proteínas de Saccharomyces cerevisiae , Acetiltransferasas/metabolismo , Animales , ADN Polimerasa II/metabolismo , Regulación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/metabolismo , Histona Acetiltransferasas , Humanos , Sustancias Macromoleculares , Modelos Biológicos , Transducción de Señal , Factor de Transcripción TFIID , Factores de Transcripción TFII/metabolismoRESUMEN
The transcriptional coactivators CBP and P/CAF are required for activation of transcription from the IFN beta enhanceosome. We show that CBP and P/CAF acetylate HMG I(Y), the essential architectural component required for enhanceosome assembly, at distinct lysine residues, causing distinct effects on transcription. Thus, in the context of the enhanceosome, acetylation of HMG I by CBP, but not by P/CAF, leads to enhanceosome destabilization and disassembly. We demonstrate that acetylation of HMG I(Y) by CBP is essential for turning off IFN beta gene expression. Finally, we show that the acetyltransferase activities of CBP and P/CAF modulate both the strength of the transcriptional response and the kinetics of virus-dependent activation of the IFN beta gene.
Asunto(s)
Proteínas Cromosómicas no Histona , Elementos de Facilitación Genéticos/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Interferón beta/genética , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Sitios de Unión/fisiología , Células COS , Proteína de Unión a CREB , Factor 1 de Ensamblaje de la Cromatina , Proteínas de Unión al ADN/metabolismo , Drosophila , Regulación Neoplásica de la Expresión Génica , Proteína HMGA1a , Proteínas del Grupo de Alta Movilidad/química , Mutagénesis , FN-kappa B/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Estructura Terciaria de Proteína , Factores de Transcripción/químicaRESUMEN
The class II transactivator (CIITA) is a key regulatory factor that controls expression of the major histocompatibility complex (MHC) class II genes that are essential components for antigen presentation and thus regulation of the immune response. We show here that the adenovirus E1A protein interferes with the action of CIITA and inhibits both B-cell-specific and gamma interferon (IFN-gamma)-induced expression of MHC class II promoters. Transfection studies provide evidence for the functional role of the CREB-binding protein (CBP) in IFN-gamma and CIITA-mediated MHC class II promoter activation. We demonstrate that the N-terminally located transcription activation domain of CIITA physically interacts with both the N-terminal and the E1A-binding (C/H3) regions of CBP. These results suggest the involvement of a multisubunit complex, which contains the gene-specific coactivator CIITA and the versatile coactivator CBP, in MHC class II gene regulation, which may be responsible for both high-level expression and modulation by different signaling pathways.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Genes MHC Clase II/genética , Proteínas Nucleares , Transactivadores/metabolismo , Proteínas E1A de Adenovirus/farmacología , Linfocitos B/fisiología , Línea Celular , Regulación de la Expresión Génica/genética , Interferón gamma/farmacología , Regiones Promotoras Genéticas/genética , Unión Proteica/fisiología , Transducción de Señal/fisiología , Activación Transcripcional/fisiologíaRESUMEN
Transcriptional activation of the IFN beta gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-kappa B, IRF1, ATF2/c-Jun, and the architectural protein HMG I(Y). The level of transcription generated by all of these activators is greater than the sum of the levels generated by individual factors, a phenomenon designated transcriptional synergy. We demonstrate that this synergy, in the context of the enhanceosome, requires a new protein-protein interaction domain in the p65 subunit of NF-kappa B. Transcriptional synergy requires recruitment of the CBP/p300 coactivator to the enhanceosome, via a new activating surface assembled from the novel p65 domain and the activation domains of all of the activators. Deletion, substitution, or rearrangement of any one of the activation domains in the context of the enhanceosome decreases both recruitment of CBP and transcriptional synergy.
Asunto(s)
Elementos de Facilitación Genéticos/fisiología , Interferón beta/genética , FN-kappa B/metabolismo , Transactivadores/metabolismo , Activación Transcripcional/fisiología , Factor de Transcripción Activador 2 , Animales , Sitios de Unión/fisiología , Células COS , Proteína de Unión a CREB , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Histona Acetiltransferasas , Factor 1 Regulador del Interferón , Leucina Zippers/genética , FN-kappa B/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Coactivador 3 de Receptor Nuclear , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Transactivadores/química , Transactivadores/genética , Factor de Transcripción ReIA , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
The biological activity of the transcription factor NF-kappaB is controlled mainly by the IkappaB alpha and IkappaB beta proteins, which restrict NF-kappaB to the cytoplasm and inhibit its DNA binding activity. Here, we carried out experiments to determine and compare the mechanisms by which IkappaB alpha and IkappaB beta inhibit NF-kappaB-dependent transcriptional activation. First, we found that in vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Second, we showed that both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. Third, the inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Animales , Células COS , Quinasa de la Caseína II , ADN/metabolismo , Proteína HMGA1a , Células HeLa , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Ratones , Inhibidor NF-kappaB alfa , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Transcripción GenéticaRESUMEN
