Title Page
Contents
List of Abbreviations 9
Abstract 11
Introduction 13
1. Structure and functions of T-bet 13
2. Transcriptional control of Th cell development. 14
3. Characterized post-transcriptional modifications of T-bet 15
4. In vivo physiological roles of T-bet 16
5. Regulation of T-bet activity 17
6. Specific aims of this study 18
Materials and Methods 24
1. Materials 24
2. Animals 24
3. Cell Culture 24
3-1. Isolation and activation of CD4+ T cells(이미지참조) 25
3-2. In vitro differentiation and retroviral transduction 26
4. ELISA 26
5. Intracellular Cytokine Staining 27
6. Reverse transcription and quantitative real-time PCR analysis 27
7. Reporter gene assay 29
8. Immunoprecipitation and Immunoblotting 29
9. DNA pull-down assay 30
10. Nuclear and Cysolic Fractionations 30
11. Immunofluorescence analysis 31
12. Statistical analysis 31
Results 32
1. T-bet protein level is decreased during Th1 cell development. 32
2. T-bet protein undergoes ubiquitin-proteasomal degradation. 36
3. Lys-313 mutant of T-bet is protected from protein degradation. 38
4. Lys-313 of T-bet is a potential ubiquitination site. 44
5. Lys-313 of T-box domain is essential for DNA binding and transcriptional activity of T-bet. 47
6. Mutation of K313 abolishes T-bet functions in CD4+ T cell development.(이미지참조) 52
6-1. T-betK313R mutant fails to produce IFN-γ.(이미지참조) 54
6-2. T-betK313R mutant is unable to suppress IL-2 & Th2 cytokine production.(이미지참조) 56
7. Lys-313 is crucial for suppression of IL-2 and Th2 cytokines through interaction with NFAT1. 58
7-1. T-betK313R is not associated with NFAT1.(이미지참조) 59
7-2. T-betK313R mutant does not suppress NFAT-induced gene promoter suppress.(이미지참조) 63
8. Threonine phosphorylation of T-bet is crucial for regulation through interaction with NFAT1. 65
8-1. T-betK313R mutant abolishes Threonine phosphorylation of T-bet.(이미지참조) 65
8-2. T302 is a threonine phosphorylation site of T-bet. 67
9. Thr-302 phosphorylation is required for suppression of NFAT activity. 69
9-1. T-betT302A fails to interact with NFAT1 but interacts with NFκB p65.(이미지참조) 69
9-2. NFAT-mediated gene promoter activity is suppressed by WT but not by T-betT302A.(이미지참조) 72
9-3. Thr-phosphorylation of T-bet is critical for inhibition of NFAT1-mediated IL-2 and Th2 cytokine production. 75
10. Threonine phosphorylation has no significant effect on IFN-γ production by T-bet. 78
10-1. T-betT302A produces comparable level of IFN-γ in T cells.(이미지참조) 78
10-2. T-betT302A mutant sustains DNA-binding activity.(이미지참조) 80
11. Threonine phosphorylation is not required for T-bet protein degradation. 82
12. Lys-313 of T-bet is also essential for the inhibition of NFAT1-mediated IL-17 expression. 84
Discussion 88
1. Putative modification of Lysine. 88
2. T-bet controls activity of lineage-specific transcription factors through physical interaction. 89
3. T-bet modification may be required for the modulation of Th17 & Treg development. 90
4. Novel post-translational modification of T-bet. 91
Concluding remark 93
References 95
Appendix (Publication) 107
국문초록 109
Figure 1. Structure of T-bet 19
Figure 2. Differentiation into effector CD4+ T cell lineages.(이미지참조) 20
Figure 3. Suppression Mechanism of T helper cells by T-bet. 21
Figure 4. T-bet is important for controlling asthma development. 22
Figure 5. The protein level of T-bet during Th cell differentiation 23
Figure 6. Ub-mediated proteasomal degradation of T-bet 34
Figure 7. Specifically proteasomal ubiquitination in T-box domain of T-bet. 37
Figure 8. Highly conserved lysine residues in the helical DNA-binding motifs. 39
Figure 9. Stabilized expression of T-betK313R.(이미지참조) 41
Figure 10. Increased nuclear and cytoplasmic expression of T-betK313R.(이미지참조) 43
Figure 11. Decreased ubiquitination of T-betK313R.(이미지참조) 45
Figure 12. No modification by either acetylation or sumoylation in T-bet. 46
Figure 13. Diminished IFN-γ activity by T-betK313R.(이미지참조) 48
Figure 14. Abolished the DNA binding of T-betK313R.(이미지참조) 50
Figure 15. Crystallography of T-bet bound to the T-box-binding element. 51
Figure 16. Retroviral transduction of T-bet into T-bet-deficient CD4+ T cells.(이미지참조) 53
Figure 17. Diminished T-bet functions by mutation of Lys-313. 55
Figure 18. Impaired IL-2 & Th2 suppression by T-betK313.(이미지참조) 57
Figure 19. Normal suppression of T-betK313R through physical interaction with GATA3 & RelA.(이미지참조) 60
Figure 20. Diminished physical association between T-betK313R and NFAT1.(이미지참조) 62
Figure 21. Abolished suppression of The T-betK313R in NFAT-induced IL-2 and Th2 promoter activities.(이미지참조) 64
Figure 22. Abolished threonine phosphorylation in T-betK313R.(이미지참조) 66
Figure 23. Identification of threonine phosphorylation site. 68
Figure 24. Abolished interaction with NFAT1 in T-betT302A.(이미지참조) 70
Figure 25. Normal interaction with NFκB in T-betT302A.(이미지참조) 71
Figure 26. The effect of T-betT302A on the ability of NFAT1-induced IL-2 and Th2 promoter activities.(이미지참조) 73
Figure 27. No inhibition of Thr-phosphorylated T-bet NFAT1-induced promoter activity in EL4 cells. 74
Figure 28. Inability of T-betT302A to suppress IL-5 cytokine production(이미지참조) 76
Figure 29. The T-betT302A abolished suppression of IL-2 and Th2 cytokines.(이미지참조) 77
Figure 30. Normal induction of IFN-γ by T-betT302A.(이미지참조) 79
Figure 31. Sustained DNA binding activity in T-betT302A.(이미지참조) 81
Figure 32. Highly expressed in nucleus and degradation in T-betT302A.(이미지참조) 83
Figure 33. Abolished the ability of T-bet to repress the production of IL-17 in T-betK313R.(이미지참조) 85
Figure 34. Diminished suppression of not only NFAT-induced IL-17 but also Foxp3 promoter activities by T-betK313R.(이미지참조) 87
Figure 35. Schematic illustration of regulatory mechanisms of T-bet Lys-313 in T cells. 94