Analyses of cytokine gene expression by helper T cell subsets : role of NFAT-mediated gene activation and subset-specific regulatory mechanism.

辅助 T 细胞亚群的细胞因子基因表达分析：NFAT 介导的基因激活的作用和亚群特异性调节机制。

基本信息

批准号：
08457103
负责人：
ARAI Ken-ichi
金额：
$ 5.18万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

The aim of this study is to elucidate the regulatory mechanism of cytokine gene expression in helper T cells and their subsets.(1) NFAT : regulation and function in T-cell activation. We have isolated human and murine genes of NFAT, a transcription factor that plays a critical role in transcriptional induction of cytokine genes. Throughout the mapping study on NFATx, a member of the NFAT family, we have defined a series of functional domains ; Rd domain critical for specific DNA-binding and association with AP-1 proteins, N-terminal domain for calcineurin-regulated activation of NFAT via nuclear translocation, and C-terminal domain for transactivation. in N-terminal domain, one of the critical segment for control of subcellular localization was narrowed down to 60-residue motif in N-terminal domain, as designated CRI.By modification of NFATx molecule (e.g., by deleting CRI or modifying calcineurin-interacting domains), we generated constitutively active of dominant negative mutants of … More NFATx with calcineurin-independent subcellular localization, that should be a useful tools for further understanding of NFAT-mediated gene regulation. We further analyzed expression and function of the NFAT family in thymocytes in which NFATx is predominantly expressed. We found that NFAT family mRNAs are developmentally regulated during the differentiation of T cells in the thymus, and that NFATx protein selectively contributes to the calcineurin-dependent NFAT-DNA binding activity in the DP stage, suggesting that NFAT is also involved in the signals required for generation of T cells in the thymus.(2) Mechanism of Th2-specific cytokine gene regulation. We have previously described the regulatory motives in the promoters of cytokine genes including GM-CSF, IL-2, IL-3, IL-4, and IL-5, leading to discovery of conserved lymphokine elements (CLE). In current study was analyzed factors that facilitate the Th-subset specific expression of cytokines. In the analyzes of IL-5 promoter, 1.2 Kb-long promoter region was shown to define transactivation of IL-5 gene only in Th2 clones. Extensive studies of this region lead to discovery of NFIL-5CLEO and NFIL-5C, and GATA-3 or related transcription factor was indicated to involved in Th2-specific IL-5 gene induction via IL-5C-driven transactivation. Furthermore, in case of IL-4, the proximal promoter gives considerably less activity than intact IL-4 gene locus in Th2 cells. We performed DNaseI hypersensitive site (HSS) analysis using Th clones and in vitro differentiated Th1/Th2 cells, and identified that two out of three HSS located in IL-4 - IL-13 intergenic region were Th2-specific and appeared only after differentiation. These results provide novel and multiple regulatory mechanism in differential expression of Th2 cytokine gees. Less

在T细胞激活中，助手T细胞及其子群中的实力元基因表达的目的功能结构域；与AP-1蛋白的特定DNA结合，N末端结构域的关键，用于钙调蛋白的核易位和C末端域的c-terminal域。通过修改NFATX分子（通过删除CRI或修饰钙调蛋白相互作用结构域），将N端结构域的缩小缩小到60个残留的基序。，这显示了一种有用的工具，可以进一步了解NFATX的NFATX。 DP的结合活性在胸腺中的T细胞中所需的信号中，我们已经描述了细胞因子基因GM-CSF的启动子中的报告动机在当前的研究中，保守的淋巴因子元素（CLE）是th-subset特异性的细胞因子的表达。 -5CLEO和NFIL-5C通过IL-5C驱动的反式激活。并确定位于IL -4-IL-13中的三个HS中的两个是Th2特异性的，只有n差分。