RESUMEN
Advanced prostate adenocarcinomas enriched in stem-cell features, as well as variant androgen receptor (AR)-negative neuroendocrine (NE)/small-cell prostate cancers are difficult to treat, and account for up to 30% of prostate cancer-related deaths every year. While existing therapies for prostate cancer such as androgen deprivation therapy (ADT), destroy the bulk of the AR-positive cells within the tumor, eradicating this population eventually leads to castration-resistance, owing to the continued survival of AR-/lo stem-like cells. In this study, we identified a critical nexus between p38MAPK signaling, and the transcription factor Forkhead Box Protein C2 (FOXC2) known to promote cancer stem-cells and metastasis. We demonstrate that prostate cancer cells that are insensitive to ADT, as well as high-grade/NE prostate tumors, are characterized by elevated FOXC2, and that targeting FOXC2 using a well-tolerated p38 inhibitor restores epithelial attributes and ADT-sensitivity, and reduces the shedding of circulating tumor cells in vivo with significant shrinkage in the tumor mass. This study thus specifies a tangible mechanism to target the AR-/lo population of prostate cancer cells with stem-cell properties.
Asunto(s)
Resistencia a Antineoplásicos , Epitelio/metabolismo , Epitelio/patología , Factores de Transcripción Forkhead/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Andrógenos/metabolismo , Animales , Benzamidas , Línea Celular Tumoral , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal/genética , Factores de Transcripción Forkhead/genética , Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Ratones , Modelos Biológicos , Clasificación del Tumor , Nitrilos , Fenotipo , Feniltiohidantoína/análogos & derivados , Feniltiohidantoína/farmacología , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Androgénicos/metabolismo , Recurrencia , Ensayos Antitumor por Modelo de Xenoinjerto , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
To understand the role of CCAAT-binding factor (CBF) in transcription in the context of chromatin-assembled DNA, we used regularly spaced nucleosomal DNA using topoisomerase IIalpha (topo IIalpha) and alpha2(1) collagen promoter templates, which were subsequently reconstituted in an in vitro transcription reaction. Binding of CBF to the nucleosomal wild-type topo IIalpha promoter containing four CBF-binding sites disrupted the regular nucleosomal structure not only in the promoter region containing the CBF-binding sites but also in the downstream region over the transcription start site. In contrast, no nucleosome disruption was observed in a mutant topo IIalpha promoter containing mutations in all CBF-binding sites. Interestingly, CBF also activated transcription from nucleosomal wild-type topo IIalpha promoter. In this experiment, a promoter containing one wild-type CBF-binding site was activated very weakly, whereas the promoter containing mutations in all sites was not activated by CBF. A truncated CBF that lacked the glutamine-rich domains did not activate transcription from nucleosomal wild-type topo IIalpha promoter but disrupted the nucleosomal structure about as much as did the binding of full-length CBF. Two nucleosomal mouse alpha2(1) collagen promoter DNAs, one containing a single and the other containing four CBF- binding sites, were also reconstituted in an in vitro transcription reaction. None of the nucleosomal collagen promoters was activated by CBF. However, both of these collagen promoters were activated by CBF when the transcription reaction was performed using naked DNA templates. Binding of CBF to the nucleosomal collagen promoter containing four binding sites disrupted the nucleosomal structure, similarly as observed in the topo IIalpha promoter. Altogether this study indicates that CBF-mediated nucleosomal disruption occurred independently of transcription activation. It also suggests that specific promoter structure may play a role in the CBF-mediated transcription activation of nucleosomal topo IIalpha promoter template.
