RESUMEN
Transcription of major histocompatibility complex (MHC) class II genes depends upon the trimeric complexes RFX and NF-Y binding to the conserved X-Y promoter elements. We produced and purified the RFX subunits from Escherichia coli, reconstituted DNA-binding to the mouse Ea X box and dissected the interactions with NF-Y. RFX and NF-Y do not interact in solution, but make cooperative interactions in EMSA: a minimal NF-Y, composed of the evolutionary conserved domains, is sufficient and the RFXAP N-terminal half is expendable. Altering the X-Y distance abolishes cooperativity, indicating that DNA imposes severe spatial constraints. When tested on a highly positioned nucleosome, RFX binds DNA well and NF-Y does not increase its affinity further. Transfections of NF-Y subunits, but not RFX, in class II negative cells improves basal transcription and coexpression of the two activators has a synergistic effect, while modestly increasing CIITA-mediated activation. These results show that interactions between the two trimers on DNA are key to MHC class II expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes MHC Clase II/genética , Proteínas Nucleares , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular , Secuencia Conservada/genética , ADN/química , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Cinética , Ratones , Mutación , Conformación de Ácido Nucleico , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes/metabolismo , Factores de Transcripción del Factor Regulador X , Elementos de Respuesta/genética , Termodinámica , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Transcripción Genética/genética , Activación Transcripcional/genética , TransfecciónRESUMEN
Histones are among the most conserved proteins in evolution, sharing a histone fold motif. A number of additional histonic proteins exist and are involved in the process of transcriptional regulation. We describe here the identification, cloning and characterization of two small members of the H2A-H2B sub-family (YBL1 and YCL1) related to the NF-YB and NF-YC subunits of the CCAAT-binding activator NF-Y and to the TATA-binding protein (TBP) binding repressor NC2. Unlike the latters, YBL1 and YCL1 have no intrinsic CCAAT or TATA-binding capacity. In nucleosome reconstitution assays, they can form complexes with histones in solution and on DNA and they are part of relatively large complexes, as determined by glycerol gradient experiments. Our data support the idea that YBL1 and YCL1 are divergent with respect to NF-YB and NF-YC for specific functions, but have coevolved the capacity to interact with nucleosomal structures.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histonas/química , Histonas/genética , Nucleosomas/metabolismo , Algoritmos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Factor de Unión a CCAAT/química , Factor de Unión a CCAAT/metabolismo , Línea Celular , Centrifugación por Gradiente de Densidad , Clonación Molecular , ADN/química , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/química , Dimerización , Embrión de Mamíferos/metabolismo , Etiquetas de Secuencia Expresada , Histonas/metabolismo , Humanos , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Peso Molecular , Nucleosomas/química , Nucleosomas/genética , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Filogenia , Unión Proteica , ARN Mensajero/análisis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Alineación de Secuencia , Soluciones , Relación Estructura-Actividad , TATA Box/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Major histocompatibility complex class II (MHC-II) molecules occupy a pivotal position in the adaptive immune system, and correct regulation of their expression is therefore of critical importance for the control of the immune response. Several regulatory factors essential for the transcription of MHC-II genes have been identified by elucidation of the molecular defects responsible for MHC-II deficiency, a hereditary immunodeficiency disease characterized by regulatory defects abrogating MHC-II expression. Three of these factors, RFX5, RFXAP, and RFXANK, combine to form the RFX complex, a regulatory protein that binds to the X box DNA sequence present in all MHC-II promoters. In this study we have undertaken a dissection of the structure and function of RFX5, the largest subunit of the RFX complex. The results define two distinct domains serving two different essential functions. A highly conserved N-terminal region of RFX5 is required for its association with RFXANK and RFXAP, for assembly of the RFX complex in vivo and in vitro, and for binding of this complex to its X box target site in the MHC-II promoter. This N-terminal region is, however, not sufficient for activation of MHC-II expression. This requires an additional domain within the C-terminal region of RFX5. This C-terminal domain mediates cooperative binding between the RFX complex and NF-Y, a transcription factor binding to the Y box sequence of MHC-II promoters. This provides direct evidence that RFX5-mediated cooperative binding between RFX and NF-Y plays an essential role in the transcriptional activation of MHC-II genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Complejo Mayor de Histocompatibilidad/genética , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B , Proteínas Potenciadoras de Unión a CCAAT , Secuencia Conservada , Proteínas de Unión al ADN/genética , Prueba de Complementación Genética , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Factores de Transcripción del Factor Regulador X , Homología de Secuencia de Aminoácido , Activación TranscripcionalRESUMEN
NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Animales , Artemia , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Pollos , ADN/metabolismo , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/metabolismo , Dimerización , Exodesoxirribonucleasas/metabolismo , Datos de Secuencia Molecular , Nucleosomas/metabolismo , Unión Proteica , Soluciones , Xenopus laevisRESUMEN
Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, a tunicate growing in mangrove roots in Caribbean. It has been shown to bind in the minor groove of DNA forming covalent adducts by reaction of the N2 of guanine with the carbinolamine moiety. We investigated ET-743 ability to inhibit the binding of different transcription factors to their consensus sequences by using gel shift assays. We have selected three types of factors: (i) oncogene products such as MYC, c-MYB and Maf; (ii) transcriptional activators regulated during the cell cycle as E2F and SRF; and (iii) general transcription factors such as TATA binding protein (TBP), Sp1 and NF-Y. We observed no inhibition of the binding of Sp1, Maf, MYB and MYC. Inhibition of DNA binding was observed for TBP, E2F, SRF at ET-743 concentrations ranging from 50 to 300 microM. The inhibition of binding of NF-Y occurs at even lower concentrations (i.e. 10-30 microM) when the recombinant subunits of NF-Y are preincubated with the drug, indicating that the inhibition of NF-Y binding does not require previous ET-743 DNA binding. Since NF-Y is a trimer containing two subunits with high resemblance to histones H2B and H2A, we have investigated the effect of ET-743 on nucleosome reconstitution. ET-743 caused a decrease of the nucleosomal band at 100 nM, with the complete disappearance of the band at 3-10 microM. These data suggest that the mode of action of this novel anticancer drug is related to its ability to modify the interaction between some DNA binding proteins and DNA.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Dioxoles/farmacología , Isoquinolinas/farmacología , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Secuencia de Consenso , Proteínas de Unión al ADN/antagonistas & inhibidores , Electroforesis , Leucemia L1210/metabolismo , Ratones , Oligonucleótidos/antagonistas & inhibidores , Oligonucleótidos/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/metabolismo , Proteína de Unión a TATA-Box , Tetrahidroisoquinolinas , Trabectedina , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
We have analysed in vivo the -2.0 kb enhancer of the human urokinase-type plasminogen activator (uPA) gene in HepG2 cells, in which gene expression can be induced by phorbol esters. The results reveal that, within the regulatory region, the enhancer, the silencer and the minimal promoter become hypersensitive to deoxyribonuclease I (DNase I) upon induction of transcription. The hypersensitivity of the enhancer can be reversed after removal of the inducer. In vivo footprinting analysis indicates that all the cis-acting elements of the enhancer, previously identified in vitro, are occupied in vivo upon 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulation of HepG2 cells. Micrococcal nuclease (MNase) cleavage of this region fails to reveal discrete nucleosomal boundaries in vivo in close proximity of the enhancer, either before or after stimulation. Furthermore, this region does not lose its nucleosomal configuration after TPA induction of transcription. An approximately 600 bp long region around the enhancer becomes more, but not fully, accessible to restriction endonucleases upon stimulation. A time-course experiment shows that this accessibility reaches a plateau after a 1 h TPA treatment suggesting the persistent presence of nucleosomes. These results indicate that TPA induces the binding of transcription factors to the uPA enhancer without chromatin remodelling of this region.
Asunto(s)
Carcinógenos/farmacología , Elementos de Facilitación Genéticos , Acetato de Tetradecanoilforbol/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Sitios de Unión , Cromatina/genética , Cromatina/ultraestructura , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Humanos , Nucleasa Microcócica/metabolismo , FN-kappa B/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Mensajero/efectos de los fármacos , Secuencias Reguladoras de Ácidos Nucleicos , Mapeo Restrictivo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcripción Genética , Activador de Plasminógeno de Tipo Uroquinasa/efectos de los fármacosRESUMEN
NF-Y is a sequence-specific evolutionary conserved activator binding to CCAAT boxes with high affinity and specificity. It is a trimer formed by NF-YA and two putative histone-like subunits, NF-YB and NF-YC, showing similarity to histones H2B and H2A, respectively. We investigated the relationships between NF-Y and chromatin using an Artemia franciscana chromatin assembly system with plasmids containing the Major HistoCompatibility complex class II Ea promoter. The NF-Y trimer, but not single subunits, protects the Y box in the presence of reconstituted chromatin, and it can bind the target sequence during and after assembly. Using reconstitution assays with purified chicken histones, we show that NF-Y associates with preformed nucleosomes. Translational analysis of various Ea fragments of identical length in which the CCAAT box is at different positions indicated that the lateral fragment was slightly more prone to NF-Y binding. In competition experiments, NF-Y is able to prevent formation of nucleosomes significantly. These data support the idea that NF-Y is a gene-specific activator with a built-in capacity to interface with chromatin structures.