RESUMEN
BACKGROUND: In multicellular organisms, precise control of cell cycle and the maintenance of genomic stability are crucial to prevent chromosomal alterations. The accurate function of the DNA damage pathway is maintained by DNA repair mechanisms including homologous recombination (HR). Herein, we show that both TFII-I and DBC1 mediate cellular mechanisms of cell-cycle regulation and DNA double strand damage repair. METHODS: Regulation of cell cycle by TFII-I and DBC1 was investigated using Trypan blue dye exclusion test, luciferase assay, and flow cytometry analysis. We also analysed the role of TFII-I and DBC1 in DNA double strand damage repair after irradiation by immunofluorescence study, clonogenicity assay, and HR assay. RESULTS: Flow cytometry analysis revealed a novel function that siRNA-mediated knockdown of endogenous DBC1 resulted in G2/M phase arrest. We also have shown that both endogenous TFII-I and DBC1 activate DNA repair mechanisms after irradiation because irradiation-induced foci formation of TFII-I-γH2AX was observed, and the depletion of endogenous TFII-I or DBC1 resulted in the inhibition of normal HR efficiency. CONCLUSION: These results reveal novel mechanisms by which TFII-I and DBC1 can modulate cellular fate by affecting cell-cycle control as well as HR pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Puntos de Control del Ciclo Celular/fisiología , Roturas del ADN de Doble Cadena , Reparación del ADN , Factores de Transcripción TFII/fisiología , Puntos de Control del Ciclo Celular/genética , División Celular/genética , División Celular/fisiología , Línea Celular , Línea Celular Tumoral , ADN/química , ADN/genética , ADN/metabolismo , ADN/efectos de la radiación , Citometría de Flujo , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Humanos , Factores de Transcripción TFII/genética , Factores de Transcripción TFII/metabolismoRESUMEN
The promyelocytic leukemia (PML) gene codes for a tumor suppressor protein that is associated with distinct subnuclear macromolecular structures called the PML bodies. The PML gene is frequently involved in the t(15;17) chromosomal translocation of acute promyelocytic leukemia (APL). The translocation results in a fusion gene product, PML-RARalpha, in which the PML gene fuses to the retinoic acid receptor alpha (RARalpha) gene. PML-RARalpha has been shown to promote transcriptional repression of genes involved in myeloid terminal differentiation and to disrupt the architecture of PML bodies, a phenotype reversed by treatment with all trans retinoic acid (ATRA). However, there are several alternatively spliced isoforms of PML-RARalpha. Here, we addressed the differences between the short and the long isoforms of PML-RARalpha (L and S) since both are associated with APL. We demonstrate that PML-RARalphaL, but not PML-RARalphaS, can directly promote cell growth by transcriptionally activating the pro-proliferative gene, c-fos, in response to mitogenic stimulation. The activity of the PML-RARalphaL is completely sensitive to ATRA. We further show that this activation is not via direct recruitment of the protein to the c-fos promoter but indirectly by altering the chromosomal environment of the c-fos gene, thereby rendering it more accessible to the signal induced transcriptional activators. Our results suggest that in addition to antagonizing the PML-tumor suppressor or the PML-pro-apoptotic activity, PML-RARalpha proteins can also directly promote cell growth by activating c-fos.
Asunto(s)
Leucemia Promielocítica Aguda/metabolismo , Proteínas de Neoplasias/química , Proteínas de Fusión Oncogénica/química , Animales , Apoptosis , Células COS , Proliferación Celular , Chlorocebus aethiops , Histona Desacetilasa 1 , Histona Desacetilasas/metabolismo , Ratones , Células 3T3 NIH , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Tretinoina/metabolismoRESUMEN
Posture-dependent trunk function data are important for appropriate normalization of submaximal trunk exertions, and is also necessary to define a more precise and specific use for strength testing in the prevention and diagnosis of spinal disorders. The aim of the current study was to quantify maximal effort trunk muscle extensor activity and trunk isometric extension torque over a functional range of sagittal standing postures. Twenty healthy, young adult male and female subjects performed isometric extension tasks over a sagittal posture range of -20 degrees extension to +50 degrees flexion, in 10 degrees increments. Erector spinae muscle activity was recorded bilaterally at the level of L3 using surface EMG electrodes. Isometric trunk extension torque was measured using a trunk dynamometer. EMG and trunk torque differed significantly between genders, but there were no differences between male and female subjects when the data were normalized with respect to the upright posture. For the combined male and female population, upright posture normalized L3 EMG activity (EMGn) and trunk extension torque (Tn) increased 1.7-fold and 3.5-fold, respectively, over the 70 degrees range of sagittal postures examined. The ratio (Tn/EMGn) increased two-fold (0.83 to 1.67) from -20 degrees extension to +50 degrees flexion, indicating that the neuromuscular efficiency increases with flexion. Trunk extension torque normalized with respect to the upright posture was linearly and positively correlated (r = 0.59, P < 0.001) to similarly normalized L3 EMG activity. This relatively weak correlation suggests that trunk muscle synergism and/or intrinsic muscle length-tension relationships are also modulated by posture. This study provides data that can be used to estimate trunk extensor muscle function over a broad range of sagittal postures. Our findings indicate that appropriate postural normalization of trunk extensor EMG activity is necessary for studies where submaximal trunk exertions are performed over a range of upright postures.
