RESUMEN
During neuronal differentiation, an exquisitely controlled program of signal transduction events takes place, leading to the temporally and spatially regulated expression of genes associated with the differentiated phenotype. A critical class of genes involved in this phenomenon is that made up of genes encoding neurotransmitter-gated ion channels that play a central role in signal generation and propagation within the nervous system. We used the well established PC12 cell line to investigate the molecular details underlying the expression of the neuronal nicotinic acetylcholine receptor class of ion channels. Neuronal differentiation of PC12 cells can be induced by nerve growth factor, leading to an increase in neuronal nicotinic acetylcholine receptor gene expression. Nerve growth factor initiates several signal transduction cascades. Here, we show that the Ras-dependent mitogen-activated protein kinase and phosphoinositide 3-kinase pathways are critical for the nerve growth factor-mediated increase in the transcriptional activity of a neuronal nicotinic acetylcholine receptor gene promoter. In addition, we show that a component of the Ras-dependent mitogen-activated protein kinase pathway, nerve growth factor-inducible c-Jun, exerts its effects on receptor gene promoter activity most likely through protein-protein interactions with Sp1. Finally, we demonstrate that the target for nerve growth factor signaling is an Sp1-binding site within the neuronal nicotinic acetylcholine receptor gene promoter.
Asunto(s)
Canales Iónicos/metabolismo , Neuronas/citología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Transducción de Señal , Factor de Transcripción Sp1/metabolismo , Animales , Sitios de Unión , Western Blotting , Diferenciación Celular , Línea Celular , Drosophila , Sistema de Señalización de MAP Quinasas , Mutación , Factor de Crecimiento Nervioso/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuronas/metabolismo , Células PC12 , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Pruebas de Precipitina , Regiones Promotoras Genéticas , Ratas , Transcripción Genética , Activación Transcripcional , Transfección , Proteínas ras/metabolismoRESUMEN
Neuronal nicotinic acetylcholine receptors (nAChR) are expressed at specific times during development and in discrete neuronal populations. Transcriptional regulation of the receptor genes clearly plays a key role in the molecular pathway underlying the expression of these critical synaptic components. In an effort to understand this regulation, we focus upon the genes encoding three receptor subunits: alpha3, alpha5 and beta4. These subunits are genomically clustered and constitute the predominant nAChR subtype expressed in the peripheral nervous system. We and others demonstrated that the general transcription factors, Sp1 and Sp3, can transactivate the promoter of each subunit gene. Further, we showed that the regulatory factor Sox10 transactivates the alpha3 and beta4 promoters and does so in a cell-type-specific manner. Interestingly, the Sp- and Sox10-binding sites on the beta4 promoter are located immediately adjacent to each other, raising the possibility that the two sets of factors functionally interact to regulate receptor gene expression. Consistent with this hypothesis, we demonstrated that the proteins can directly interact. Here, we extend these observations and show that Sox10 and the Sp factors functionally interact, leading to synergistic transcriptional activation in a cholinergic cell line. Finally, evidence for the existence of cell-type-specific co-regulators for Sp1 and Sox10 is presented.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Receptores Nicotínicos/genética , Factor de Transcripción Sp1/genética , Animales , Células Cultivadas , Quimera/genética , Ratones , Plásmidos , Pruebas de Precipitina , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas , Factores de Transcripción SOXE , Factor de Transcripción Sp3 , Factores de Transcripción/genética , Transcripción Genética/genética , TransfecciónRESUMEN
The genes encoding the alpha3, alpha5 and beta4 subunits of nicotinic acetylcholine receptors are tightly clustered within the genome. As these three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system, their genomic proximity suggests a regulatory mechanism ensuring their coordinate expression. We previously identified two transcriptional regulatory elements within the beta4 promoter. One of these elements, a CT box, interacts with the regulatory factors heterogeneous nuclear ribonucleoprotein K and Puralpha. Another element, a CA box, interacts with Sp1 and Sp3. The binding site for a fifth factor, Sox10, overlaps the CT and CA boxes. As the CT and CA boxes are adjacent, we postulated that the proteins that bind to the elements interact. Here we report that the CT box-binding factors interact with each other as do the CA box-binding factors. However, there are no direct associations between the two pairs of proteins. Interestingly though, Sox10 directly interacts with all four proteins, suggesting a central role in beta4 gene expression for this member of the Sox family of regulatory factors.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/fisiología , Regiones Promotoras Genéticas , Receptores Nicotínicos/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , ADN/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Unión Proteica , Ribonucleoproteínas/fisiología , Factores de Transcripción SOXE , Factor de Transcripción Sp1/fisiología , Factor de Transcripción Sp3 , Factores de Transcripción/fisiologíaRESUMEN
JAB1 was originally described as a transcriptional coactivator of c-Jun and Jun D. Recent data suggests that JAB1 is a component of a large protein complex, the JAB1 signalosome in mammals and the COP9 complex in plants. The JAB1 signalosome is implicated in the phosphorylation of selected transcription factors, while the COP9 complex is involved in repression of photomorphogenesis in Arabidopsis. In this study, we describe the partial characterization of mouse JAB1 (mJAB1). The murine JAB1 protein is encoded by a gene located on mouse chromosome 1. mJAB1 mRNA is abundantly expressed in a variety of adult tissues as well as in mouse embryos. The JAB1 protein was readily detectable in many cell types and localized to both the nucleus and cytoplasm. Endogenous JAB1 protein is relatively stable and its degradation is not perturbed by blocking 26S proteasome activity, suggesting that this protein is not degraded by the ubiquitin-mediated proteolytic pathway.
