RESUMEN
Intercellular adhesion molecule-1 (ICAM-1) is up-regulated on numerous cell types in response to inflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha) activates the ICAM-1 promoter through a variant nuclear factor-kappaB (NF-kappaB) site at -187/-178 bp upstream of the transcription start site. In this investigation, we provide biochemical and functional evidence that an adjacent CCAAT/enhancer binding protein (C/EBP) site and this variant NF-kappaB site define a composite element for activation of the ICAM-1 promoter in certain cell lines. We detected an endogenous TNF-alpha-inducible DNA-protein complex in nuclear extracts from A549, HeLa, and EVC304 cells that contained both RelA and C/EBPbeta but not other family members. Complex formation required intact C/EBP and NF-kappaB sites and was absolutely dependent on translocation of RelA into the nucleus. Complex formation and cooperative binding were also demonstrated using recombinant proteins, and as above, both binding sites were necessary. Interestingly, the RelA/C/EBPbeta complex was not detected in either Jurkat or Raji cells, indicating cell type specificity. Functional studies with various reporter gene constructs revealed that both binding sites were required for maximal activation of the ICAM-1 promoter in response to TNF-alpha and for synergistic activation by RelA and C/EBPbeta. This is the first detailed analysis of how RelA and C/EBPbeta function to regulate ICAM-1 expression, and this study has important implications for how this gene is activated in specific cell types.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Molécula 1 de Adhesión Intercelular/genética , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Sitios de Unión , Transporte Biológico , Proteínas Potenciadoras de Unión a CCAAT , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Sinergismo Farmacológico , Células HeLa , Humanos , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-rel , Factor de Transcripción ReIA , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
This study examines the biochemical properties of two members of the murine MSX family, MSX-1 and MSX-2, which have been implicated to have partially overlapping functions during embryogenesis. Our analyses show that MSX-1 and MSX-2 share many features in common including their DNA binding and transcriptional properties. In particular, MSX-1 and MSX-2 interact with a common consensus DNA site, and exhibit similar DNA binding site preferences. However, MSX-2 has a higher apparent affinity for DNA, and the distinction between MSX-1 and MSX-2 resides in their differing sequences N-terminal to the homeodomain. With respect to their transcriptional properties, both MSX-1 and MSX-2 function as repressors and share the distinct property that they do so independently of their consensus DNA binding sites. However, MSX-1 is a more potent repressor, and the difference between these proteins also maps to their N-terminal regions. Similarly, the expression patterns of Msx-1 and Msx-2 as examined by whole mount in situ hybridization are related but not identical. Thus, Msx-1 and Msx-2 are co-expressed in the limbs, neural tube, and branchial arches; however, Msx-1 has a broader expression pattern overall and is expressed uniquely in certain embryonic regions. These features suggest that these members of the Msx family are 'equivalent but not equal' and that their proposed redundancy may be achieved via distinct biochemical mechanisms that yield a similar functional outcome.
Asunto(s)
Proteínas de Unión al ADN/análisis , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/análisis , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Sitios de Unión , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factor de Transcripción MSX1 , Ratones , Datos de Secuencia Molecular , Embarazo , Alineación de SecuenciaRESUMEN
In a previous study we showed that the murine homeodomain protein Msx-1 is a potent transcriptional repressor and that this activity is independent of its DNA binding function. The implication of these findings is that repression by Msx-1 is mediated through its association with certain protein factors rather than through its interaction with DNA recognition sites, which prompted investigation of the relevant protein factors. Here we show that Msx-1 interacts directly with the TATA binding protein (TBP) but not with several other general transcription factors. This interaction is mediated by the Msx-1 homeodomain, specifically through residues in the N-terminal arm. These same N-terminal arm residues are required for repression by Msx-1, suggesting a functional relationship between TBP association and transcriptional repression. This is further supported by the observation that addition of excess TBP blocks the repressor action of Msx-1 in in vitro transcription assays. Finally, DNA binding activity is separable from both TBP interaction and repression, which further shows that these other activities of the Msx-1 homeodomain are distinct. Therefore, these findings define a role for the Msx-1 homeodomain, particularly the N-terminal arm residues in protein-protein interaction and transcriptional repression, and implicate a more complex role overall for homeodomains in transcriptional regulation.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , TATA Box , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Factor de Transcripción MSX1 , Datos de Secuencia Molecular , Proteínas Represoras/metabolismo , Relación Estructura-Actividad , Factor de Transcripción TFIID , Transcripción GenéticaRESUMEN
This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.