Asunto(s)
Factor de Unión a CCAAT/fisiología , ADN-Topoisomerasas de Tipo II/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional/fisiología , Células 3T3 , Animales , Antígenos de Neoplasias , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al ADN , RatonesRESUMEN
Scaffold molecules interact with multiple effectors to elicit specific signal transduction pathways. CIITA, a non-DNA-binding regulator of class II major histocompatibility complex (MHC) gene transcription, may serve as a transcriptional scaffold. Regulation of the class II MHC promoter by CIITA requires strict spatial-helical arrangements of the X and Y promoter elements. The X element binds RFX (RFX5/RFXANK-RFXB/RFXAP) and CREB, while Y binds NF-Y/CBF (NF-YA, NF-YB, and NF-YC). CIITA interacts with all three. In vivo analysis using both N-terminal and C-terminal deletion constructs identified critical domains of CIITA that are required for interaction with NF-YB, NF-YC, RFX5, RFXANK/RFXB, and CREB. We propose that binding of NF-Y/CBF, RFX, and CREB by CIITA results in a macromolecular complex which allows transcription factors to interact with the class II MHC promoter in a spatially and helically constrained fashion.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteínas de Unión al ADN/genética , Genes MHC Clase II , Proteínas Nucleares , Regiones Promotoras Genéticas/genética , Transactivadores/genética , Factores de Transcripción/genética , Activación Transcripcional , Animales , Proteínas Potenciadoras de Unión a CCAAT , Células COS , Factores de Transcripción del Factor Regulador XRESUMEN
The heterotrimeric CCAAT-binding factor CBF specifically interacts with the CCAAT motif present in the proximal promoters of numerous mammalian genes. To understand the in vivo function of CBF, a dominant negative mutant of CBF-B subunit that inhibits DNA binding of wild type CBF was stably expressed in mouse fibroblast cells under control of tetracycline-responsive promoter. Expression of the mutant CBF-B but not the wild-type CBF-B resulted in retardation of fibroblast cell growth. The analysis of cell growth using bromodeoxyuridine labeling showed that expression of the mutant CBF-B decreased the number of cells entering into S phase, and also delayed induction of S phase in the quiescent cells after serum stimulation, thus indicating that the inhibition of CBF binding prolonged the progression of S phase in fibroblasts. These results provide direct evidence for the first time that CBF is an important regulator of fibroblast growth. The inhibition of CBF binding reduced expression of various cellular genes including the alpha2(1) collagen, E2F1, and topoisomerase IIalpha genes which promoters contain the CBF-binding site. This result implied that expression of many other genes which promoters contain CBF-binding site was also decreased by the inhibition of CBF binding, and that the decreased expression of multiple cellular genes possibly caused the retardation of fibroblast cell growth.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , División Celular/genética , Proteínas de Unión al ADN/genética , Transcripción Genética/genética , Animales , Bromodesoxiuridina/metabolismo , Proteínas Potenciadoras de Unión a CCAAT , Colágeno/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Fibroblastos , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Mutación , Factores de Transcripción NFI , Proteínas Nucleares , Unión Proteica/genética , ARN Mensajero/metabolismo , Proteína 1 de Unión a Retinoblastoma , Fase S/genética , Tetraciclina/farmacología , Factor de Transcripción DP1 , Factores de Transcripción/metabolismo , Transfección , Proteína 1 de Unión a la Caja YRESUMEN
We report that the heterotrimeric transcription factor NFY or "CAAT-binding factor" binds the -60 region of the human H ferritin promoter, the B site. DNA binding analysis with specific antibodies demonstrates that NFY/B/C subunits tightly bind this site and that NFY/C subunit is masked in vivo by binding with other protein(s). NFY binds the co-activator p300. Specifically, the NFY/B subunit interacts with the central segment of p300 in vivo and in vitro. cAMP substantially increases the formation of the NFY.p300 complex. Taken together these data provide a general model of cAMP induction of non-CRE-containing promoters and suggest that the NFY-B.p300 complex is located at the 5' end of the promoter and the NFY-B.C. TFIIB on the 3' end toward the transcription start site.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Ferritinas/genética , Células HeLa , Humanos , Regiones Promotoras Genéticas , Conformación ProteicaRESUMEN