Asunto(s)
Contracción Isométrica/fisiología , Movimiento/fisiología , Músculo Esquelético/fisiología , Esfuerzo Físico/fisiología , Postura/fisiología , Adulto , Análisis de Varianza , Fenómenos Biomecánicos , Electromiografía , Femenino , Humanos , Masculino , Valores de Referencia , Distribución por Sexo , TorqueRESUMEN
An animal cell has the capability to respond to a variety of external signals through cell surface receptors. The response is usually manifested in terms of altered gene expression in the nucleus. Thus, in modern molecular and cell biology, it has become important to understand how the communication between extracellular signals and nuclear gene transcription is achieved. Originally discovered as a basal factor required for initiator-dependent transcription in vitro, recent evidence suggests that TFII-I is also an inducible multifunctional transcription factor that is activated in response to a variety of extracellular signals and translocates to the nucleus to turn on signal-induced genes. Here I review the biochemical and biological properties of TFII-I and related proteins in nuclear gene transcription, signal transduction and genetic disorders.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Animales , Regulación de la Expresión Génica , Genes/genética , Humanos , Modelos Biológicos , Isoformas de Proteínas/genética , Transducción de SeñalRESUMEN
TFII-I is an unusual transcription factor possessing both basal and signal-induced transcriptional functions. Here we report the characterization of a TFII-I-related factor (MusTRD1/BEN) that regulates transcriptional functions of TFII-I by controlling its nuclear residency. MusTRD1/BEN has five or six direct repeats, each containing helix--loop--helix motifs, and, thus, belongs to the TFII-I family of transcription factors. TFII-I and MusTRD1/BEN, when expressed individually, show predominant nuclear localization. However, when the two proteins are coexpressed ectopically, MusTRD1/BEN locates almost exclusively to the nucleus, whereas TFII-I is largely excluded from the nucleus, resulting in a loss of TFII-I-dependent transcriptional activation of the c-fos promoter. Mutation of a consensus nuclear localization signal in MusTRD1/BEN results in a reversal of nuclear residency of the two proteins and a concomitant gain of c-fos promoter activity. These data suggest a means of transcriptional repression by competition at the level of nuclear occupancy.
