RESUMEN
Helix-loop-helix proteins are essential factors for lymphocyte development and function. In particular, E-proteins are crucial for commitment of lymphoid progenitors to the B- and T-cell lineages. E-proteins are negatively regulated by the Id class of helix-loop-helix proteins. The Id proteins function as dominant-negative inhibitors of E-proteins by inhibiting their ability to bind DNA. Here, we review the role of E-proteins and their Id protein antagonists in lymphocyte proliferation and developmental progression. In addition, we discuss how E-protein activity and Id gene expression are regulated by T-cell receptor (TCR) and pre-TCR-mediated signalling.
Asunto(s)
Proteínas de Unión al ADN/genética , Linfocitos/inmunología , Proteínas Represoras , Factores de Transcripción/genética , Animales , Linfocitos B/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , División Celular , Supervivencia Celular , Secuencias Hélice-Asa-Hélice/genética , Humanos , Proteína 1 Inhibidora de la Diferenciación , Activación de Linfocitos , Linfocitos/citología , Linfocitos/metabolismo , Modelos Inmunológicos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
To determine why the H-2Dd antigen is expressed on the surface of transfected cells at a rate several-fold higher than an analogously transfected H-2Ld molecule, we analyzed both previously described and new H-2 hybrid genes. These genes were constructed by exchanging domains between H-2 genes. Quantitative radioimmunoassay indicates that the region of the H-2Dd molecule responsible for its enhanced expression resides in the polymorphic N domain, the first domain of the mature class I molecule.
Asunto(s)
Genes MHC Clase II , Antígenos H-2/genética , Animales , Clonación Molecular , Antígenos H-2/análisis , Antígeno de Histocompatibilidad H-2D , Células L/inmunología , Metalotioneína/genética , Ratones , Operón , ARN Mensajero/análisisRESUMEN
The transcription factors encoded by the E2A and early B cell factor (EBF) genes are required for the proper development of B lymphocytes. However, the absence of B lineage cells in E2A- and EBF-deficient mice has made it difficult to determine the function or relationship between these proteins. We report the identification of a novel model system in which the role of E2A and EBF in the regulation of multiple B lineage traits can be studied. We found that the conversion of 70Z/3 pre-B lymphocytes to cells with a macrophage-like phenotype is associated with the loss of E2A and EBF. Moreover, we show that ectopic expression of the E2A protein E12 in this macrophage line results in the induction of many B lineage genes, including EBF, IL7Ralpha, lambda5, and Rag-1, and the ability to induce kappa light chain in response to mitogen. Activation of EBF may be one of the critical functions of E12 in regulating the B lineage phenotype since expression of EBF alone leads to the activation of a subset of E12-inducible traits. Our data demonstrate that, in the context of this macrophage line, E12 induces expression of EBF and together these transcription factors coordinately regulate numerous B lineage-associated genes.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Proteínas Represoras , Transactivadores/genética , Factores de Transcripción/metabolismo , Animales , Antígenos CD/genética , Linfocitos B/efectos de los fármacos , Sitios de Unión , Línea Celular , Linaje de la Célula , Membrana Celular/metabolismo , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Cadenas Ligeras de Inmunoglobulina , Inmunoglobulina de Cadenas Ligeras Subrogadas , Cadenas kappa de Inmunoglobulina/metabolismo , Cadenas mu de Inmunoglobulina/metabolismo , Proteína 2 Inhibidora de la Diferenciación , Lipopolisacáridos/farmacología , Antígeno de Macrófago-1/metabolismo , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Mitógenos/farmacología , Proteínas Nucleares/genética , Factor de Transcripción PAX5 , Fenotipo , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptores de Interleucina/genética , Receptores de Interleucina-7 , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
During specific stages of thymocyte development, the T cell receptor (TCR) locus is assembled from variable (V), diversity (D), and joining (J) gene segments. Proper TCR gamma and delta V(D)J rearrangement during thymocyte development requires the presence of the E2A proteins. Here we show that E2A and a closely related protein, HEB, in the presence of recombination activating gene (RAG)1 and RAG2, each have the ability to activate TCR gamma and delta rearrangement in human kidney cells. The coding joints are diverse, contain nucleotide deletions, and occasionally show the presence of P nucleotides. Interestingly, only a subset of V, D, and J segments are targeted by the E2A and HEB proteins. Thus, E2A and HEB permit localized accessibility of the TCR gamma and delta loci to the recombination machinery. These data indicate that a distinct but diverse TCR repertoire can be induced in nonlymphoid cells by the mere presence of the V(D)J recombinase and the transcriptional regulators, E2A and HEB.