繆希椿臺灣大學：免疫學研究所曹孝瑋Tsao, Hsiao-WeiHsiao-WeiTsao2007-11-292018-07-092007-11-292018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/63315Ets-1 has been demonstrated to play some roles in the regulation of T lymphocytes, which including T cell development, differentiation and apoptosis. Recently, Ets-1 has been reported to be a modulator of T helper cell cytokine secreting profiles which include interleukin-2, interleukin-4, interleukin-10 and interferon-gamma. Although the regulation of Ets-1 has been extensively studied, the role of sumoylation on the function of Ets-1 remains elusive. Here, we demonstrated that abolishing the consensus SUMO conjugation motif in Ets-1 did not alter the transcription of IFN-gamma by Ets-1 and T-bet. Furthermore, we demonstrated that the transactivation ability of Ets-1 on the reporter carrying Ets-1-binding region had been no changed when we mutated the sumoylation sites. Moreover, wild-type and sumoylated Ets-1 have the similar transactivation ability in the presence of PIAS proteins, SUMO E3 ligase. These results imply that sumoylation may play no role on Ets-1’s transcriptional activities. However, since the sumoylation of Ets-1 is real, the functions of sumoylated Ets-1 need to be further discovered .Acknowledgement i 摘要 ii Abstract iii CHAPTER I Introduction 1 1.1 The role of T helper cells in immunity 1 1.1.1 T helper cell development and differentiation 1 1.2 An overview of Ets-1 transcription factor 2 1.2.1 The discovery of Ets-1 2 1.2.2 Protein structure of Ets-1 2 1.2.3 Biological roles of Ets-1 4 1.2.4 Regulation of Ets-1 5 1.3 An overview of sumoylation 5 1.3.1 SUMO: small ubiquitin-like modifier 5 1.3.2 The SUMO conjugation pathway 6 1.3.3 Biological functions of SUMO 7 1.4 Rationale to study the relationship between Ets-1 and sumoylation in T helper cells 7 CHAPTER II Material and Methods 9 2.1 Experimental procedures 9 2.1.1Constructions 9 2.1.2 Sumoylation sites prediction 11 2.1.3 Site-directed mutagenesis 12 2.1.4 Cell culture 13 2.1.5 HEKHEK293T cells transfection 13 2.1.6 Luciferase assay 13 2.1.7 Purification of GST-Ets1 fusion protein for the production of Ets-1 polyclonal antibodies 14 2.1.8 Co-immunoprecipitation 15 2.2 Experimental materials 16 2.2.1 Enzymes 16 2.2.2 Kits 17 2.2.3 Chemicals, reagents and materials 17 2.2.4 Instruments and softwares 19 2.2.5 Media, solutions and buffers 20 CHAPTER III Results 25 3.1 The functional synergy between T-bet and wild-type or sumoylation sites mutated Ets-1 is comparable 25 3.2 The role of PIAS proteins in IFNγ transactivation by T-bet 25 3.3 The Ets-1 minimal reporter luciferase assay system 26 3.4 The transactivation abilities of wild type or sumoylation sites mutated Ets-1 27 3.5 The transcriptional activities of wild type Ets-1 in the presence of PIAS proteins. 27 3.6 The transcriptional activities of Ets-1 mutants in the presence of PIAS family proteins. 28 CHAPTER IV Discussions 29 REFERENCES 33 FIGURES AND TABLES 36355543 bytesapplication/pdfen-US細胞激素輔助型T細胞Ets-1SUMOotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63315/1/ntu-95-R93449001-1.pdf