In this study, we used a photocross-linking method to identify specific contact of CCAAT-binding factor (CBF) subunits in a CBF-DNA complex. The analysis showed that all three subunits in the CBF-DNA complex were cross-linked to DNA and that CBF-B and CBF-C were cross-linked more strongly than CBF-A. None of the CBF-A and CBF-C subunits, which together formed a CBF-A/CBF-C heterodimer, were cross-linked without CBF-B; in contrast, CBF-B was cross-linked in the absence of CBF-A/CBF-C. No subunit of heterotrimeric CBF containing DNA-binding domain mutant of either CBF-B or CBF-C was cross-linked to DNA, and interestingly, cross-linking of CBF-B that occurred without CBF-A/CBF-C was inhibited in presence of mutant CBF-C/CBF-A heterodimer. Altogether, these results indicated that the specific DNA contact surface of each CBF subunit is generated as a result of interaction between CBF-B and CBF-A/CBF-C heterodimer and that the three CBF subunits interact interdependently with DNA to form a CBF-DNA complex. Equilibrium interactions among the three CBF subunits and between CBF subunits and DNA were studied by electrophoretic mobility shift assay. This showed that at equilibrium DNA-binding conditions, the CBF-A/CBF-C heterodimer is very stable, but association between CBF-B and CBF-A/CBF-C is very weak. The nature of the association of CBF-B with CBF-A/CBF-C was also revealed by studying the inhibition of CBF-DNA complex formation by the mutant CBF-B. This study indicated that the association between CBF-B and CBF-A/CBF-C is stabilized upon interaction with DNA, a process likely to favor formation of a high-affinity CBF-DNA complex.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Bromodesoxiuridina , Proteínas Potenciadoras de Unión a CCAAT , Reactivos de Enlaces Cruzados , ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/efectos de la radiación , Dimerización , Modelos Químicos , Mutación , Fotoquímica , Unión Proteica , Conformación Proteica , Propiedades de Superficie , Rayos UltravioletaRESUMEN
The CCAAT motif is one of the common promoter elements present in the proximal promoter of numerous mammalian genes transcribed by RNA polymerase II. CBF (also called NF-Y and CP1) consists of three different subunits and interacts specifically with the CCAAT motif. In each CBF subunit, the segment needed for formation of the CBF-DNA complex is conserved from yeast to human and, interestingly, the conserved segment of two CBF subunits, CBF-A and CBF-C, are homologous to the histone-fold motif of eukaryotic histones and archaebacterial histone-like protein HMf-2. The histone fold motifs of CBF-A and CBF-C interact with each other to form a heterodimer that associates with CBF-B to form a heterotrimeric CBF molecule, which then binds to DNA.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Neoplasias , Regiones Promotoras Genéticas , Factores de Transcripción/fisiología , Transcripción Genética , Secuencia de Aminoácidos , Animales , Factores de Unión al Sitio Principal , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The CCAAT-binding factor CBF is a heterotrimeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes. Previous studies have shown that CBF contains two transcription activation domains: a glutamine-rich, serine-threonine-rich domain present in the CBF-B subunit and a glutamine-rich domain in the CBF-C subunit. In this study, by using a series of deletion mutations of CBF-B and CBF-C in transcription assay in vitro, we further delineated smaller segments in these domains that were sufficient to support transcriptional activation by CBF. To test whether transcription activation by CBF requires co-activators, we examined the interaction between CBF and dTAF110, a component of the Drosophila TFIID complex. Recent work has demonstrated that glutamine-rich domains of the Sp1 transcription factor interact with dTAF110 and that this interaction has an important role in mediating transcription activation. Here we first demonstrate in a direct interaction assay in vitro that CBF binds dTAF110. By using a yeast two-hybrid system we show that both of the transcription activation domains of CBF interact with dTAF110. A deletion analysis suggests that a segment of CBF-B needed for transcription activation is also involved in interaction with dTAF110. In CBF-C the C-terminal portion of the molecule seems to be needed for these two activities. Our results suggest that TAF110 might represent one of the co-activators that mediate transcriptional activation by CBF.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Proteínas de Neoplasias , Factores Asociados con la Proteína de Unión a TATA , Factores de Transcripción TFII/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión/genética , Factores de Unión al Sitio Principal , Proteínas de Unión al ADN/genética , Drosophila , Unión Proteica , Factor de Transcripción TFIID , Factores de Transcripción/genética , Factores de Transcripción TFII/genéticaRESUMEN
