RESUMEN
Models of mammalian regulatory networks controlling gene expression have been inferred from genomic data but have largely not been validated. We present an unbiased strategy to systematically perturb candidate regulators and monitor cellular transcriptional responses. We applied this approach to derive regulatory networks that control the transcriptional response of mouse primary dendritic cells to pathogens. Our approach revealed the regulatory functions of 125 transcription factors, chromatin modifiers, and RNA binding proteins, which enabled the construction of a network model consisting of 24 core regulators and 76 fine-tuners that help to explain how pathogen-sensing pathways achieve specificity. This study establishes a broadly applicable, comprehensive, and unbiased approach to reveal the wiring and functions of a regulatory network controlling a major transcriptional response in primary mammalian cells.
Asunto(s)
Bacterias/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Inflamación/metabolismo , Virus/inmunología , Animales , Ensamble y Desensamble de Cromatina , ADN de Cadena Simple/inmunología , Retroalimentación Fisiológica , Perfilación de la Expresión Génica , Inflamación/inmunología , Lipopéptidos/inmunología , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos C57BL , Poli I-C/inmunología , Proteínas de Unión al ARN/metabolismo , Receptores Toll-Like/agonistas , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Foxp3 has been shown to be both necessary and sufficient for the development and function of naturally arising CD4+ CD25+ regulatory T cells in mice. Mutation of Foxp3 in Scurfy mice and FOXP3 in humans with IPEX results in fatal, early onset autoimmune disease and demonstrates the critical role of FOXP3 in maintaining immune homeostasis. The FOXP3 protein encodes several functional domains, including a C2H2 zinc finger, a leucine zipper, and a winged-helix/forkhead (FKH) domain. We have shown previously that FOXP3 functions as a transcriptional repressor and inhibits activation-induced IL-2 gene transcription. To characterize the role of each predicted functional domain on the in vivo activity of FOXP3, we have evaluated the location of point mutations identified in a large cohort of patients with the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) and found them to cluster primarily within the FKH domain and the leucine zipper, but also present within the poorly defined N-terminal portion of the protein. The molecular functions of each of the IPEX-targeted domains were investigated. We show that FOXP3 is constitutively localized to the nucleus and this localization requires sequences at both the amino and C-terminal ends of its FKH domain. Moreover, FOXP3 was found to homodimerize through its leucine zipper. We also identify a novel functional domain within the N-terminal half of FOXP3, which is required for FOXP3-mediated repression of transcription from both a constitutively active and a NF-AT-inducible promoter. Furthermore, we demonstrate that IPEX mutations in these domains correlate with deficiencies in FOXP3 repressor function, corroborating their in vivo relevance.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Transcripción Genética/genética , Transporte Activo de Núcleo Celular , Línea Celular , Dimerización , Factores de Transcripción Forkhead/genética , Humanos , Leucina Zippers , Mutación/genética , Poliendocrinopatías Autoinmunes/genética , Poliendocrinopatías Autoinmunes/metabolismo , Regiones Promotoras Genéticas/genética , Activación TranscripcionalRESUMEN
During development in the thymus, mature CD4+ or CD8+ cells are derived from immature CD4+CD8+ cells through a series of selection events. One of the hallmarks of this maturation process is the expression of CD69, which first appears on thymocytes as they begin positive selection. We have used blockade and overexpression of CD69 to determine the role of CD69 in thymocyte development. Blockade of CD69 led to a reduction in single-positive cells and a concomitant increase in double-positive cells in the thymus. Overexpression of a CD69 transgene in the thymus resulted in a dramatic increase in both CD8SP and CD4SP cells. Coexpression with a TCR transgene demonstrated that both positive and negative selection were enhanced by the increased levels of CD69 on thymocytes. Finally, mice overexpressing CD69 displayed a sharp reduction in the number of T cells in the spleen and lymph node. Taken as a whole, these data suggest the involvement of CD69 in the process of selection and maturation during the trafficking of thymocytes to the medulla.
Asunto(s)
Antígenos CD/metabolismo , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígenos de Diferenciación de Linfocitos T/fisiología , Subgrupos de Linfocitos T/inmunología , Timo/inmunología , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales/farmacología , Antígenos CD/genética , Antígenos de Diferenciación de Linfocitos T/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Linaje de la Célula , Citometría de Flujo , Inmunofenotipificación , Lectinas Tipo C , Ganglios Linfáticos/inmunología , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Eliminación de Secuencia , Bazo/inmunología , Subgrupos de Linfocitos T/clasificación , Timo/crecimiento & desarrolloRESUMEN
We observed that the human CD40 ligand (CD40L) gene 5'-flanking region conferred weak promoter activity in activated CD4 T cells, suggesting that additional regions are required for optimal CD40L gene transcription. We therefore examined a 3'-flanking segment of the CD40L gene, which contained a putative NF-kappaB/Rel cis-element, for its ability to enhance CD40L promoter function. This segment augmented CD40L promoter activity in an orientation-independent manner in CD4 T-lineage cells but not in human B cell or monocyte cell lines. Mapping of CD4 T-lineage cell nuclei identified a DNase I-hypersensitive site in the flanking region near the NF-kappaB/Rel sequence, suggesting a transcriptional regulatory role. This was further supported by truncation analysis and site-directed mutagenesis, which indicated that the CD40L 3'-flanking NF-kappaB/Rel cis-element was critical for enhancer function. Electrophoretic mobility shift assays showed that the cis-element preferentially bound the p50 form of the NF-kappaB1 gene contained in human T cell nuclear protein extracts. This binding also appeared to occur in vivo in CD4 T cells based on chromatin immunoprecipitation assays using NF-kappaB p50-specific antiserum. Together, these results suggest that the CD40L gene 3'-flanking region acts as a T cell-specific classical transcriptional enhancer by a NF-kappaB p50-dependent mechanism.