Asunto(s)
Núcleo Celular/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Transporte Biológico/genética , Células COS , Células HeLa , Humanos , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
When mammalian cells are subjected to stress targeted to the endoplasmic reticulum (ER), such as depletion of the ER Ca(2+) store, the transcription of a family of glucose-regulated protein (GRP) genes encoding ER chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE), consisting of a unique tripartite structure, CCAAT(N(9))CCACG. Within a subset of mammalian ERSEs, N(9) represents a GC-rich sequence of 9 bp that is conserved across species. A novel complex (termed ERSF) exhibits enhanced binding to the ERSE of the grp78 and ERp72 promoters using HeLa nuclear extracts prepared from ER-stressed cells. Optimal binding of ERSF to ERSE and maximal ERSE-mediated stress inducibility require the conserved GGC motif within the 9-bp region. Through chromatographic purification and subsequent microsequencing, we have identified ERSF as TFII-I. Whereas TFII-I remains predominantly nuclear in both nontreated NIH 3T3 cells and cells treated with thapsigargin (Tg), a potent inducer of the GRP stress response through depletion of the ER Ca(2+) store, the level of TFII-I transcript was elevated in Tg-stressed cells, correlating with an increase in TFII-I protein level in the nuclei of Tg-stressed cells. Purified recombinant TFII-I isoforms bind directly to the ERSEs of grp78 and ERp72 promoters. The stimulation of ERSE-mediated transcription by TFII-I requires the consensus tyrosine phosphorylation site of TFII-I and the GGC sequence motif of the ERSE. We further discovered that TFII-I is an interactive protein partner of ATF6 and that optimal stimulation of ERSE by ATF6 requires TFII-I.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico , Chaperonas Moleculares/genética , Elementos de Respuesta , Factores de Transcripción/metabolismo , Células 3T3 , Factor de Transcripción Activador 6 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Cromatografía/métodos , Secuencia Conservada , Proteínas de Unión al ADN/genética , Chaperón BiP del Retículo Endoplásmico , Células HeLa , Homeostasis , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
The transcription factor TFII-I can bind specifically to several DNA sequence elements and is implicated in both basal and activated transcription. There are four alternatively spliced isoforms of TFII-I, all characterized by the presence of six I-repeats, R1-R6, each containing a potential helix-loop-helix motif implicated in protein-protein interactions. These isoforms exhibit both homomeric and heteromeric interactions that lead to nuclear localization. In this study we mapped two distinct regions in TFII-I that affect its DNA binding. Deletion of either of these regions led to abrogation of DNA binding and transcriptional activation from both the Vbeta and c-fos promoters. The I-repeats, as expected, were capable of mediating homomeric interactions either individually or in combination. Unexpectedly, an additional homomeric interaction domain was found within the N-terminal end of TFII-I that includes a putative leucine zipper motif. These data suggest a model in which TFII-I undergoes regulated homomeric interaction mediated by both the N-terminal end and the I-repeats.
Asunto(s)
Proteínas de Unión al ADN/química , Secuencias Hélice-Asa-Hélice , Secuencias Repetitivas de Aminoácido , Factores de Transcripción/química , Animales , Células COS , Núcleo Celular/química , ADN/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/fisiología , Relación Estructura-Actividad , Factores de Transcripción/análisis , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
Müllerian-inhibiting substance (MIS) plays an essential role in mammalian male sexual development; thus, it is important to determine how the tightly regulated expression of the MIS gene is transcriptionally controlled. Transcription of eukaryotic genes is dependent on regulatory elements in the enhancer and one or both distinct elements in the core promoter: the TATA box, and the initiator (Inr) element. Because the human MIS gene does not contain a consensus TATA and has not been reported to contain an Inr element, we hypothesized that the initiator region of the core promoter was essential for promoter activity. Transient transfection assays were conducted using an immortalized Embryonic Day 14.5 male rat urogenital ridge cell line (CH34) that expresses low levels of MIS. These studies revealed that promoter activity is dependent on the region around the start site (-6 to +10) but not on the nonconsensus TATA region. Electrophoretic mobility shift assays demonstrated that the human MIS initiator sequence forms a specific DNA-protein complex with CH34 cell nuclear extract, HeLa cell nuclear extract, and purified TFII-I. This complex could be blocked or supershifted by the addition of antibodies directed against TFII-I. These data suggest that the human MIS gene contains a functional initiator that is specifically recognized by TFII-I.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Glicoproteínas , Inhibidores de Crecimiento/genética , Secuencias Reguladoras de Ácidos Nucleicos , Hormonas Testiculares/genética , Factores de Transcripción/metabolismo , Animales , Hormona Antimülleriana , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/inmunología , Electroforesis/métodos , Inhibidores de Crecimiento/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Ratas , Homología de Secuencia de Ácido Nucleico , TATA Box , Hormonas Testiculares/metabolismo , Factores de Transcripción/inmunología , Transcripción GenéticaRESUMEN