CBF is a heterotrimeric protein that binds to DNA containing CCAAT motifs. Here we have analyzed interactions of recombinant CBF with DNA using hydroxyl radical footprinting and methylation interference assays. In the CBF-DNA complex, three separate DNA regions are protected from hydroxyl radical cleavage, one located over and immediately adjacent to the CCAAT motif itself and the other two located on both sides of the CCAAT motif. The methylation interference assay showed, however, that only in the CCAAT motif region methylation of bases was able to interfere with the formation of a CBF-DNA complex, suggesting that CBF makes sequence-specific contacts only in the CCAAT motif region. To further determine the specific DNA sequences necessary for CBF binding, we employed a polymerase chain reaction-mediated random binding site selection method. This analysis showed that CBF binding to DNA requires the CCAAT sequence and other specific sequences immediately flanking both ends of the CCAAT motif. We also showed that the nature of the flanking nucleotide sequences affects the affinity of CBF for DNA. Interestingly, most of the CCAAT motifs present in various higher eukaryotic promoters correspond to the CBF binding sites that were selected, consistent with the hypothesis that these motifs are binding sites for CBF and, hence, that CBF could regulate transcription of numerous eukaryotic genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Huella de ADN , Metilación de ADN , Células Eucariotas , Regulación de la Expresión Génica , Radical Hidroxilo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
We performed in vivo dimethylsulfate footprinting of the 220 bp mouse proximal proalpha1(I) collagen promoter and the 350 bp mouse proximal proalpha2(I) collagen promoter in BALB/3T3 fibroblasts, primary mouse skin fibroblasts, S-194 B cells, NMuLi liver epithelial cells and RAG renal adenocarcinoma cells and in vitro DNase I footprinting of these promoters using nuclear extracts of these different cell types. Whereas proalpha1(I) and proalpha2(I) collagen RNAs were present in BALB/3T3 fibroblasts and primary fibroblasts, these RNAs could not be detected in the three other cell lines. Comparison of in vitro DNase I footprints for each of the two proximal collagen promoters indicated that the patterns of protection were very similar with the different nuclear extracts, suggesting that the DNA binding proteins binding to these promoters were present in all cell types tested. In contrast, in vivo footprints over these proximal promoters were cell-specific, occurring only in fibroblast cells and not in the other three cell types. The in vivo footprints were generally located within the in vitro footprinted regions. Our results suggest that although all cell types tested contained nuclear proteins that can bind to the proximal proalpha1(I) and proalpha2(I) collagen promoters in vitro , it is only in fibroblasts that these proteins bind to their cognate sites in vivo . We discuss possible regulatory mechanisms in type I collagen genes that can contribute to the cell-specific in vivo protein-DNA interactions at the proximal promoters.
Asunto(s)
Colágeno/genética , Proteínas de Unión al ADN/química , Procolágeno/genética , Regiones Promotoras Genéticas , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/fisiología , Huella de ADN , Desoxirribonucleasa I , Fibroblastos/fisiología , Hígado/fisiología , Ratones , Datos de Secuencia Molecular , Ésteres del Ácido Sulfúrico/químicaRESUMEN
The mammalian CCAAT-binding factor CBF (NFY) consists of three subunits, CBF-A, CBF-B, and CBF-C. All three subunits are evolutionarily conserved and are essential for DNA binding of CBF. In this study we report the identification of human and plant homologs of CBF-C. Northern analysis revealed that, like the other two subunits, CBF-C was produced at equal levels in all rat tissues that were examined. We assigned the mouse CBF-C gene (designated Nfyc) to chromosome 4 with tight linkage to Lmyc. Our mouse linkage data suggest that the human NFYC homolog will map to 1p32.
