RESUMEN
It has been proposed that differential kinetics of CD4/CD8 coreceptors regulate fate choice of selected thymocytes. Sustained signals by interaction between MHC class II and TCR/CD4 is required for commitment to the CD4 helper lineage. Although prematurely terminated MHC-TCR/CD4 interaction in transgenic mouse models results in lineage redirection, it is unclear whether CD4 expression is actively maintained by endogenous cis-elements to facilitate prolonged signaling under physiological conditions. In this article, we show that sustained CD4 expression in postselection thymocytes requires an intronic sequence containing an uncharacterized DNase I hypersensitivity (DHS) site located 3' to the silencer. Despite normal CD4 expression before selection, thymocytes lacking a 1.5-kb sequence in intron 1 including the 0.4-kb silencer and the DHS, but not the 0.4-kb silencer alone, failed to maintain CD4 expression upon positive selection and are redirected to the CD8 lineage after MHC class II-restricted selection. Furthermore, CpG dinucleotides adjacent to the DHS are hypermethylated in CD8(+) T cells. These results indicate that the 1.5-kb cis-element is required in postselection thymocytes for helper lineage commitment, presumably mediating the maintenance of CD4 expression, and suggest that inactivation of the cis-element by DNA methylation may contribute to epigenetic Cd4 silencing.
Asunto(s)
Antígenos CD4/inmunología , Regulación de la Expresión Génica/inmunología , Silenciador del Gen/inmunología , Intrones/inmunología , Elementos Silenciadores Transcripcionales/inmunología , Timocitos/inmunología , Animales , Antígenos CD4/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Metilación de ADN/inmunología , Regulación de la Expresión Génica/genética , Ratones , Ratones Noqueados , Timocitos/citologíaRESUMEN
Differentiation of MHC class II-selected thymocytes toward the CD4(+) helper lineage depends on function of the transcription factor ThPOK, whose expression is repressed in CD8(+) cytotoxic lineage cells by a transcriptional silencer activity within the distal regulatory element (DRE) in the Thpok gene. Interestingly, the DRE also functions as a transcriptional enhancer. However, how the DRE exerts such dual functionality remains obscure. In this study, we dissected the DRE and identified DNA sequences specifically responsible for enhancer activity, and designated this as the thymic enhancer. Removal of the thymic enhancer from the murine Thpok locus resulted in inefficient ThPOK induction, thereby inducing a redirection toward alternative CD8(+) cytotoxic lineage in a proportion of MHC class II-selected cells, even when they express monoclonal MHC class II-restricted transgenic TCR. Thus, regulation of contiguous but separable sequences with opposite function in the DRE plays an important role in precise coupling of TCR signaling with the selection process of two opposite lineages.
Asunto(s)
Elementos de Facilitación Genéticos/inmunología , Elementos Silenciadores Transcripcionales/inmunología , Factores de Transcripción/genética , Activación Transcripcional/inmunología , Animales , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Elementos de Facilitación Genéticos/genética , Genes Reporteros , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia de ADN , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Timo/citología , Timo/inmunología , Timo/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/deficienciaAsunto(s)
Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/inmunología , Proteínas de Neoplasias/inmunología , Proteínas Represoras/inmunología , Animales , Antígenos CD8/genética , Antígenos CD8/inmunología , Diferenciación Celular , Linaje de la Célula , Redes Reguladoras de Genes , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteínas Represoras/genética , Elementos Silenciadores Transcripcionales/inmunología , Factores de Transcripción/inmunología , Activación Transcripcional/inmunología , Dedos de Zinc/inmunologíaRESUMEN
The CD4 versus CD8 lineage specification of thymocytes is linked to coreceptor expression. The transcription factor MAZR has been identified as an important regulator of Cd8 expression. Here we show that variegated CD8 expression by loss of Cd8 enhancers was reverted in MAZR-deficient mice, which confirms that MAZR negatively regulates the Cd8 loci during the transition to the double-positive (DP) stage. Moreover, loss of MAZR led to partial redirection of major histocompatibility complex (MHC) class I-restricted thymocytes into CD4(+) helper-like T cells, which correlated with derepression of Th-POK, a central transcription factor for helper-lineage development. MAZR bound the silencer of the gene encoding Th-POK, which indicated direct regulation of this locus by MAZR. Thus, MAZR is part of the transcription factor network that regulates the CD8 lineage differentiation of DP thymocytes.
