SATO Noriko Inst.of Medical Science, The University of Tokyo, Assistant Professor, 医科学研究所, 助手 (70280956)
WATANABE Sumiko Inst.of Medical Science, The University of Tokyo, Assistant Professor, 医科学研究所, 助手 (60240735)
MASAI Hisao Inst.of Medical Science, The University of Tokyo, Associate Professor, 医科学研究所, 助教授 (40229349)
YOKOTA Takashi Inst.of Medical Science, The University of Tokyo, Professor, 医科学研究所, 客員教授 (50134622)
|Budget Amount *help
¥8,100,000 (Direct Cost : ¥8,100,000)
Fiscal Year 1998 : ¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1997 : ¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1996 : ¥2,500,000 (Direct Cost : ¥2,500,000)
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
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.
Th2特異的に発現しその機能において重要なサイトカインであるIL-4,IL-13は、染色体上で近接して発現する。またIL-4は近位プロモーター単独の機能では十分な発現が保証されず、より高位の制御機構が示唆された。我々はT細胞クローン、およびTCRトランスジェニックマウス由来のナイーブT細胞を用いたTh1/Th2/のin vitro分化系を用いた解析で、IL-4とIL-13各遺伝子座の間にTh2細胞特異的なDNasel hypersensitive siteを同定し、この出現がTh2への分化に従い新たに誘導されることを示した。また、IL-5プローターの解析において、GATA-3がth2特異的なIL-5遺伝子の発現に重要であることを見い出した。 Less