Asunto(s)
ADN Complementario/genética , Proteínas de Unión al ADN/genética , Complejo de la Endopetidasa Proteasomal , Factores de Transcripción/genética , Animales , Complejo del Señalosoma COP9 , Línea Celular , Núcleo Celular/química , Mapeo Cromosómico , Citoplasma/química , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Hidrólisis , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Muridae , Oligopéptidos , Péptido Hidrolasas/metabolismo , Péptidos/genética , ARN/genética , ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Distribución Tisular , Factores de Transcripción/metabolismo , Ubiquitinas/metabolismoRESUMEN
Simple analytical methods are proposed for calculating the reflection function of a semi-infinite and conservative scattered layer, the value of which is needed to solve many atmospheric optics problems. The methods are based on approximations of the exact values obtained with a strict numerical method. For a Henyey-Greenstein phase function, knowledge of the zeroth and sixth higher harmonics appears to be sufficient for a quite accurate approximation of the angle range, which is acceptable for solution of direct and inverse problems in atmospheric optics when a plane atmosphere is assumed. An error estimation and a comparison with the exact solution are presented.
RESUMEN
The regulatory factor Sox10 is expressed in neural crest derivatives during development as well as in the adult CNS and peripheral nervous system. Mutations of the human Sox10 gene have been identified in patients with Waardenburg-Hirschsprung syndrome that is characterized by defects in neural crest development. Previous studies suggested that Sox10 might function as an important transcriptional regulator of neural crest development. No natural target genes of Sox10 have yet been identified. Although human Sox10 activates a synthetic promoter consisting of a TATA box and multiple Sox consensus sequences, no transcriptional activity of the rat Sox10 homolog has been detected. Here we report that the neuronal nicotinic acetylcholine receptor beta4 and alpha3 subunit gene promoters are transactivated by rat Sox10 in a cell type-specific manner. The alpha3 and beta4 subunits, in combination with the alpha5 subunit, make up the predominant nicotinic receptor subtype expressed in the peripheral nervous system. Transfections using Sox10 mutants indicate that the C-terminal region is dispensable for its ability to activate the beta4 and alpha3 promoters. Rat Sox10 was originally identified as an accessory protein of the POU domain protein Tst-1/Oct6/SCIP in glial cells. Tst-1/Oct6/SCIP was shown previously to activate the alpha3 promoter. We now demonstrate that it can transactivate the beta4 promoter as well. However, we were unable to detect any synergistic effects of Sox10 and Tst-1/Oct6/SCIP on beta4 or alpha3 promoter activity. Finally, we present data suggesting that recombinant Sox10 protein can directly interact with a previously characterized regulatory region of the beta4 gene.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas del Grupo de Alta Movilidad/metabolismo , Neuronas/metabolismo , Nervios Periféricos/metabolismo , Regiones Promotoras Genéticas , Receptores Nicotínicos/genética , Activación Transcripcional , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/genética , Células HeLa , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Neuroglía/metabolismo , Células PC12 , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , Factores de Transcripción SOXE , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
The Id proteins are a family of related mammalian helix-loop-helix (HLH) proteins which can interact with other HLH proteins but lack a basic region and are thus not thought to bind to DNA. Instead, they are hypothesized to act as dominant negative regulators of DNA-binding basic HLH (bHLH) proteins, by forming inactive heterodimers with these proteins. All four Id family proteins possess related HLH dimerization domains and can interact with similar bHLH proteins, although with differing affinities. The functions of the largely unrelated N- and C-terminal regions of the proteins are unknown. In this study, we have identified a novel transcriptional activity of the mammalian Id proteins. We show that when fused to the heterologous GAL4 DNA-binding domain, all four of the mammalian Id proteins can activate GAL4-dependent transcription. The HLH domain is necessary for the transactivation activity observed, suggesting that interaction with a cellular HLH protein is required. Co-transfection with exogenous Class A bHLH proteins (E-proteins) greatly potentiates the transactivation, which is abolished upon co-transfection with Class B bHLH proteins. These results are consistent with the idea that the Id proteins have a transcriptional activity when present in a DNA-binding complex.
