RESUMEN
A TCRß enhancer, known as the Eß enhancer, plays a critical role in V(D)J recombination and transcription of the Tcrb gene. However, the coordinated action of trans-acting factors in the activation of Eß during T cell development remains uncharacterized. Here, we characterized the roles of Runx complexes in the regulation of the Eß function. A single mutation at one of the two Runx binding motifs within the Eß severely impaired Tcrb activation at the initiation phase in immature thymocytes. However, TCRß expression level in mature thymocytes that developed under such a single Runx site mutation was similar to that of the control. In contrast, mutations at two Runx motifs eliminated Eß activity, demonstrating that Runx complex binding is essential to initiate Eß activation. In cells expressing Tcrb harboring rearranged V(D)J structure, Runx complexes are dispensable to maintain TCRß expression, whereas Eß itself is continuously required for TCRß expression. These findings imply that Runx complexes are essential for Eß activation at the initiation phase, but are not necessary for maintaining Eß activity at later developmental stages. Collectively, our results indicate that the requirements of trans-acting factor for Eß activity are differentially regulated, depending on the developmental stage and cellular activation status.
Asunto(s)
Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Animales , Eliminación de Gen , Células Germinativas/metabolismo , Cinética , Activación de Linfocitos/genética , Ratones , Mutación/genética , Unión Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Recombinación Genética , Transcripción GenéticaRESUMEN
UNLABELLED: Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2(kd/kd) ) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal cross-linker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na(+) /H(+) exchanger regulatory factor were disturbed in bile ducts of Vil2(kd/kd) mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl(-) efflux activity. Furthermore, bile flow and biliary HCO3 (-) concentration were also significantly reduced in Vil2(kd/kd) mice. CONCLUSION: Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2(kd/kd) mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies.
Asunto(s)
Conductos Biliares/patología , Bilis , Colestasis Intrahepática/genética , Colestasis Intrahepática/patología , Proteínas del Citoesqueleto/genética , Animales , Epitelio/patología , Fibrosis , Ratones , Ratones NoqueadosRESUMEN
Mouse CD4+CD8+ double-positive (DP) thymocytes differentiate into CD4+ helper-lineage cells upon expression of the transcription factor Th-POK but commit to the CD8+ cytotoxic lineage in its absence. We report the redirected differentiation of class I-restricted thymocytes into CD4+CD8- helper-like T cells upon loss of Runx transcription factor complexes. A Runx-binding sequence within the Th-POK locus acts as a transcriptional silencer that is essential for Th-POK repression and for development of CD8+ T cells. Thus, Th-POK expression and genetic programming for T helper cell development are actively inhibited by Runx-dependent silencer activity, allowing for cytotoxic T cell differentiation. Identification of the transcription factors network in CD4 and CD8 lineage choice provides insight into how distinct T cell subsets are developed for regulating the adaptive immune system.
Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/fisiología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/fisiología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Linaje de la Célula , Inmunoprecipitación de Cromatina , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Elementos Silenciadores Transcripcionales , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Factores de Transcripción/genéticaRESUMEN
Interferon gamma (IFN gamma) is the hallmark cytokine produced by T helper type 1 (Th1) cells, whereas interleukin (IL)-4 is the hallmark cytokine produced by Th2 cells. Although previous studies have revealed the roles of cytokine signaling and of transcription factors during differentiation of Th1 or Th2 cells, it is unclear how the exclusive expression pattern of each hallmark cytokine is established. The DNaseI hypersensitivity site IV within the mouse Il4 locus plays an important role in the repression of Il4 expression in Th1 cells, and it has been named the Il4 silencer. Using Cbf beta- or Runx3-deficient T cells, we show that loss of Runx complex function results in derepression of IL-4 in Th1 cells. Binding of Runx complexes to the Il4 silencer was detected in naive CD4(+) T cells and Th1 cells, but not in Th2 cells. Furthermore, enforced expression of GATA-3 in Th1 cells inhibited binding of Runx complexes to the Il4 silencer. Interestingly, T cell-specific inactivation of the Cbf beta gene in mice led to elevated serum immunoglobulin E and airway infiltration. These results demonstrate critical roles of Runx complexes in regulating immune responses, at least in part, through the repression of the Il4 gene.