Asunto(s)
ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción , Transcripción Genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , ADN/química , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/aislamiento & purificación , Factor de Transcripción MSX1 , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Proteínas Recombinantes de Fusión , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Supresión Genética , Moldes Genéticos , TransfecciónRESUMEN
The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series of related specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Osteocalcina/genética , Factores de Transcripción , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Células Cultivadas , Técnicas In Vitro , Factor de Transcripción MSX1 , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos/química , Osteoblastos/citología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Ratas , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for minimalist design and alanine scanning mutagenesis) which contains the consensus residues and in which all nonconsensus residues have been replaced with alanine. The murine homeodomain Msx served as the prototype for the minimAl homeodomain, Ala-Msx. We show that Ala-Msx binds to DNA specifically, albeit with lower affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx(NT), which contains a native rather than an alanine-substituted N-terminal arm, has similar DNA binding affinity as Msx. We show that the native N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an intact N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appropriate tertiary structure of Msx. Therefore, the homeodomain "scaffold" consists of consensus residues, which are sufficient for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Homeodominio , Factores de Transcripción , Alanina/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Secuencia de Consenso , Análisis Mutacional de ADN , Genes Homeobox , Factor de Transcripción MSX1 , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
The hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing murine embryo is colinear with their chromosomal organization. Members of the hox gene family share a conserved DNA binding domain, termed the homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes. In this study, we characterized the DNA binding properties of five representative members of the Hox family: HoxA5, HoxB4, HoxA7, HoxC8, and HoxB1. To facilitate a comparative analysis of their DNA binding properties, we produced the homeodomain regions of these Hox proteins in Escherichia coli and obtained highly purified polypeptides. We showed that these Hox proteins interact in vitro with a common consensus DNA site that contains the motif (C/G)TAATTG. We further showed that the Hox proteins recognize the consensus DNA site in vivo, as determined by their ability to activate transcription through this site in transient transfection assays. Although they interact optimally with the consensus DNA site, the Hox proteins exhibit subtle, but distinct, preferences for DNA sites that contain variations of the nucleotides within the consensus motif. In addition to their modest differences in DNA binding specificities, the Hox proteins also vary in their relative affinities for DNA. Intriguingly, their relative affinities correlate with the positions of their respective genes on the hox cluster. These findings suggest that subtle differences in DNA binding specificity combined with differences in DNA binding affinity constitute features of the "Hox code" that contribute to the selective functions of Hox proteins during murine embryogenesis.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Genes Homeobox , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Secuencia Conservada , ADN/genética , Proteínas de Unión al ADN/química , Drosophila/genética , Embrión de Mamíferos , Embrión no Mamífero , Expresión Génica , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes Homeobox , Proteínas de Homeodominio , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Cinética , Factor de Transcripción MSX1 , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
The expression of c-myb mRNA is differentially regulated in murine B lymphoid tumors such that B cell lymphomas and plasmacytomas contain significantly less c-myb mRNA than pre-B cell lymphomas. To examine the low level of c-myb mRNA expression in the murine B cell lymphoma cell line BCL1, nonessential amino acid starvation was used to block these cells in a G1 state. When BCL1 cells were released from this block, a 7- to 10-fold increase in c-myb mRNA was detected in late G1 and S phase cells relative to that detected in exponentially growing BCL1 cells. This increase was not inhibited by aphidicolin. To determine whether cell cycle regulation of c-myb mRNA expression occurred during exponential growth in both murine pre-B cell lymphoma and B cell lymphoma cell lines, elutriation was used to separate exponentially growing cell populations. An increase in c-myb mRNA expression was seen in late G1 and S phase fractions from B cell lymphoma cell lines. In contrast, c-myb mRNA levels remained constant in elutriation fractions isolated from pre-B cell lymphoma cell lines. Expression of c-myb mRNA was not detected in exponentially growing or in Go serum-stimulated murine fibroblasts. These results indicate that constitutive vs cell cycle regulation of c-myb mRNA expression is related to the state of differentiation in murine B lymphoid tumors and suggest that a switch in regulation may occur during normal B cell development.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia de Células B/genética , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas/genética , Animales , Linfocitos B/fisiología , Ciclo Celular , Separación Celular , Genes , Genes myc , Histonas/genética , Ratones , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-myb , ARN Mensajero/genética , ARN Neoplásico/genética , Transcripción Genética , Células Tumorales CultivadasAsunto(s)
Proto-Oncogenes , Animales , Linfocitos B , Intrones , Linfoma/genética , Ratones , Regiones Promotoras Genéticas , ARN/genética , ARN sin Sentido , Transcripción GenéticaRESUMEN
A mutation within the ompC structural gene of Escherichia coli K-12 which affects expression of outer membrane proteins was characterized. The mutation consisted of a 6-base-pair deletion near the 3' end of the gene which removed the amino acids Val-300 and Gly-301 of the mature coding sequence but otherwise left the reading frame intact. The deletion occurred within a region highly conserved among the porins. No protein product was detected from a single copy of the mutant gene. The mutation caused a trans-dominant decrease in the expression of a wild-type ompC allele. The mutation caused a similar decrease in the amounts of OmpA, OmpF, LamB, and Lc proteins, yet it did not appear to affect the minor outer membrane proteins. It had no significant effect on transcription from either ompF or ompC promoters as measured with lacZ operon fusions. The effects of the mutation on other proteins were completely eliminated when the signal sequence was disrupted so that the mutant protein no longer interacted with the secretion machinery of the cell but instead accumulated as precursor in the cytoplasm. A model is proposed involving the translocation of proteins to the outer membrane and the importance of protein conformation in this process.