TFII-I is a multifunctional phosphoprotein with roles in transcription and signal transduction. Here we report characterization of three additional alternatively spliced isoforms of TFII-I. Employing isoform-specific antibodies, we show that the isoforms form a stable complex in vivo preferentially in the nucleus compared with the cytoplasm. We further show that both homomeric and heteromeric interactions are possible and that the heteromeric interactions between a wild type and a nuclear localization-deficient mutant result in nuclear translocation of the complex, leading us to postulate that complex formation might aid in nuclear translocation. In functional assays all four isoforms individually bind to DNA and transactivate reporter genes to a similar extent. However, although co-expression of different TFII-I isoforms leads to enhanced basal activity, it results in attenuated signal responsive activity. Thus, TFII-I might differentially regulate its target genes via complex or subcomplex formation.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Células COS , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/química , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Transducción de Señal , Relación Estructura-Actividad , Factores de Transcripción/químicaRESUMEN
Bruton's tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B cells, nuclear TFII-I is greater in xid B cells. Most strikingly, receptor cross-linking of wild-type (but not xid) B cells results in increased nuclear import of TFII-I. Taken together, these data suggest that although the PH domain of Btk is primarily responsible for its physical interaction with TFII-I, an intact kinase domain of Btk is required to enhance transcriptional activity of TFII-I in the nucleus. Thus, mutations impairing the physical and/or functional association between TFII-I and Btk may result in diminished TFII-I-dependent transcription and contribute to defective B-cell development and/or function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Agammaglobulinemia Tirosina Quinasa , Animales , Linfocitos B/enzimología , Linfocitos B/fisiología , Células COS , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Ratones , Ratones Endogámicos BALB C , Mutagénesis , Proteínas Tirosina Quinasas/genética , Fracciones Subcelulares , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Intestinal toxicity exerted by indomethacin was compared to that induced by copper-indomethacinate, free or associated to zwitterionic phospholipids. A single high dose of indomethacin (15 or 20 mg/kg), copper-indomethacinate (15 or 20 mg/kg), or copper-indomethacinate liposomes or nanocapsules (15 mg/kg) was orally administered. Then 24 hr later jejunoileal tissue was taken for macroscopic observation, ex vivo nitrite production, and determination of myeloperoxydase and iNOS activities. Antiinflammatory activity of the drugs was investigated using the carrageenan-induced paw edema model. Indomethacin induced penetrating ulcerations of the intestine that were maximal at hour 24. Copper-indomethacinate induced significantly less ulceration than indomethacin with no significant difference in MPO and iNOS activities. The injurious action of indomethacin on the small intestine was further reduced when copper-indomethacinate was administered as the phospholipid-associated state while similar anti-inflammatory action was observed on rat paw edema. The antiulcerogen effect of copper-indomethacinate seems to be linked to its free radical scavenging effect without any modification of nitric oxide release.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Cobre/farmacología , Indometacina/farmacología , Intestino Delgado/efectos de los fármacos , Óxido Nítrico Sintasa/metabolismo , Compuestos Organometálicos/farmacología , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Indometacina/efectos adversos , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Liposomas , Masculino , Óxido Nítrico Sintasa de Tipo II , Peroxidasa/metabolismo , Fosfolípidos , Ratas , Ratas Sprague-Dawley , Úlcera/inducido químicamenteRESUMEN
This study aimed to evaluate the role of L-type calcium channels in the increased renal vascular resistance (RVR) of the Lyon hypertensive (LH) rat before and after normalization of its blood pressure (BP) by angiotensin-converting enzyme inhibition with perindopril. Concentration-response curves to Bay K 8644 (from 0.1 nM to 1 microM), a selective agonist of L-type calcium channels, were obtained in single-pass perfused kidneys isolated from groups of eight untreated or perindopril-treated (3 mg/kg/day, p.o., from age 3 to 7 weeks) LH and low-BP (LL) control rats. In untreated rats, the negative logarithm of the molar concentration of Bay K 8644 required to produce a half-maximal effect (pD2) values for Bay K 8644 did not differ between the two strains, whereas the maximal RVR response was higher in LH than in LL kidneys (28.0+/-4.9 vs. 12.9+/-0.8 mm Hg/ml/min/g, respectively). Perindopril normalized BP and RVR in LH rats and suppressed the interstrain differences in the maximal RVR responses (11.4+/-0.6 vs. 10.5+/-1.2 mm Hg/ml/min/g in LH and LL rats, respectively). These results demonstrate that LH rats exhibit an increased maximal contractile response to Bay K 8644 compared with LL controls, and that this alteration is not a primary defect because it disappears after normalization of BP level.
