RESUMEN
All metazoans use insulin to control energy metabolism, but they secrete it from different cells: neurons in the central nervous system in invertebrates and endocrine cells in the gut or pancreas in vertebrates. Despite their origins in different germ layers, all of these insulin-producing cells share common functional features and gene expression patterns. In this study, we tested the role in insulin-producing cells of the vertebrate homologues of Dachshund, a transcriptional regulator that marks the earliest committed progenitors of the neural insulin-producing cells in Drosophila. Both zebrafish and mice expressed a single dominant Dachshund homologue in the pancreatic endocrine lineage, and in both species loss of this homologue reduced the numbers of all islet cell types including the insulin-producing ß-cells. In mice, Dach1 gene deletion left the pancreatic progenitor cells unaltered, but blocked the perinatal burst of proliferation of differentiated ß-cells that normally generates most of the ß-cell mass. In ß-cells, Dach1 bound to the promoter of the cell cycle inhibitor p27Kip1, which constrains ß-cell proliferation. Taken together, these data demonstrate a conserved role for Dachshund homologues in the production of insulin-producing cells.
Asunto(s)
Islotes Pancreáticos/embriología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Embrión no Mamífero/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Ratones , Pez Cebra/embriología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
In various mammalian species, including humans, water restriction leads to an acute increase in urinary sodium excretion. This process, known as dehydration natriuresis, helps prevent further accentuation of hypernatremia and the accompanying rise in extracellular tonicity. Serum- and glucocorticoid-inducible kinase (Sgk1), which is expressed in the renal medulla, is regulated by extracellular tonicity. However, the mechanism of its regulation and the physiological role of hypertonicity-induced SGK1 gene expression remain unclear. Here, we identified a tonicity-responsive enhancer (TonE) upstream of the rat Sgk1 transcriptional start site. The transcription factor NFAT5 associated with TonE in a tonicity-dependent fashion in cultured rat renal medullary cells, and selective blockade of NFAT5 activity resulted in suppression of the osmotic induction of the Sgk1 promoter. In vivo, water restriction of rats or mice led to increased urine osmolality, increased Sgk1 expression, increased expression of the type A natriuretic peptide receptor (NPR-A), and dehydration natriuresis. In cultured rat renal medullary cells, siRNA-mediated Sgk1 knockdown blocked the osmotic induction of natriuretic peptide receptor 1 (Npr1) gene expression. Furthermore, Npr1-/- mice were resistant to dehydration natriuresis, which suggests that Sgk1-dependent activation of the NPR-A pathway may contribute to this response. Collectively, these findings define a specific mechanistic pathway for the osmotic regulation of Sgk1 gene expression and suggest that Sgk1 may play an important role in promoting the physiological response of the kidney to elevations in extracellular tonicity.
Asunto(s)
Deshidratación/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Natriuresis , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Deshidratación/genética , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces/genética , Soluciones Isotónicas , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Ratas , Receptores del Factor Natriurético Atrial/deficiencia , Receptores del Factor Natriurético Atrial/genética , Receptores del Factor Natriurético Atrial/metabolismoRESUMEN
Pancreatic islet cells and neurons share common functions and similar ontogenies, but originate in different germ layers. To determine whether ectoderm-derived cells contribute instructive signals to the developing endoderm-derived pancreas, we defined the chronology of migration and differentiation of neural crest cells in the pancreas, and tested their role in the development of the islets. The homeodomain transcription factor Phox2b marks the neural precursors from the neural crest that colonize the gut to form the enteric nervous system. In the embryonic mouse pancreas, we found Phox2b expressed briefly together with Sox10 along the epithelial-mesenchymal border at E12.5 in cells derived from the neural crest. Downregulation of Phox2b shortly thereafter was dependent upon Nkx2.2 expressed in the adjacent pancreatic epithelium. In Phox2b(-/-) embryos, neurons and glia did not develop in the pancreas, and Nkx2.2 expression was markedly upregulated in the epithelium. In addition, the number and replication rate of insulin-expressing beta-cells increased in the Phox2b(-/-) mice. We conclude that, during pancreatic development, Phox2b and Nkx2.2 form a non-cell-autonomous feedback loop that links the neural crest with the pancreatic epithelium, regulates the size of the beta-cell population, and thereby impacts insulin-secretory capacity and energy homeostasis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Homeodominio/metabolismo , Células Secretoras de Insulina/fisiología , Cresta Neural/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Inmunohistoquímica , Ratones , Ratones Noqueados , Modelos Biológicos , Cresta Neural/citología , Cresta Neural/embriología , Páncreas/citología , Páncreas/embriología , Páncreas/metabolismo , Factores de Transcripción SOXE , Transducción de Señal , Factores de Transcripción/genética , Proteínas de Pez Cebra , beta-Galactosidasa/metabolismoRESUMEN
The type II bare lymphocyte syndrome (BLS) or major histocompatibility complex class II (MHCII) deficiency is a severe combined immunodeficiency (SCID) that is characterized by the absence of constitutive and inducible expression of MHCII determinants on immune cells. Four complementation groups of BLS have been defined, and they result from mutations in DNA-bound activators and the coactivator for MHCII transcription. Recently, all complementation groups of BLS patients have been accounted for. Studies of the syndrome and specific mutations reveal important lessons for the genetics of the immune response.