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T , Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T , Secuencias Hélice-Asa-Hélice , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Proteínas de Unión al ADN/genética , Humanos , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The E2A gene encodes the E47 and E12 basic helix-loop-helix (bHLH) transcription factors. T cell development in E2A-deficient mice is partially arrested before lineage commitment. Here we demonstrate that E47 expression becomes uniformly high at the point at which thymocytes begin to commit towards the T cell lineage. E47 protein levels remain high until the double positive developmental stage, at which point they drop to relatively moderate levels, and are further downregulated upon transition to the single positive stage. However, stimuli that mimic pre-T cell receptor (TCR) signaling in committed T cell precursors inhibit E47 DNA-binding activity and induce the bHLH inhibitor Id3 through a mitogen-activated protein kinase kinase-dependent pathway. Consistent with these observations, a deficiency in E2A proteins completely abrogates the developmental block observed in mice with defects in TCR rearrangement. Thus E2A proteins are necessary for both initiating T cell differentiation and inhibiting development in the absence of pre-TCR expression. Mechanistically, these data link pre-TCR mediated signaling and E2A downstream target genes into a common pathway.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Linfocitos T/citología , Timo/citología , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Antígenos CD8/metabolismo , Diferenciación Celular , Linaje de la Célula , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Noqueados , Ratones SCID , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Factores de Transcripción TCF , Timo/metabolismo , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Accessibility of immunoglobulin (Ig) gene segments to V(D)J recombination is highly regulated and is normally only achieved in B cell precursors. We previously showed that ectopic expression of E2A or early B cell factor (EBF) with recombination activating gene (RAG) induces rearrangement of IgH and IgL genes in nonlymphoid cells. VkappaI genes throughout the locus were induced to rearrange after transfection with E2A, suggesting that the entire Vkappa locus was accessible. However, here we show that Ig loci are not opened globally but that recombination is localized. Gene families are interspersed in the D(H), Vkappa, and Vlambda loci, and we show that certain families and individual genes undergo high levels of recombination after ectopic expression of E2A or EBF, while other families within the same locus are not induced to rearrange. Furthermore, in some families, induction of germline transcription correlates with the level of induced recombination, while in others there is no correlation, suggesting that recombination is not simply initiated by induction of germline transcription. The induced repertoire seen at 24 hours does not change significantly over time indicating the absence of many secondary rearrangements and also suggesting a direct targeting mechanism. We propose that accessibility occurs in a local manner, and that binding sites for factors facilitating accessibility are therefore likely to be associated with individual gene segments.
Asunto(s)
Genes de Inmunoglobulinas , Genes RAG-1 , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Familia de Multigenes , Recombinación Genética , Factores de Transcripción/metabolismo , Animales , Linfocitos B/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , ADN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Reordenamiento Génico , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Transfección , VDJ RecombinasasRESUMEN
The E2A proteins, E12 and E47, are required for progression through multiple developmental pathways, including early B and T lymphopoiesis. Here, we provide in vitro and in vivo evidence demonstrating that E47 activity regulates double-positive thymocyte maturation. In the absence of E47 activity, positive selection of both major histocompatibility complex (MHC) class I- and class II-restricted T cell receptors (TCRs) is perturbed. Additionally, development of CD8 lineage T cells in an MHC class I-restricted TCR transgenic background is sensitive to the dosage of E47. Mice deficient for E47 display an increase in production of mature CD4 and CD8 lineage T cells. Furthermore, ectopic expression of an E2A inhibitor helix-loop-helix protein, Id3, promotes the in vitro differentiation of an immature T cell line. These results demonstrate that E2A functions as a regulator of thymocyte positive selection.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Activación de Linfocitos , Linfocitos T/inmunología , Factores de Transcripción , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Supervivencia Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Citometría de Flujo , Dosificación de Gen , Secuencias Hélice-Asa-Hélice , Linfoma de Células T/inmunología , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Transfección , Células Tumorales Cultivadas , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genética , Microglobulina beta-2/fisiologíaRESUMEN
A key feature of B and T lymphocyte development is the generation of antigen receptors through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products are essential for the proper coordinated temporal regulation of V(D)J rearrangements within the T cell receptor (TCR) gamma and delta loci. Specifically, we show that E2A is required during adult thymocyte development to inhibit rearrangements to the gamma and delta V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal Vgamma3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of Vgamma3 germline transcripts and increased levels of double-stranded DNA breaks at the recombination signal sequence bordering Vgamma3. Additionally, rearrangements to a number of Vgamma and Vdelta gene segments used predominantly during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular Vdelta gene segments is highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of the E2A proteins is rate limiting for the recombination reaction involving these Vdelta regions.