Asunto(s)
Canales de Calcio/fisiología , Hipertensión/fisiopatología , Riñón/irrigación sanguínea , Resistencia Vascular , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Agonistas de los Canales de Calcio/farmacología , Técnicas In Vitro , Indoles/farmacología , Riñón/efectos de los fármacos , Pruebas de Función Renal , Masculino , Perindopril , Ratas , Resistencia Vascular/fisiologíaRESUMEN
The transcription factor TFII-I binds to distinct promoter sequences including an initiator element in several eukaryotic genes. Here we demonstrate that TFII-I is phosphorylated in vivo at serine/threonine and tyrosine residues in the absence of any apparent extracellular signals. This "basal" phosphorylation of TFII-I is not required and does not affect its specific DNA binding, but is critical for its in vitro transcriptional properties via the Vbeta promoter. To better assess the functional role of phosphorylation in regulating TFII-I activity, we focused on tyrosine phosphorylation of TFII-I. Ectopically expressed recombinant TFII-I, like its native counterpart, exhibits tyrosine phosphorylation in the absence of distinct extracellular signals. More important, mutation of a potential consensus tyrosine phosphorylation site in TFII-I leads to severe reduction in its basal transcriptional activation of the Vbeta promoter in vivo. Taken together, these data suggest that tyrosine phosphorylation of TFII-I is important for its initiator-dependent transcriptional activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Cartilla de ADN , Células HeLa , Humanos , Fosforilación , Regiones Promotoras Genéticas , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Tirosina/metabolismoRESUMEN
In our effort to understand the transcriptional regulation of naturally occurring TATA-less but initiator (Inr)-containing genes, we have employed the murine T-cell receptor Vbeta 5.2 promoter as a model. Here we show by transient-transfection assays that the Inr binding transcription factor TFII-I is required for efficient expression of the Vbeta promoter in vivo. Mutations in the Inr element that reduced binding of TFII-I also abolished the Vbeta promoter activity by ectopic TFII-I. We further biochemically identified a protease-resistant N-terminal DNA binding fragment of TFII-I, p70. When ectopically expressed, recombinant p70 bound to the Vbeta Inr element with a specificity similar to that of wild-type TFII-I. More importantly, p70, which lacks independent activation functions, behaved as a dominant negative mutant that inhibited Inr-specific function of wild-type TFII-I. However, the activation functions of p70 were restored when fused to the heterologous activation domain of the yeast activator protein GAL4. Taken together, these data suggest that TFII-I functions in vivo require an intact Inr element and that the Inr-specific transcriptional functions of TFII-I are solely dictated by its N-terminal DNA binding domain and do not require its own C-terminal activation domain.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Factores de Transcripción/metabolismo , Animales , Bacterias/metabolismo , Sitios de Unión , Células COS , ADN/metabolismo , Proteínas de Unión al ADN/genética , Ratones , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Trombina/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The transcription factor TFII-I was initially isolated as a factor that can bind to initiator elements in core promoters. Recent evidence suggests that TFII-I may also have a role in signal transduction. We have found that overexpression of TFII-I can enhance the response of the wild-type c-fos promoter to a variety of stimuli. This effect depends on the c-fos c-sis-platelet-derived growth factor-inducible factor binding element (SIE) and serum response element (SRE). There is no effect of cotransfected TFII-I on the TATA box containing the c-fos basal promoter. Three TFII-I binding sites can be found in c-fos promoter. Two of these overlap the c-fos SIE and SRE, and another is located just upstream of the TATA box. Mutations that distinguish between serum response factor (SRF), STAT, and TFII-I binding to the c-fos SIE and SRE suggest that the binding of TFII-I to these elements is important for c-fos induction in conjunction with the SRF and STAT transcription factors. Moreover, TFII-I can form in vivo protein-protein complexes with the c-fos upstream activators SRF, STAT1, and STAT3. These results suggest that TFII-I may mediate the functional interdependence of the c-fos SIE and SRE elements. In addition, the ras pathway is required for TFII-I to exert its effects on the c-fos promoter, and growth factor stimulation enhances tyrosine phosphorylation of TFII-I. These results indicate that TFII-I is involved in signal transduction as well as transcriptional activation of the c-fos promoter.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Células 3T3 , Animales , Sitios de Unión , ADN/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Ratones , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , TATA Box , Transactivadores/metabolismoRESUMEN
The human homeodomain protein Phox1 interacts functionally with serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay. However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro. Here, we report the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1. We show that this protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo. Furthermore, SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE). SPIN is identical to the initiator-binding protein TFII-I. Consistent with this hypothesis, SPIN exhibits modest affinity for a characterized initiator sequence in vitro. We propose that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Western Blotting , Clonación Molecular , ADN/metabolismo , Sondas de ADN , ADN Complementario , Proteínas de Unión al ADN/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Genes fos/genética , Células HeLa , Proteínas de Homeodominio/metabolismo , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factor de Respuesta Sérica , Factores de Transcripción/aislamiento & purificaciónRESUMEN
Transcription initiation in eukaryotic mRNA coding genes is brought about by a host of general transcription factors, which assemble into a functional preinitiation complex (PIC) at the core promoter region, and gene-specific factors, which exert their effects on the rate and/or stability of the PIC. The core promoter region consists of a well-characterized TATA box and/or a less well-characterized pyrimidine-rich initiator element (Inr). While the biochemical mechanisms of TATA-mediated transcription initiation are extensively studied and known to be directed by the TATA binding protein, the mechanisms via the Inr element are poorly understood, as several factors have been shown to bind to an Inr. Here, we describe the biochemical properties of an Inr binding protein, TFII-I, employing the naturally occurring TATA-less but Inr-containing promoter derived from the T-cell receptor beta chain gene (V beta).