Asunto(s)
Genes MHC Clase II , Inmunodeficiencia Combinada Grave/genética , Factores de Transcripción/genética , Repetición de Anquirina , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Mutación , Inmunodeficiencia Combinada Grave/inmunología , Factores de Transcripción/química , Transcripción GenéticaRESUMEN
MHC class II (MHCII) determinants play a crucial role in the immune response by presenting antigenic peptides to T cells. Their expression is controlled from compact promoters at the transcriptional level. Pre-assembled regulatory factor X (RFX) and nuclear factor Y (NFY) complexes form a platform on DNA. The class II transactivator (CIITA) can then be recruited through multiple protein-protein interactions. In this report, we defined domains of CIITA that are responsible for its interactions with these DNA-bound factors. Furthermore, using DNA-affinity precipitation, we demonstrated that although CIITA binds at least five activators, RFX5, RFXAP, RFXANK/B, NFYB and NFYC, its assembly on the promoter requires the addition of nuclear extracts. We conclude that not only does the platform bind DNA via multiple, spatially constrained nteractions, but that it can recruit only modified and/or complexed CIITA to MHCII promoters.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Genes MHC Clase II , Proteínas Nucleares , Transactivadores/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Factor de Unión a CCAAT/metabolismo , Células COS , Chlorocebus aethiops , Sustancias Macromoleculares , Modelos Genéticos , Estructura Terciaria de Proteína , Conejos , Factores de Transcripción del Factor Regulador X , Eliminación de Secuencia , Transactivadores/química , Transactivadores/fisiología , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Bare lymphocyte syndrome (BLS) is an autosomal recessive severe-combined immunodeficiency that can result from mutations in four different transcription factors that regulate the expression of major histocompatibility complex (MHC) class II genes. We have identified here the defective gene that is responsible for the phenotype of the putative fifth BLS complementation group. The mutation was found in the regulatory factor that binds X-box 5 (RFX5) and was mapped to one of the arginines in a DNA-binding surface of this protein. Its wild-type counterpart restored binding of the RFX complex to DNA, transcription of all MHC class II genes and the appearance of these determinants on the surface of BLS cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Antígenos HLA-D/metabolismo , Inmunodeficiencia Combinada Grave/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arginina/química , Sitios de Unión , Linfoma de Burkitt/patología , Línea Celular Transformada , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Genes MHC Clase II , Prueba de Complementación Genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Factores de Transcripción del Factor Regulador X , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Inmunodeficiencia Combinada Grave/clasificación , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Major histocompatibility complex class II (MHC-II) genes are regulated in a B-cell-specific and gamma interferon-inducible manner. Conserved upstream sequences (CUS) in their compact promoters bind nuclear factor Y (NFY) and regulatory factor X (RFX) complexes. These DNA-bound proteins form a platform that attracts the class II transactivator, which initiates and elongates MHC-II transcription. In this report, we analyzed the complex assembly of these DNA-bound proteins. First, we found that NFY can interact with RFX in cells. In particular, NFYA and NFYC bound RFXANK/B in vitro. Next, RFX5 formed dimers in vivo and in vitro. Within a leucine-rich stretch N-terminal to the DNA-binding domain in RFX5, the leucine at position 66 was found to be critical for this self-association. Mutant RFX5 proteins that could not form dimers also did not support the formation of higher-order DNA-protein complexes on CUS in vitro or MHC-II transcription in vivo. We conclude that the MHC-II transcriptional platform begins to assemble off CUS and then binds DNA via multiple, spatially constrained interactions. These findings offer one explanation of why in the Bare Lymphocyte Syndrome, which is a congenital severe combined immunodeficiency, MHC-II promoters are bare when any subunit of RFX is mutated or missing.