Asunto(s)
Mapeo Cromosómico , Proteínas de Unión al ADN/genética , Secuencia de Aminoácidos , Animales , Proteínas Potenciadoras de Unión a CCAAT , Cromosomas Humanos Par 1 , Humanos , Ratones , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
In vivo transient expression and in vitro transcription experiments indicated that a segment between -170 and -40 bp upstream of the start of transcription of the mouse proalpha2(I) collagen gene was essential to activate transcription. DNase I protection experiments identified three strong footprints in this segment. Experiments with deletion mutants encompassing the sequences defined by these three footprints indicated that each of the three elements contributed to the transcriptional activity of the promoter. All three elements are GC-rich, redundant sites for a complex set of DNA binding proteins that includes SP1, other proteins that bind to an SP1 consensus site and proteins that bind to a Krox consensus site. In addition, the segment corresponding to the most proximal footprint also binds the multimeric CCAAT binding protein CBF. Addition of an excess amount of oligo- nucleotides corresponding to either of the two distal footprints significantly inhibited in vitro transcription of the -350 bp proalpha2(I) collagen promoter. Anti-SP1 antibodies that completely inhibited transcription of the early SV40 promoter had little effect on transcription of the wild-type -350 bp promoter, suggesting that SP1 has only a minor role in activity of this promoter. Our results show that the segment between base pairs -170 and -40 of the proalpha2(I) collagen promoter, which contains redundant binding sites for a complex set of nuclear proteins, is essential in the transcriptional activity of this promoter in fibroblasts.
Asunto(s)
Regulación de la Expresión Génica , Procolágeno/genética , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Secuencia de Consenso , Huella de ADN , Análisis Mutacional de ADN , Proteínas de Unión al ADN/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Eliminación de Secuencia , Factor de Transcripción Sp1/metabolismo , TransfecciónRESUMEN
The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF-C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A-CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.
Asunto(s)
Proteínas de Unión al ADN/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Proteínas Potenciadoras de Unión a CCAAT , Reactivos de Enlaces Cruzados , Secuencias Hélice-Asa-Hélice , Sustancias Macromoleculares , Datos de Secuencia Molecular , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.
Asunto(s)
Cartílago/metabolismo , Colágeno/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Regiones Promotoras Genéticas , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
CBF-A, CBF-B, and CBF-C together form the heterotrimeric mammalian CCAAT-binding factor, CBF, which binds to DNA to form a CBF-DNA complex. Here we examined the transcription activation function of CBF in an in vitro reconstituted system using the three purified recombinant CBF subunits expressed in Escherichia coli. Two of the subunits, CBF-A and CBF-C, were coexpressed and purified as a CBF-A/CBF-C complex. Addition of the three wild-type recombinant CBF subunits to EL4 cell nuclear extracts depleted of CBF stimulated transcription 5-20-fold from proalpha2(1) collagen promoters and 10-fold from the Rous sarcoma virus long terminal repeat. Two CBF deletion mutants, one containing full-length CBF-A and CBF-C and a CBF-B lacking the NH2-terminal residues 1-224, and the other containing full- length CBF-A and CBF-B and a CBF-C lacking the COOH-terminal residues 114-309, also stimulated transcription from these promoters, but the level of activation was reduced to half that obtained with the full-length CBF subunits. In contrast, a CBF deletion mutant protein containing full-length CBF-A and deleted forms of both CBF-B and CBF-C showed very little transcription activation from these promoters. Hence, this study demonstrates that the heterotrimeric CBF protein consists of two transcription activation domains, one present in CBF-B and the other in CBF-C, and that the two domains act additively in the in vitro assay. The activation domains of both CBF-B and CBF-C, which are rich in glutamine and hydrophobic residues, showed amino acid sequence similarities with each other and with the glutamine-rich activation domain of transcription factor Sp1.