Asunto(s)
Secuencias Hélice-Asa-Hélice , Proteínas de Neoplasias , Proteínas Represoras , Factores de Transcripción/metabolismo , Transcripción Genética , Línea Celular , Humanos , Proteína 1 Inhibidora de la Diferenciación , Proteínas Inhibidoras de la DiferenciaciónRESUMEN
Id proteins are helix-loop-helix (HLH) transcription factors that lack DNA-binding domains. These proteins form inactive heterodimers with basic HLH (bHLH) factors, inhibiting their DNA-binding and transcriptional activities. Consistent with a proposed role for Id proteins as inhibitors of terminal differentiation, Id1 and Id3 have been shown to negatively regulate myogenesis in cultured muscle cells. Here we have investigated the possibility that Id2 and/or Id4 can act in a similar manner. Surprisingly, while overexpression of Id2 resulted in inhibition of differentiation of Sol 8 myoblast cells, overexpression of Id4 did not. Sol 8 cells stably transfected with Id4 showed no apparent changes in expression of muscle-specific genes upon differentiation. DNA-binding activities present at the muscle creatine kinase (MCK) enhancer E-box and transcription of the MCK enhancer were not altered in Id4-overexpressing cells, compared with vector-transfected cells. Id2 is also a more potent inhibitor of protein/DNA complex formation at the MCK-R enhancer E-box than Identified in vitro. Therefore, our data support the notion that members of the Id family might be involved in the regulation of distinct developmental pathways.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Factores Reguladores Miogénicos/fisiología , Proteínas/fisiología , Proteínas Represoras , Factores de Transcripción/fisiología , Animales , Células COS , Diferenciación Celular , Células Cultivadas , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteína 1 Inhibidora de la Diferenciación , Proteína 2 Inhibidora de la Diferenciación , Proteínas Inhibidoras de la Diferenciación , Masculino , Ratones , Ratones Endogámicos C57BL , Músculos/citología , Factores Reguladores Miogénicos/biosíntesis , Proteínas Nucleares/fisiologíaRESUMEN
The neuronal nicotinic acetylcholine receptor gene family consists of 11 members, alpha2-alpha9 and beta2-beta4. Three of the genes, those encoding the alpha3, alpha5, and beta4 subunits, are clustered tightly within the genome. These three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system. The genomic proximity of the three genes suggests a regulatory mechanism ensuring their coordinate expression. However, it is likely that gene-specific regulatory mechanisms are also functioning because the expression patterns of the three genes, although similar, are not identical. Previously we identified regulatory elements within the beta4 promoter region and demonstrated that these elements interact specifically with nuclear proteins. One of these elements, E1, interacts with the regulatory factor Puralpha as well as three other unidentified DNA-binding proteins with molecular masses of 31, 65, and 114 kDa. Another element, E2, interacts with Sp1 and Sp3. Because E1 and E2 are immediately adjacent to one another, we postulated that the proteins that bind to the elements interact to regulate beta4 gene expression. Here we report the identification of the 65-kDa E1-binding protein as heterogeneous nuclear ribonucleoprotein K and demonstrate that it affects the transactivation of beta4 promoter activity by Sp1 and Sp3 differentially.
Asunto(s)
Regiones Promotoras Genéticas/genética , Receptores Colinérgicos/genética , Ribonucleoproteínas/fisiología , Activación Transcripcional/fisiología , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Drosophila , Regulación de la Expresión Génica/fisiología , Ribonucleoproteínas Nucleares Heterogéneas , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/metabolismo , Ratas , Factores de Transcripción/fisiología , Transfección/genéticaRESUMEN
Neuronal nicotinic acetylcholine receptors play important roles in signal transduction within the nervous system. The receptors exist in a variety of functionally distinct subtypes that are determined by their subunit structures. The subunits are encoded by 11 genes, alpha2-alpha9 and beta2-beta4. Three of the genes, alpha3, alpha5, and beta4, are tightly clustered, and their encoded proteins make up the predominant receptor subtype in the peripheral nervous system. The tight linkage of the genes suggests there may be a common regulatory mechanism underlying their expression. However, although their expression patterns significantly overlap, they are not identical, indicating that independent regulatory mechanisms must also exist. Our studies have focused upon the gene encoding the beta4 subunit for which we have identified several transcriptional regulatory elements. One of these elements, E2, specifically interacts with the general transcription factor Sp1. Here we show that another member of the Sp family of factors, Sp3, can specifically interact with E2 whereas two other members, Sp2 and Sp4, cannot. Co-transfection experiments indicate that Sp3 can transactivate a beta4 promoter/reporter gene construct and, furthermore, that Sp1 and Sp3 can transactivate the beta4 reporter construct synergistically. The transactivation is dependent upon an intact E2 and may involve direct interactions between Sp1 and Sp3.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica , Neuronas/metabolismo , Receptores Nicotínicos/genética , Factor de Transcripción Sp1/fisiología , Factores de Transcripción/fisiología , Dedos de Zinc , Animales , Drosophila , Regiones Promotoras Genéticas , Ratas , Análisis de Secuencia de ADN , Factor de Transcripción Sp3 , Activación TranscripcionalRESUMEN