Asunto(s)
Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Linaje de la Célula , Proteínas de Neoplasias/metabolismo , Proteínas Represoras/metabolismo , Animales , Trasplante de Médula Ósea , Antígenos CD4/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/genética , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Transdiferenciación Celular/genética , Transdiferenciación Celular/inmunología , Células Cultivadas , Redes Reguladoras de Genes , Antígenos H-2/genética , Antígenos H-2/metabolismo , Linfopoyesis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Quimera por Radiación , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/inmunología , Elementos Silenciadores Transcripcionales/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Activación Transcripcional/inmunologíaRESUMEN
Activation-induced cytidine deaminase (AID) is essential for the generation of antibody memory but also targets oncogenes, among other genes. We investigated the transcriptional regulation of Aicda (which encodes AID) in class switch-inducible CH12F3-2 cells and found that Aicda regulation involved derepression by several layers of positive regulatory elements in addition to the 5' promoter region. The 5' upstream region contained functional motifs for the response to signaling by cytokines, the ligand for the costimulatory molecule CD40 or stimuli that activated the transcription factor NF-kappaB. The first intron contained functional binding elements for the ubiquitous silencers c-Myb and E2f and for the B cell-specific activator Pax5 and E-box-binding proteins. Our results show that Aicda is regulated by the balance between B cell-specific and stimulation-responsive elements and ubiquitous silencers.
Asunto(s)
Linfocitos B/inmunología , Citidina Desaminasa/genética , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/inmunología , Genes de Inmunoglobulinas/genética , Elementos Silenciadores Transcripcionales/genética , Animales , Citidina Desaminasa/inmunología , Elementos de Facilitación Genéticos/inmunología , Expresión Génica , Perfilación de la Expresión Génica , Genes de Inmunoglobulinas/inmunología , Humanos , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Ratones , Mutagénesis Sitio-Dirigida , Análisis de Secuencia por Matrices de Oligonucleótidos , Oncogenes/genética , Oncogenes/inmunología , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Elementos Silenciadores Transcripcionales/inmunología , Hipermutación Somática de Inmunoglobulina/genética , Hipermutación Somática de Inmunoglobulina/inmunologíaRESUMEN
CD4 and the transcription factor ThPOK are essential for the differentiation of major histocompatibility complex class II-restricted thymocytes into the helper T cell lineage; their genes (Cd4 and Zbtb7b (called 'ThPOK' here)) are repressed by transcriptional silencer elements in cytotoxic T cells. The molecular mechanisms regulating expression of these genes during helper T cell lineage differentiation remain unknown. Here we showed that inefficient upregulation of ThPOK, induced by removal of the proximal enhancer from the ThPOK locus, resulted in the transdifferentiation of helper lineage-specified cells into the cytotoxic T cell lineage. Furthermore, direct antagonism by ThPOK of the Cd4 and ThPOK silencers generated two regulatory loops that initially inhibited Cd4 downregulation and later stabilized ThPOK expression. Our results show how an initial lineage-specification signal can be amplified and stabilized during the lineage-commitment process.