Asunto(s)
Reordenamiento Génico de Linfocito T/inmunología , Secuencias Hélice-Asa-Hélice/inmunología , Receptores de Antígenos de Linfocitos T/genética , Recombinación Genética/inmunología , Linfocitos T/inmunología , Timo/inmunología , Factores de Transcripción , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Citometría de Flujo , Regulación de la Expresión Génica/inmunología , Reordenamiento Génico de Linfocito T/genética , Secuencias Hélice-Asa-Hélice/genética , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunología , Recombinación Genética/genética , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Transcripción Genética/genéticaRESUMEN
A novel H-2 gene in which the first external (N) domain of the H-2Ld antigen was replaced with that of the H-2Dd antigen was constructed and introduced into L cells. A transformant expressing the products of the hybrid gene was studied for binding to monoclonal antibodies specific for H-2Ld and H-2Dd antigens. It was found that serological determinants are distributed both in the N (Dd) and Cl (Ld) domains. Determinants recognized by allospecific cytotoxic T lymphocytes (CTLs) and virus-specific CTLs also mapped to the N and Cl domains. Determinants recognized by vesicular stomatitis virus (VSV)-specific effect cells, however, were not present on the recombinant molecule. These results show that a recombinant gene of two H-2 antigens in which the first external domain has been reshuffled can express a functional H-2 antigen that can then be used to map serological and CTL determinants to specific domains.
Asunto(s)
Epítopos/genética , Genes MHC Clase II , Antígenos H-2/genética , Linfocitos T Citotóxicos/inmunología , Animales , Reacciones Antígeno-Anticuerpo , Precipitación Química , Epítopos/inmunología , Genes , Antígenos H-2/inmunología , Antígenos H-2/aislamiento & purificación , Virus de la Influenza A/inmunología , Células L/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Transformación Genética , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
The T-cell antigen receptor is a cell surface molecule vital in mediating the cellular immune response. The arrangement and rearrangement of the gene segments encoding the beta-chain polypeptide of the receptor are similar to those of immunoglobulin gene segments. The two constant region genes of the human T-cell antigen receptor are 8 kilobases apart with a cluster of joining segments located 5' of each constant region gene. Although most beta-chain gene rearrangements involve the variable, diversity, and joining segments, analysis of a beta-chain complementary DNA clone suggests the occasional occurrence of another type of rearrangement.
Asunto(s)
Receptores de Antígenos de Linfocitos T/genética , Secuencia de Bases , ADN/genética , Genes , Humanos , Sustancias Macromoleculares , Recombinación GenéticaRESUMEN
The human T-cell gamma chain genes have been characterized in an attempt to better understand their role in immune response. These immunoglobulin-like genes are encoded in the genome in variable, joining, and constant segments. The human gamma genes include at least six variable region genes, two joining segments, and two constant-region genes in germline DNA. Variable and joining segments recombine during the development of T cells to form rearranged genes. The diversity of human gamma genes produced by this recombinational mechanism is greater than that produced by the murine genome but is more limited than that of other immunoglobulin-like genes.
Asunto(s)
Genes MHC Clase II , Linfocitos T/fisiología , Animales , ADN/genética , Humanos , Cadenas J de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas gamma de Inmunoglobulina/genética , Ratones , Hibridación de Ácido NucleicoRESUMEN
The gene (E2A) that codes for proteins with the properties of immunoglobulin enhancer binding factors E12/E47 was mapped to chromosome region 19p13.2-p13.3, a site associated with nonrandom translocations in acute lymphoblastic leukemias. The majority of t(1;19)(q23;p13)-carrying leukemias and cell lines studied contained rearrangements of E2A as determined by DNA blot analyses. The rearrangements altered the E2A transcriptional unit, resulting in the synthesis of a transcript larger than the normal-sized E2A mRNAs in one of the cell lines with this translocation. These observations indicate that the gene for a transcription factor is located at the breakpoint of a consistently recurring chromosomal translocation in many acute leukemias and suggest a direct role for alteration of such factors in the pathogenesis of some malignancies.