The mammalian high-mobility-group protein I(Y) [HMG I(Y)], while not a typical transcriptional activator, is required for the expression of many eukaryotic genes. HMG I(Y) appears to recruit and stabilize complexes of transcriptional activators through protein-DNA and protein-protein interactions. The protein binds to the minor groove of DNA via three short basic repeats, preferring tracts of adenines and thymines arranged on the same face of the DNA helix. However, the mode by which these three basic repeats function together to recognize HMG I(Y) binding sites has remained unclear. Here, using deletion mutants of HMG I(Y), DNase I footprinting, methylation interference, and in vivo transcriptional assays, we have characterized the binding of HMG I(Y) to the model beta-interferon enhancer. We show that two molecules of HMG I(Y) bind to the enhancer in a highly cooperative fashion, each molecule using a distinct pair of basic repeats to recognize the tandem AT-rich regions of the binding sites. We have also characterized the function of each basic repeat, showing that only the central repeat accounts for specific DNA binding and that the presence of a second repeat bound to an adjacent AT-rich region results in intramolecular cooperativity in binding. Surprisingly, the carboxyl-terminal acidic tail of HMG I(Y) is also important for specific binding in the context of the full-length protein. Our results present a detailed examination of HMG I(Y) binding in an important biological context, which can be extended not only to HMG I(Y) binding in other systems but also to the binding mode of many other proteins containing homologous basic repeats, which have been conserved from bacteria to humans.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas del Grupo de Alta Movilidad/metabolismo , Interferón beta/genética , Regiones Promotoras Genéticas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Huella de ADN , Drosophila melanogaster , Regulación de la Expresión Génica , Proteína HMGA1a , Células HeLa , Humanos , Sustancias Macromoleculares , Modelos Biológicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
An unresolved aspect of current understanding of erythroid cell-specific gene expression relates to how a limited number of transcriptional factors cooperate to direct high-level expression mediated by cis-regulatory elements separated over large distances within globin loci. In this report, we provide evidence that GATA-1, the major erythroid transcription factor, activates transcription in a synergistic fashion with two Krüppel family factors, the ubiquitous protein Sp1 and the erythroid-restricted factor EKLF (erythroid Krüppel-like factor), which recognize GC and/or GT/CACC motifs. Binding sites for both GATA-1 and these Krüppel proteins (especially Sp1) are found in close association in the promoters and enhancers of numerous erythroid cell-expressed genes and appear to cooperate in directing their expression. We have shown that GATA-1 interacts physically with Sp1 and EKLF and that interactions are mediated through their respective DNA-binding domains. Moreover, we show that GATA-1 and Sp1 synergize from a distance in constructs designed to mimic the architecture of globin locus control regions and downstream globin promoters. Finally, the formation of GATA-1-SP1 complexes was demonstrated in vivo by the ability of Sp1 to recruit GATA-1 to a promoter in the absence of GATA-binding sites. These experiments provide the first evidence for functionally important protein-protein interactions involved in erythroid cell-specific expression and suggest a mechanism by which DNA loops between locus control regions and globin promoters (or enhancers) might be formed or stabilized.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Línea Celular , ADN/genética , ADN/metabolismo , Drosophila melanogaster , Eritrocitos/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Globinas/genética , Factores de Transcripción de Tipo Kruppel , Ratones , Modelos Genéticos , Regiones Promotoras GenéticasRESUMEN
GATA-1, the founding member of a distinctive family of transcription factors, is expressed predominantly in erythroid cells and participates in the expression of numerous erythroid cell-expressed genes. GATA-binding sites are found in the promoters and enhancers of globin and nonglobin erythroid genes as well as in the alpha- and beta-globin locus control regions. To elucidate how GATA-1 may function in a variety of regulatory contexts, we have examined its protein-protein interactions. Here we show that GATA-1 self-associates in solution and in whole-cell extracts and that the zinc finger region of the molecule is sufficient to mediate this interaction. This physical interaction can influence transcription, as GATA-1 self-association is able to recruit a transcriptionally active but DNA-binding-defective derivative of GATA-1 to promoter-bound GATA-1 and result in superactivation. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, we have localized the minimal domain required for GATA-1 self-association to 40 amino acid residues within the C-terminal zinc finger region. Finally, we have detected physical interaction of GATA-1 with other GATA family members (GATA-2 and GATA-3) also mediated through the zinc finger domain. These findings have broad implications for the involvement of GATA factors in transcriptional control. In particular, the interaction of GATA-1 with itself and with other transcription factors may facilitate its function at diverse promoters in erythroid cells and also serve to bring together, or stabilize, loops between distant regulatory elements, such as the globin locus control regions and downstream globin promoters. We suggest that the zinc finger region of GATA-1, and related proteins, is multifunctional and mediates not only DNA binding but also important protein-protein interactions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Sondas de ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila melanogaster , Eritrocitos/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Haplorrinos , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Eliminación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/genética , Dedos de Zinc/genéticaRESUMEN
GATA-binding proteins constitute a family of transcription factors that recognize a target site conforming to the consensus WGATAR (W = A or T and R = A or G). Here we have used the method of polymerase chain reaction-mediated random site selection to assess in an unbiased manner the DNA-binding specificity of GATA proteins. Contrary to our expectations, we show that GATA proteins bind a variety of motifs that deviate from the previously assigned consensus. Many of the nonconsensus sequences bind protein with high affinity, equivalent to that of conventional GATA motifs. By using the selected sequences as probes in the electrophoretic mobility shift assay, we demonstrate overlapping, but distinct, sequence preferences for GATA family members, specified by their respective DNA-binding domains. Furthermore, we provide additional evidence for interaction of amino and carboxy fingers of GATA-1 in defining its binding site. By performing cotransfection experiments, we also show that transactivation parallels DNA binding. A chimeric protein containing the finger domain of areA and the activation domains of GATA-1 is capable of activating transcription in mammalian cells through GATA motifs. Our findings suggest a mechanism by which GATA proteins might selectively regulate gene expression in cells in which they are coexpressed.