RESUMEN
Tumor necrosis factor (TNF) is one of the key primary response genes in the immune system that can be activated by a variety of stimuli. Previous analysis of chromatin accessibility to DNaseI demonstrated open chromatin conformation of the TNF proximal promoter in T cells. Here, using chromatin probing with restriction enzyme EcoNI and micrococcal nuclease we show that in contrast to the proximal promoter, the TNF transcription start site remains in a closed chromatin configuration in primary T helper (Th) cells, but acquires an open state after activation or polarization under Th1 and Th17 conditions. We further demonstrate that transcription factor c-Jun plays a pivotal role in the maintenance of open chromatin conformation at the transcription start site of the TNF gene.
Asunto(s)
Cromatina/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Células Th17/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Microambiente Celular , Ratones , Ratones Endogámicos C57BL , Nucleasa Microcócica/metabolismo , Regiones Promotoras Genéticas/genética , Conformación Proteica , Proteínas Proto-Oncogénicas c-jun/genética , Sitio de Iniciación de la Transcripción , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The Src-family tyrosine kinase Lck is an enzyme associated with the CD4 and CD8 co-receptors and promoting signaling through the T cell receptor (TCR) complex. The levels of Lck expression and activity change during the development and differentiation of T cells. Here we show that Lck expression is higher in Th1 cells as compared to Th2 cells. Ectopic overexpression of Lck in Th2 cells results in increased expression of CD4 co-receptor and enhanced S73 phosphorylation of transcription factor c-Jun. Our findings indicate that TCR-mediated signaling in Th2 cells may be directly attenuated by Lck protein expression level.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Células Th2/metabolismo , Animales , Células Cultivadas , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Ratones Endogámicos C57BL , Fosforilación , Serina/metabolismo , Transducción de Señal , Células TH1/metabolismoRESUMEN
This chapter provides protocols for in vitro and in vivo analysis of TNF-producing cells from a novel TNF reporter mouse. In these transgenic mice, genetic sequence encoding far-red reporter protein Katyushka (FRFPK) was placed under control of the same regulatory elements as TNF, thus providing the basis for detection, isolation, and visualization of TNF-producing cells.