探討在輔助型T細胞中酪胺酸磷酸化之c-Maf對於介白素四與二十一啟動子的結合機轉

Abstract

Maf家族的蛋白質，參與了細胞當中各種不同的生物反應與機制，包括水晶體的發育，免疫細胞的分化以及腫瘤的形成。c-Maf是large Maf家族成員的一員，在免疫系統中扮演著多樣且十分重要的角色。c-Maf被發現可以在第二型輔助性T細胞中轉錄活化介白素四的表現，也可在第十七型輔助性T細胞中轉錄活化介白素二十一的表現。在我們之前的研究當中發現，c-Maf在第21,92,131號酪胺酸的位置可以被磷酸化。而且在這三個位置的磷酸化有助於c-Maf結合到介白素四的啟動子，也可促進第二型輔助性T細胞分泌介白素四。在本篇當中，為了進一步了解c-Maf 在第十七型輔助性T細胞中，對介白素二十一的調控。我們首先確認了c-Maf可以在第十七型輔助性T細胞中被磷酸化。接著我們將野生型c-Maf(WT c-Maf)和磷酸化位置被突變的c-Maf(Y3F c-MAf)過度表現在第十七型輔助性T細胞。發現介白素二十一的表現在Y3F c-Maf當中有下降的趨勢。我們也同時使用染色質免疫沉澱分析，發現相較於WT c-Maf，Y3F c-Maf結合到介白素二十一的啟動子的能力較差。總結來說，我們發現c-Maf的磷酸化會影響c-Maf結合到啟動子的能力，進而也影響到對介白素轉錄活化的能力。

Maf (Musculoaponeurotic fibrosarcoma) proteins are involved in a variety of biological processes such as oncogenesis, lens development and differentiation. c-Maf, one of the large Maf proteins, plays important roles in immune system. c-Maf is the specific transcription factor of the IL-4 and IL-21 genes in type 2 helper T cell (Th2) and type 17 helper T cell (Th17), respectively. In our previous study, we found that c-Maf undergoes phosphorylation, a post-translational modification, in Th2 cells. The Tyrosine 21, 92, and 131 residues are identified as sites for tyrosine phosphorylation in c-Maf. We previously reported that the tyrosine-phosphorylation of c-Maf enhances its transactivity on IL-4 gene expression in Th2 cells. To understand the regulation of IL-21 cytokine gene expression by tyrosine-phosphorylation of c-Maf in Th17 cells, we showed c-Maf is subjected to tyrosine-phosphorylation in Th17 cells. We introduced WT c-Maf or c-Maf mutant carrying deficiency in tyrosine-phosphorylation (Y3F) into primary murine Th17 cells via retrovirus transduction and measured the gene expression of IL-21 with quantitive PCR and ELISA assay. We demonstrated that IL-21 production is reduced by c-Maf Y3F. Importantly, We performed chromatin immunoprecipitation to analyzed the binding ability of WT c-Maf or c-Maf Y3F to IL-21 promoter and found that c-Maf Y3F binds relatively weakly to IL-21 promoter compared to WT c-Maf.