Asunto(s)
Virus del Sarcoma Aviar/genética , Proteínas de Unión al ADN/metabolismo , Procolágeno/biosíntesis , Procolágeno/genética , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción , Transcripción Genética , Animales , Proteínas Potenciadoras de Unión a CCAAT , Núcleo Celular/metabolismo , Clonación Molecular , Colágeno/biosíntesis , Escherichia coli , Proteínas Fúngicas/biosíntesis , Células HeLa , Humanos , Luciferasas/biosíntesis , Linfoma , Sustancias Macromoleculares , Ratones , Mutagénesis , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Eliminación de Secuencia , Moldes Genéticos , Activación Transcripcional , Transfección , Células Tumorales CultivadasAsunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Proteínas de Unión al ADN/fisiología , Hígado/metabolismo , Mamíferos/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genéticaRESUMEN
The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consists of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and present in the CBF-DNA complex. In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molecule of each subunit is present in the complex. To begin to understand the interactions between the CBF subunits and DNA, we performed a mutational analysis of the CBF-A subunit. This analysis identified three classes of mutations in the segment of CBF-A that is conserved in Saccharomyces cerevisiae and mammals. Analysis of the first class of mutants revealed that a major part of the conserved segment was essential for interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex. The second class of mutants identified a segment of CBF-A that is necessary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer. The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA. The second and third classes of mutants acted as dominant negative mutants inhibiting the formation of a complex between the wild-type CBF subunits and DNA. The segment of CBF-A necessary for DNA binding showed sequence homology to a segment of CBF-C. Interestingly, these sequences in CBF-A and CBF-C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein HMf-2. We discuss the functional domains of CBF-A and the properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the formation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Mutación , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteínas Potenciadoras de Unión a CCAAT , Secuencia Conservada , Proteínas de Unión al ADN/química , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Conformación Proteica , Ratas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The CCAAT binding factor CBF is a heteromeric transcription factor, which binds to functional CCAAT motifs in many eukaryotic promoters. cDNAs for the A and B subunits of CBF (CBF-A and CBF-B) and for their yeast homologues HAP3 and HAP2 have been previously isolated, but the purified recombinant CBF-A and CBF-B together are unable to bind to CCAAT motifs in DNA. Here we report the isolation of a cDNA coding for rat CBF-C, demonstrate that recombinant CBF-C is required together with CBF-A and CBF-B to form a CBF-DNA complex, and show that CBF-C is present in this protein-DNA complex together with the other two subunits. We further show that CBF-C allows formation of a complex between the purified recombinant yeast HAP2 and HAP3 polypeptides and a CCAAT-containing DNA and is present in this complex, implying the existence of a CBF-C homologue in yeast. We show that CBF-A and CBF-C interact with each other to form a CBF-A-CBF-C complex and that CBF-B does not interact with CBF-A or CBF-C individually but that it associates with the CBF-A-CBF-C complex. Our results indicate that CBF is a unique evolutionarily conserved DNA binding protein.
Asunto(s)
Factor de Unión a CCAAT , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Neoplasias , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Factores de Unión al Sitio Principal , ADN Complementario/genética , Proteínas de Unión al ADN/química , Sustancias Macromoleculares , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Mapeo Peptídico , Péptidos/química , Unión Proteica , Ratas , Proteínas Recombinantes , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae , Factores de Transcripción/químicaRESUMEN
The CCAAT-binding factor CBF is a heteromeric transcription factor that specifically binds to CCAAT sequences in many eukaryotic genes. CBF consists of three subunits, CBF-A, CBF-B, and CBF-C, all three of which are necessary for DNA binding. In this study we examined the transcription activation function of CBF by two different approaches. We first used a heterologous system in which a series of deletion mutations of CBF-B, fused to the bacterial LexA DNA binding domain, were transfected into HeLa cells together with a reporter gene driven by a minimal promoter containing LexA binding sites. These experiments showed that CBF-B needed both a glutamine-rich domain and an adjacent serine/threonine-rich domain to activate the reporter gene optimally. The glutamine-rich domain by itself activated transcription only modestly. We also set up an in vitro transcription reconstituted system in which trans-activation by CBF occurred through a physiological CCAAT motif. Nuclear extracts from NIH 3T3 cells were first depleted of CBF and then complemented with recombinant CBF-B and a highly purified fraction containing native CBF-A and CBF-C. Recombinant full-length CBF-B together with CBF-A and CBF-C activated transcription of several alpha 2(I) collagen gene promoter constructs. We then tested whether in this system the glutamine- and serine/threonine-rich domains of CBF-B were needed for trans-activation by CBF. We generated a truncated form of CBF-B that was still able to bind DNA in the presence of CBF-A and CBF-C. Even in the absence of the glutamine- and serine/threonine-rich domains of CBF-B, reconstituted CBF did activate transcription, suggesting that CBF transcriptional activation can also be mediated by the other subunits of CBF or by another transcription factor present in the nuclear extracts that interacts with CBF. Taken together our results suggest a model in which CBF has the potential to activate transcription either through the glutamine- and serine/threonine-rich domains of CBF-B or through the other subunits of CBF or through another component recruited by CBF.