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

本研究旨在阐明辅助性T细胞及其亚群中细胞因子基因表达的调控机制。(1) NFAT：在t细胞活化中的调控和作用。我们已经分离出人类和小鼠的NFAT基因，NFAT是一种转录因子，在细胞因子基因的转录诱导中起着关键作用。在对NFAT家族成员NFATx的图谱研究中，我们定义了一系列功能域；Rd结构域对特异性dna结合和与AP-1蛋白的结合至关重要，n端结构域通过核易位调节NFAT的激活，c端结构域用于反激活。在n端区域，一个控制亚细胞定位的关键片段被缩小到n端区域的60个基序，称为CRI。通过修饰NFATx分子（例如，通过删除CRI或修饰钙调磷酸酶相互作用结构域），我们产生了具有组成活性的显性负突变体…更多具有钙调磷酸酶独立亚细胞定位的NFATx，这应该是进一步了解NFATx介导的基因调控的有用工具。我们进一步分析了NFAT家族在NFATx主要表达的胸腺细胞中的表达和功能。我们发现NFAT家族mrna在胸腺T细胞分化过程中受到发育调控，并且NFATx蛋白在DP阶段选择性地参与钙调磷酸酶依赖的NFAT- dna结合活性，这表明NFAT也参与了胸腺T细胞生成所需的信号。(2) th2特异性细胞因子基因调控机制。我们之前已经描述了包括GM-CSF、IL-2、IL-3、IL-4和IL-5在内的细胞因子基因启动子的调控动机，从而发现了保守淋巴因子（CLE）。本研究分析了促进th亚群细胞因子特异性表达的因素。在对IL-5启动子的分析中，1.2 kb长的启动子区域仅在Th2克隆中定义IL-5基因的反激活。对该区域的广泛研究发现了NFIL-5CLEO和NFIL-5C，并表明GATA-3或相关转录因子通过il - 5c驱动的转激活参与th2特异性IL-5基因诱导。此外，在IL-4的情况下，Th2细胞中近端启动子的活性明显低于完整的IL-4基因位点。我们利用Th克隆和体外分化的Th1/Th2细胞进行了DNaseI超敏感位点（HSS）分析，发现位于IL-4 - IL-13基因间区的3个HSS中有2个是Th2特异性的，仅在分化后出现。这些结果为Th2细胞因子基因的差异表达提供了新的多种调控机制。少

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