Asunto(s)
Cromosomas Humanos Par 19 , Cromosomas Humanos Par 1 , Proteínas de Unión al ADN/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factores de Transcripción/genética , Translocación Genética/fisiología , Niño , Mapeo Cromosómico , Humanos , Células Tumorales CultivadasRESUMEN
The advent of gene targeting in the mouse has led to rapid advances in the identification of factors controlling gene expression that are essential for normal hematopoietic development. Recent work has also uncovered roles for some of these factors in leukemogenesis and in the global regulation of chromatin structure.
Asunto(s)
Hematopoyesis/fisiología , Factores de Transcripción/fisiología , Animales , Linaje de la Célula , Cromatina/fisiología , Células Asesinas Naturales/citología , Ratones , Linfocitos T/citologíaRESUMEN
The past year has provided insight into the mechanisms by which multipotent progenitors commit to differentiation through the B lymphocyte lineage. Mice lacking the Pax5 gene develop pro-B lymphocytes but these cells are not uniquely committed to the B lineage as they can give rise to all hematopoietic cell types if cultured under appropriate conditions. Regulators of lymphocyte proliferation and survival have also been identified that may allow lymphocytes to respond to information provided by the external environment.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Factores de Transcripción/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Linaje de la Célula , Proteínas de Unión al ADN/fisiología , Humanos , Factor de Unión 1 al Potenciador Linfoide , Proteínas Nucleares/fisiología , Factor de Transcripción PAX5 , Proteínas Proto-Oncogénicas/fisiología , Transactivadores/fisiologíaRESUMEN
A class of helix-loop-helix (HLH) proteins, including E2A (E12 and E47), E2-2, and HEB, that bind in vitro to DNA sequences present in the immunoglobulin (Ig) enhancers has recently been identified. E12, E47, E2-2, and HEB are each present in B cells. The presence of many different HLH proteins raises the question of which of the HLH proteins actually binds the Ig enhancer elements in B cells. Using monoclonal antibodies specific for both E2A and E2-2, we show that both E2-2 and E2A polypeptides are present in B-cell-specific Ig enhancer-binding complexes. E2-box-binding complexes in pre-B cells contain both E2-2 and E2A HLH subunits, whereas in mature B cells only E2A gene products are present. We show that the difference in E2-box-binding complexes in pre-B and mature B cells may be caused by differential expression of E2A and E2-2.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Factores de Transcripción , Especificidad de Anticuerpos , Northern Blotting , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Electroforesis , Humanos , Immunoblotting , Sustancias Macromoleculares , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Recent studies have identified a family of DNA-binding proteins that share a common DNA-binding and dimerization domain with the potential to form a helix-loop-helix (HLH) structure. Various HLH proteins can form heterodimers that bind to a common DNA sequence, termed the E2-box. We demonstrate here that E2-box-binding B-cell- and myocyte-specific nuclear factors contain subunits which are identical or closely related to ubiquitously expressed (E12/E47) HLH proteins. These biochemical function for E12/E47-like molecules in mammalian differentiation, similar to the genetically defined function of daughterless in Drosophila development.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Músculos/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Elementos de Facilitación Genéticos , Células HeLa/metabolismo , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Conformación Proteica , Transcripción GenéticaRESUMEN
We have used a binding site selection strategy to determine the optimal binding sites for Pbx proteins by themselves and as heterodimeric partners with various Hox gene products. Among the Pbx proteins by themselves, only Pbx3 binds with high affinity, as a monomer or as a homodimer, to an optimal binding site, TGATTGATTTGAT. An inhibitory domain located N terminal of the Pbx1 homeodomain prevents intrinsic Pbx1 binding to this sequence. When complexed with Hoxc-6, each of the Pbx gene products binds the same consensus sequence, TGATTTAT, which differs from the site bound by Pbx3 alone. Three members of the Antennapedia family, Hoxc-6, Hoxb-7, and Hoxb-8, select the same binding site in conjunction with Pbx1. The affinities of these proteins as heterodimeric partners with Pbx1 for the selected optimal binding site are similar. However, the binding specificity of Hox proteins for optimal binding sites is increased, compared to nonspecific DNA, in the presence of Pbx proteins. Thus, while cooperative DNA binding involving heterodimers of Pbx and Hox gene products derived from members within the Antennapedia family does not increase binding site selectivity, DNA binding specificity of the Hox gene products is significantly enhanced in the presence of Pbx.