Asunto(s)
Transdiferenciación Celular/inmunología , Regulación de la Expresión Génica/inmunología , Elementos Silenciadores Transcripcionales/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/citología , Factores de Transcripción/genética , Animales , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Citometría de Flujo , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Timo/citología , Timo/inmunología , TransfecciónRESUMEN
The transcription factor ThPOK is necessary and sufficient to trigger adoption of the CD4 lymphocyte fate. Here we investigate the regulation of ThPOK expression and its subsequent control of CD4+ T cell commitment. Treatment of immature thymocytes with anti-TCR (T cell receptor) showed that TCR signals were important in ThPOK induction and that the CD4+8lo stage was the likely target of the inductive TCR signal. We identified at the ThPOK locus a key distal regulatory element (DRE) that mediated its differential expression in class I- versus II-restricted CD4+8lo thymocytes. The DRE was both necessary for suppression of ThPOK expression in class I-restricted thymocytes and sufficient for its induction in class II-restricted thymocytes. Mutagenesis analysis defined an essential 80bp core DRE sequence and its potential regulatory motifs. We propose a silencer-dependent model of lineage choice, whereby inactivation of the DRE silencer by a strong TCR signal leads to CD4 commitment, whereas continued silencer activity leads to CD8 commitment.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Linaje de la Célula/genética , Regulación de la Expresión Génica , Elementos Silenciadores Transcripcionales/genética , Factores de Transcripción/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Citometría de Flujo , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Elementos Silenciadores Transcripcionales/inmunología , Timo/citología , Timo/inmunología , Factores de Transcripción/inmunologíaRESUMEN
The Bcl6 proto-oncogene, which encodes a transcriptional repressor, is ubiquitously expressed and predominantly in germinal center (GC) B cells. Although the promoter region of the human Bcl6 gene has been reported, enhancer molecules for its high expression in GC B cells were largely unknown. Here we show that transcriptional start sites of the murine Bcl6 gene were different from the reported human one. DNA sequence around the new promoter region is highly conserved between mice and humans and has no canonical TATA or CCAAT box. Two AP-1-binding elements in the promoter region were the major enhancer elements in GC-derived B lymphoma cells, and JunD/AP-1 was detected in GC B cells. In addition, we identified the silencer region with three Bcl6-binding elements around the start site. Bcl6 bound to the silencer elements and its over-expression repressed the promoter activity through the elements. Activated STAT factors (STATs), especially activated STAT3, also bound to the silencer elements in GC B cells and competed with Bcl6 for the binding, suggesting that JunD/AP-1 and activated STATs drive high Bcl6 expression in GC B cells. Since stimulation of splenic B cells with IL-4 or IL-21 induced high Bcl6 expression with induction of junD and activation of STATs, these cytokines may be inducers for its high expression in GC B cells. However, IL-21 but not IL-4 stimulation activated STAT3 in splenic B cells. Thus, IL-21 may be a major inducer for high Bcl6 expression in GC B cells.
Asunto(s)
Linfocitos B/inmunología , Proteínas de Unión al ADN/inmunología , Regulación de la Expresión Génica/inmunología , Genes jun/inmunología , Centro Germinal/inmunología , Factor de Transcripción AP-1/inmunología , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Centro Germinal/citología , Centro Germinal/metabolismo , Humanos , Interleucina-4/inmunología , Interleucina-4/farmacología , Interleucinas/inmunología , Interleucinas/farmacología , Ratones , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-bcl-6 , Factor de Transcripción STAT3/inmunología , Factor de Transcripción STAT3/metabolismo , Elementos Silenciadores Transcripcionales/inmunología , Especificidad de la Especie , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , TATA Box/inmunología , Factor de Transcripción AP-1/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunologíaRESUMEN
A characteristic of the secondary response of CD8(+) T cells that distinguishes it from the primary response is the generation of greater numbers of effector cells. Because effector CD8(+) T cells are derived from a pool of less differentiated, replicating cells in secondary lymphoid organs, and because IL-2 mediates effector differentiation, the enhanced secondary response may reflect the enlargement of this generative pool by the transient repression of IL-2-mediated differentiation. We have examined for this function the transcriptional repressor BCL6b, a homologue of BCL6 that represses IL-2-induced B cell differentiation. BCL6b is expressed in a small subset of antigen-experienced CD8(+) T cells. Ectopic expression of BCL6b in CD8(+) T cells diminishes their growth in response to IL-2 in vitro. Female mice in which the BCL6b gene has been interrupted have normal primary responses of CD8(+) T cells to infection with vaccinia expressing the H-Y epitope, Uty, but Uty-specific, BCL6b(-/-), memory CD8(+) T cells have diminished recall proliferative responses to this epitope in vitro. BCL6b(-/-) mice also have normal primary CD8(+) T cell responses to influenza infection, but nucleoprotein peptide-specific, BCL6b(-/-), memory CD8(+) T cells have a cell autonomous defect in the number of effector cells generated in response to reinfection. Therefore, BCL6b is required for the enhanced magnitude of the secondary response of memory CD8(+) T cells.