Id3 (originally named HLH462) belongs to the Id family of the helix-loop-helix transcription factors. Members of the Id family do not contain basic DNA binding regions adjacent to the helix-loop-helix dimerization domain and are, therefore, hypothesized to act as negative regulators of other helix-loop-helix proteins by preventing the formation of functional dimers. We have investigated the potential role of Id3 in the control of muscle cell differentiation. Id3 mRNA is expressed at a high level in proliferating myoblasts and is down-regulated following induction of differentiation. We show that stable overexpression of Id3 mRNA inhibits differentiation of the Sol 8 muscle cell line. Both the HLH and COOH-terminal domains of Id3 are necessary and sufficient for its dominant-negative activity in muscle cells. DNA-binding activity present in nuclear extracts prepared from Id3-overexpressing cells was significantly reduced when compared to the wild-type or vector-transfected cells. Finally, we show by in situ hybridization that the Id3 mRNA is co-expressed with the myogenic regulatory factor myogenin in somites and developing muscle during embryogenesis, although unlike the myogenic regulatory factors, Id3 is also expressed in many other locations in the embryo. These data support a model in which Id3 negatively regulates muscle differentiation by inhibiting the DNA-binding activities of the myogenic regulatory factors.
Asunto(s)
Diferenciación Celular/fisiología , Fibras Musculares Esqueléticas/fisiología , Proteínas de Neoplasias , Factores de Transcripción/farmacología , Secuencia de Aminoácidos , Animales , Northern Blotting , División Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Dimerización , Regulación hacia Abajo , Secuencias Hélice-Asa-Hélice/genética , Inmunohistoquímica , Hibridación in Situ , Proteínas Inhibidoras de la Diferenciación , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Somitos/fisiología , Factores de Transcripción/genética , Transcripción Genética/fisiologíaRESUMEN
Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major determinant of the thymic disease specificity of Moloney virus genetically maps to the conserved viral core motif in the Moloney virus enhancer. Point mutations introduced into the core site significantly shifted the disease specificity of the Moloney virus from thymic leukemia to erythroid leukemia (N.A. Speck, B. Renjifo, E. Golemis, T.N. Fredrickson, J.W. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We previously reported the purification of core-binding factors (CBF) from calf thymus nuclei (S. Wang and N.A. Speck, Mol. Cell. Biol. 12:89-102, 1992). CBF binds to core sites in murine leukemia virus and T-cell receptor enhancers. Affinity-purified CBF contains multiple polypeptides. In this study, we sequenced five tryptic peptides from two of the bovine CBF proteins and isolated three cDNA clones from a mouse thymus cDNA library encoding three of the tryptic peptides from the bovine proteins. The cDNA clones, which we call CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6, encode three highly related but distinct proteins with deduced molecular sizes of 22.0, 21.5, and 17.6 kDa that appear to be translated from multiply spliced mRNAs transcribed from the same gene. CBF beta p22.0, CBF beta p21.5, and CBF beta p17.6 do not by themselves bind the core site. However, CBF beta p22.0 and CBF beta p21.5 form a complex with DNA-binding CBF alpha subunits and as a result decrease the rate of dissociation of the CBF protein-DNA complex. Association of the CBF beta subunits does not extend the phosphate contacts in the binding site. We propose that CBF beta is a non-DNA-binding subunit of CBF and does not contact DNA directly.
Asunto(s)
Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Virus de la Leucemia Murina de Moloney/genética , Proteínas de Neoplasias , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Factores de Transcripción/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Núcleo Celular/fisiología , Cromatografía Líquida de Alta Presión , Clonación Molecular/métodos , Factores de Unión al Sitio Principal , ADN/genética , ADN/aislamiento & purificación , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Fragmentos de Péptidos/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/inmunología , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The core-binding factor (CBF) binds the conserved core motif in mammalian type C retrovirus enhancers. We analyzed the phosphate contacts made by CBF on the Moloney murine leukemia virus enhancer by ethylation interference assay. The phosphate contacts span 9 bp centered around the consensus core site. To examine the sequence preferences for CBF binding, we employed the technique of selected and amplified binding sequence footprinting (T. K. Blackwell and H. Weintraub, Science 250:1104-1110, 1990). The consensus binding site for CBF defined by selected and amplified binding sequence footprinting is PyGPyG GTPy.