指導教授：繆希椿臺灣大學：免疫學研究所曹孝瑋Tsao, Hsiao-WeiHsiao-WeiTsao2014-11-262018-07-092014-11-262018-07-092013http://ntur.lib.ntu.edu.tw//handle/246246/262037E26轉型特異性序列1(Ets-1)是ETS轉錄因子家族蛋白之原型，也是第一個被發現的ETS家族成員，其對於小鼠體內輔助型T細胞(Th cells)的介白素二(Interleukin-2, IL-2)表現非常重要，然而目前對於Ets-1如何促進介白素二的表現，其機制仍然不明。 我們的研究顯示，Ets-1不僅對小鼠的介白素二表現重要，它也是人類介白素二重要的調控因子。雖然Ets-1可負調控一已知的介白素二抑制者Blimp-1，但在Ets-1基因剔除的輔助型T細胞中再剔除Blimp-1並無法讓介白素二的生成恢復正常。 另一方面，Ets-1可以和NFAT蛋白產生交互作用，除了促進NFAT蛋白的功能之外，也是NFAT聚集並活化至介白素二之啟動子所必須。除此之外，在未活化的輔助型T細胞中，Ets-1同時分布於細胞核和細胞中，當受到鈣離子的訊號刺激之後，核中的Ets-1會快速的離開細胞核並和細胞質中的NFAT蛋白競爭與NRON 複合體的結合，讓NFAT蛋白離開NRON 複合體。這個Ets-1在細胞受到刺激後從細胞核進入細胞質的過程是發生在NFAT蛋白進入核之前，所以當細胞缺少Ets-1時，NFAT蛋白進入核的能力就會受到影響，但其被去磷酸化的能力則沒有變化。 因此，我們認為Ets-1調控介白素二的主要機制乃是透過控制NFAT蛋白的活性而達成的。這篇論文不只增進了我們對於介白素二轉錄機制的了解，另一方面更指出了Ets-1控制下游基因的特殊機制。Ets-1, the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 in murine and human T cells. Although IL-2 is a well characterized cytokine, how Ets-1 regulates the expression of IL-2 remains unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by murine CD8+ T cells and primary human T helper (Th) cells. We also observed an elevated Blimp-1 expression in Ets-1 KO T cells. Although Blimp-1 plays as a suppressor for IL-2, our data indicated that Blimp-1 is not responsible for the impaired IL-2 production in Ets-1 KO cells. The defects we observed in Ets-1-deficient cells cannot be rescued by Blimp-1 deficiency in Ets-1 KO cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) through multiple domains and is required for the recruitment of NFAT to the IL-2 promoter. In resting Th cells, Ets-1 is located both in nucleus and cytoplasm. When receiving calcium dependent signals from T cell receptors (TCR), nuclear Ets-1 starts to exit the nucleus and competes with NFAT proteins for binding to protein components of NRON (non-coding RNA repressor of NFAT) complex. NRON complex serves as a cytoplasmic trap for phosphorylated NFAT proteins in cytoplasm and this competition results in the release of NFAT from the complex. Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. We also found that the nuclear resident Ets-1 can interact with NFAT proteins to facilitate the transcription activity of NFAT in nucleus. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT. To the best of our knowledge, Ets-1 is the first transcription factor that is known to interact with NRON complex and facilitate nuclear entry of NFAT proteins.中文摘要 7 ABSTRACT 8 CHARPTER I. Introduction 10 1.E26 transformation-specific sequence (Ets) family genes 10 2.Protein structure of Ets-1 10 3.Biological roles of Ets-1 13 4.Ets-1 and autoimmune diseases 14 5.Role of IL-2 in immune system 16 6.Transcriptional regulation of IL-2 16 7.Role of Ets-1 in the regulation of IL-2 18 8.NFAT proteins and NRON complex 19 9.Summary 21 10.Specific aims 22 CHARPTER II. Materials and methods 23 1.Materials 23 1-1 Buffers 23 1-2 Antibodies 28 1-3 Primers 29 2.Experimental procedures 31 2-1 Mice 31 2-2 Purification and differentiation of Th cells 31 2-3 Preparation of cytoplasmic and nuclear extract of Th cells 32 2-4 Luciferase assay 32 2-5 Co-immunoprecipitation 32 2-6 Intracellular cytokine staining 33 2-7 Western blot analysis 34 2-8 Real-time PCR 34 2-9 Chromatin immunoprecipitation (ChIP) 35 2-10 siRNA 36 2-11 Size-exclusion chromatography (Gel-filtration assay) 36 2-12 Concentration of Lentiviral/Retroviral Supernatants by Precipitation Using polyethylene glycol (PEG) 37 2-13 Spin Infection 37 CHARPTER III. Regulation of IL-2 by Ets-1 38 Results 38 1.Kinetics of Ets-1 and IL-2 expression in T cells 38 2.Ets-1 is required for IL-2 production in mouse CD8+ T cells and human T cells 38 3.Overexpression of Ets-1 increases IL-2 production 40 4.Ets-1 can be recruited to the proximal promoter of IL-2 41 5.Ets-1 can transactivate IL-2 promoter 42 6.Reduced IL-2-luciferase activity in Ets-1 KO Th1 cells 45 7.Expression of multiple transcription factors in Ets-1 KO Th1 cells 45 8.Suppression of Blimp-1 by Ets-1 in T cells 46 9.Blimp-1 is not the major cause of IL-2 deficiency in Ets-1 KO T cells 47 10.Runx1 and Runx3 suppress Ets-1 induced IL-2 promoter activity 49 11.Functionally synergy between Ets-1 and NFAT in transactivating IL-2 50 12.Ets-1 interacts with Nfatc1 and Nfatc2 in Th1 cells 52 13.Multiple domains of Ets-1 interact with NFAT 52 14.ETS domain of Ets-1 is required for Ets-1/NFAT in transactivating IL-2 promoter 54 15.Recruitment of NFAT was reduced in the absence of Ets-1 in Th1 cells 55 16.Synergistic effect between Ets-1 and Nfat proteins when the EBS was abolished 56 17.Defect in nuclear entry of NFAT in Ets-1 KO Th1 cells 57 18.Calcium dependent signals drive nuclear exit of Ets-1 58 19.mRNA expression of NRON complex components in Ets-1 KO cells 60 20.Ets-1 interacts with NRON complex 63 21.Ets-1 is required for efficient nuclear entry of NFAT 65 Discussion 67 CHARPTER IV. Phenotypic characterization of CD8+ T cells in P054 mice 75 Results 75 1.Phenotypic characterization of CD8+ T cells in P054 mice 75 2.Control of IL-2, granzyme B and IFNgamma expression in CD8+ T cells by Runx3 77 3.Runx3 regulates the expression of Eomesodermin 78 4.Regulation of cytokine productions by Eomes 79 5.Ets-1 drives Eomes promoter 80 Discussion 82 REFERENCES 84 FIGURES AND FIGURE LEGENDS 974466284 bytesapplication/pdf論文公開時間：2014/02/25論文使用權限：同意有償授權(權利金給回饋學校)介白素二基因調控thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/262037/1/ntu-102-D95449004-1.pdf