Asunto(s)
ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción , Proteína con Homeodominio Antennapedia , Secuencia de Bases , Sitios de Unión , ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Proteínas de Homeodominio/genética , Humanos , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Proteínas Proto-Oncogénicas/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Two classes of homeodomain proteins, Hox and Pbx gene products, have the ability to bind cooperatively to DNA. In Hox proteins, the homeodomain and a highly conserved hexapeptide are required for cooperative DNA binding. In Pbx, the homeodomain and a region immediately C terminal of the homeodomain are essential for cooperativity. Using fluorescence and circular dichroism spectroscopy, we demonstrated that Hox and Pbx proteins interact in the absence of DNA. The interaction in solution is accompanied by conformational changes. Furthermore, upon interaction with specific DNA, additional conformational changes are induced in the Pbx-1/Hoxb-8 heterodimer. These data indicate that prior to DNA binding, Hox-Pbx interaction in solution is accompanied by structural alterations. We propose that these conformational changes modulate the DNA binding properties of these proteins, ultimately resulting in cooperative DNA binding.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Dimerización , Escherichia coli , Modelos Moleculares , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Conformación Proteica , Soluciones , Espectrometría de FluorescenciaRESUMEN
A conserved region, designated the AD1 domain, is present in a class of helix-loop-helix (HLH) proteins, E proteins, that includes E12, E47, HEB, E2-2, and a Xenopus laevis HLH protein closely related to E12. We demonstrate that the AD1 domain in E2A and the conserved region of E2-2 activate transcription in both yeast and mammalian cells. The AD1 domain contains a highly conserved putative helix that is crucial for its transactivation properties. Circular dichroism spectroscopy data show that AD1 is structured and contains distinctive helical properties. In addition, we show that a synthetic peptide corresponding to the conserved region is unstructured in aqueous solution at neutral pH but can adopt an alpha-helical conformation in the presence of the hydrophobic solvent trifluoroethanol. Amino acid substitutions that destabilize the helix abolish the transactivation ability of the AD1 domain. Both structural and functional analyses of AD1 reveal striking similarities to the acidic class of activators. Remarkably, when wild-type and mutant proteins are expressed in mammalian cells and Saccharomyces cerevisiae, identical patterns of transactivation are observed, suggesting that the target molecule is conserved between S. cerevisiae and mammals. These data show that transactivation by E proteins is mediated, in part, by a strikingly conserved peptide that has the ability to form a helix in a hydrophobic solvent. We propose that the unstructured domain may become helical upon interaction with its cellular target molecule.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Secuencia de Consenso , Secuencia Conservada , Cartilla de ADN/genética , Células HeLa , Secuencias Hélice-Asa-Hélice/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Xenopus laevisRESUMEN
Pancreatic beta-cell-type-specific transcription of the insulin gene is principally controlled by trans-acting factors which influence insulin control element (ICE)-mediated expression. The ICE activator is composed, in part, of the basic helix-loop-helix proteins E12, E47, and E2-5 encoded by the E2A gene. Previous experiments showed that ICE activation in beta cells was repressed in vivo by the c-jun proto-oncogene (E. Henderson and R. Stein, Mol. Cell. Biol. 14:655-662, 1994). Here we focus on the mechanism by which c-Jun inhibits ICE-mediated activation. c-Jun was shown to specifically repress the transactivation potential of the E2A proteins. Thus, we found that the activity of GAL4:E2A fusion constructs was inhibited by c-Jun. The transrepression capabilities of c-Jun were detected only in pancreatic islet cell lines that contained a functional ICE activator. Repression of GAL4:E2A was mediated by the basic leucine zipper regions of c-Jun, which are also the essential regions of this protein necessary for controlling ICE activator-stimulated expression in vivo. The specific target of c-Jun repression was the transactivation domain (located between amino acids 345 and 408 in E12 and E47) conserved in E12, E47, and E2-5. In contrast, the activation domain unique to the E12 and E47 proteins (located between amino acids 1 and 99) was unresponsive to c-Jun. Our results indicate that c-Jun inhibits insulin gene transcription in beta cells by reducing the transactivation potential of the E2A proteins present in the ICE activator complex.