Asunto(s)
Proteínas de Unión al ADN/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética , Western Blotting , Electroforesis en Gel de Poliacrilamida , Células HeLa , Calor , Humanos , Células Jurkat , Proteínas Nucleares/inmunología , Proteínas Nucleares/aislamiento & purificación , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
The transcription factor TFII-I has been shown to bind independently to two distinct promoter elements, a pyrimidine-rich initiator (Inr) and a recognition site (E-box) for upstream stimulatory factor 1 (USF1), and to stimulate USF1 binding to both of these sites. Here we describe the isolation of a cDNA encoding TFII-I and demonstrate that the corresponding 120 kDa polypeptide, when expressed ectopically, is capable of binding to both Inr and E-box elements. The primary structure of TFII-I reveals novel features that include six directly repeated 90 residue motifs that each possess a potential helix-loop/span-helix homology. These unique structural features suggest that TFII-I may have the capacity for multiple protein-protein and, potentially, multiple protein-DNA interactions. Consistent with this hypothesis and with previous in vitro studies, we further demonstrate that ectopic TFII-I and USF1 can act synergistically, and in some cases independently, to activate transcription in vivo through both Inr and the E-box elements of the adenovirus major late promoter. We also describe domains of USF1 that are necessary for its independent and synergistic activation functions.
Asunto(s)
Adenoviridae/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , ADN Complementario , Proteínas de Unión al ADN/aislamiento & purificación , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Transcripción/aislamiento & purificación , Transfección , Factores Estimuladores hacia 5'RESUMEN
We show that the binding of Rel p50 and p52 homodimers at sites within the transcriptional initiation region of HIV-1 provides for their ability to interact with other proteins that bind the initiator. The binding of one such protein, the initiator protein TFII-I, to the initiation region of HIV-1 is augmented in the presence of Rel p50 and Rel p52 homodimers. Consistent with this, in vitro Rel homodimers potentiate HIV-1 transcription in a manner dependent upon TFII-I. The findings suggest that Rel dimers may regulate HIV-1 transcription in two ways. First, through binding at the kappa B enhancer sites at (-104 to -80), NF-kappa B p50:p65 participates in classical transcriptional activation. Second, Rel dimers such as p50 or p52 might bind at initiator sequences to regulate the de novo binding of components of certain preinitiation complexes. These findings, and the existence of Rel binding sites at the initiators of other genes, suggest roles for Rel proteins in early events determining transcriptional control.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Duplicado del Terminal Largo de VIH , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases , Sitios de Unión , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , FN-kappa B/genética , Subunidad p50 de NF-kappa B , Iniciación de la Cadena Peptídica Traduccional , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Many biological processes are controlled, spatially and temporally, at the level of transcription. Thus, understanding the mechanisms of transcriptional regulation of gene expression is critical in deciphering the molecular modes of differentiation and development of a eukaryotic cell. Transcriptional control is mediated largely through interactions of regulatory transcription factors with their cognate enhancer elements. The regulatory signals generated at enhancer elements are communicated to the general transcription machinery formed at the core promoter elements of all genes. Recent observations suggest that the general transcription machinery can also generate regulatory signals independent of enhancer-generated interactions. Thus, the transcriptional regulation of gene expression, both in time and in space, may result from appropriate interfacing of independent signals generated at the core promoter and at the enhancer.