在CD4輔助T細胞中，轉錄因子 c-Maf 是一重要的調控因子。c-Maf 會週控介白素4 (IL-4) 在第二型輔助T細胞 (TH2 cells) 和介白素21 (IL-21) 在17型輔助T細胞 (TH17 cells) 以及濾泡輔助型T細胞 (TFH cells) 中的基因表現。介白素4是第二型輔助T細胞的特徵細胞激素，並對於第二型輔助T細胞之功能有重要的影響。而 c-Maf 則藉由對介白素4的影響對第二型輔助T細胞的分化有重要的功能。最近，c-Maf 被發現也會表現在新發現的17型輔助T細胞以及濾泡輔助型T細胞中並調控介白素21的表現。介白素21對發炎反應和B細胞的生發中心反應 (Germinal center reaction) 有重要的影響。為了驗證 c-Maf 鹼性區域 (basic region) 在功能上的角色，我們利用了幾個在鹼性區域中的突變。我們發現鹼性區域在 c-Maf 調控介白素4上有重要性但在調控介白素21上則沒有影響。這些突變影響了 c-Maf 結合 DNA 的能力，但卻增強了 c-Maf 蛋白質的穩定度。而造成在 IL-4 和 IL-21 上有不同影響的機制則需要進一步的研究。

論文口試委員審定書 i
Acknowledgment ii
摘要 iii
Abstract iv
Chapter Ⅰ Introduction 1
1. CD4+ Helper T Cells 1
2. The Maf Family 3
3. Overview of Transcription Factor c-Maf 4
4. Transcription Factor c-Maf And Its Function In Immune System 6
5. Overviews of Cytokine IL-4 and IL-21 7
6. Overview of c-Maf Basic Region and Mutations in Basic Region 8
Chapter Ⅱ Materials and Methods 10
1. Experimental Materials 10
1.1 Chemicals and Reagents 10
1.2 Enzymes And Antibodies 12
1.3 Kits 12
1.4 Instruments 13
1.5 Medium, Solutions and Buffers 13
2. Experimental Procedure 15
2.1 Plasmid construction and preparation 15
2.2 Cell Culture and Transfection 16
2.3 Luciferase Assay 17
2.4 Western Blotting Analysis 17
2.5 Nucleus and Cytoplasmic Fractionation 17
2.6 Electrophoretic Mobility Shift Assay (EMSA) 17
2.7 Confocal Micorscopy 18
2.8 Stability Assay 19
Chapter Ⅲ Results 20
1. c-Maf with mutation in basic region affects its transactivity on IL-4 promoter but not IL-21 promoter. 20
2. The DNA binding abilities of Mutation A, Mutation B and Mutation C are impaired. 21
3. The cellular localization of Mutation A, Mutation B and Mutation C c-Maf remains in the nucleus. 22
4. c-Maf Mutation A, Mutation B and Mutation C is more stable than wild-type. 23
5. The DNA binding domain of c-Maf is indispensable in IL-21 transactivation. 23
6. Batf does not cooperate with c-Maf to regulate IL-21 expression. 24
Chapter Ⅳ Discussion 26
1. The c-Maf basic region plays significant roles in DNA binding and MARE recognition of c-Maf Proteins. 26
2. c-Maf may cooperate with other protein to regulate IL-21 expression. 27
3. The c-Maf instability may be related to DNA binding and regulation of gene expression control. 29
4. The subcellular localization of Mutation A, Mutation B and Mutation C c-Maf need to be further examined. 29
Figures 31
Reference 45

1.Zhu, J., H. Yamane, and W.E. Paul. 2010. Differentiation of effector CD4 T cell populations (*). Annu Rev Immunol 28:445-489.
2.Ho, I.C., and L.H. Glimcher. 2002. Transcription: tantalizing times for T cells. Cell 109 Suppl:S109-120.
3.Romagnani, S. 1999. Th1/Th2 cells. Inflamm Bowel Dis 5:285-294.
4.Korn, T., E. Bettelli, M. Oukka, and V.K. Kuchroo. 2009. IL-17 and Th17 Cells. Annu Rev Immunol 27:485-517.
5.King, C. 2009. New insights into the differentiation and function of T follicular helper cells. Nat Rev Immunol 9:757-766.
6.Nurieva, R.I., Y. Chung, G.J. Martinez, X.O. Yang, S. Tanaka, T.D. Matskevitch, Y.H. Wang, and C. Dong. 2009. Bcl6 Mediates the Development of T Follicular Helper Cells. Science
7.Yu, D., M. Batten, C.R. Mackay, and C. King. 2009. Lineage specification and heterogeneity of T follicular helper cells. Curr Opin Immunol 21:619-625.
8.Nishizawa, M., K. Kataoka, N. Goto, K.T. Fujiwara, and S. Kawai. 1989. v-maf, a viral oncogene that encodes a "leucine zipper" motif. Proc Natl Acad Sci U S A 86:7711-7715.
9.Kataoka, K., M. Nishizawa, and S. Kawai. 1993. Structure-function analysis of the maf oncogene product, a member of the b-Zip protein family. J Virol 67:2133-2141.
10.Yang, Y., and A. Cvekl. 2007. Large Maf Transcription Factors: Cousins of AP-1 Proteins and Important Regulators of Cellular Differentiation. Einstein J Biol Med 23:2-11.
11.Fujiwara, K.T., K. Kataoka, and M. Nishizawa. 1993. Two new members of the maf oncogene family, mafK and mafF, encode nuclear b-Zip proteins lacking putative trans-activator domain. Oncogene 8:2371-2380.
12.Kotkow, K.J., and S.H. Orkin. 1995. Dependence of globin gene expression in mouse erythroleukemia cells on the NF-E2 heterodimer. Mol Cell Biol 15:4640-4647.
13.Blank, V., M.J. Kim, and N.C. Andrews. 1997. Human MafG is a functional partner for p45 NF-E2 in activating globin gene expression. Blood 89:3925-3935.
14.Motohashi, H., J.A. Shavit, K. Igarashi, M. Yamamoto, and J.D. Engel. 1997. The world according to Maf. Nucleic Acids Res 25:2953-2959.
15.Blank, V. 2008. Small Maf proteins in mammalian gene control: mere dimerization partners or dynamic transcriptional regulators? J Mol Biol 376:913-925.
16.Ho, I.C., M.R. Hodge, J.W. Rooney, and L.H. Glimcher. 1996. The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 85:973-983.
17.Zhang, C., T. Moriguchi, M. Kajihara, R. Esaki, A. Harada, H. Shimohata, H. Oishi, M. Hamada, N. Morito, K. Hasegawa, T. Kudo, J.D. Engel, M. Yamamoto, and S. Takahashi. 2005. MafA is a key regulator of glucose-stimulated insulin secretion. Mol Cell Biol 25:4969-4976.
18.Moriguchi, T., M. Hamada, N. Morito, T. Terunuma, K. Hasegawa, C. Zhang, T. Yokomizo, R. Esaki, E. Kuroda, K. Yoh, T. Kudo, M. Nagata, D.R. Greaves, J.D. Engel, M. Yamamoto, and S. Takahashi. 2006. MafB is essential for renal development and F4/80 expression in macrophages. Mol Cell Biol 26:5715-5727.
19.Mears, A.J., M. Kondo, P.K. Swain, Y. Takada, R.A. Bush, T.L. Saunders, P.A. Sieving, and A. Swaroop. 2001. Nrl is required for rod photoreceptor development. Nat Genet 29:447-452.
20.Kataoka, K. 2007. Multiple mechanisms and functions of maf transcription factors in the regulation of tissue-specific genes. J Biochem 141:775-781.
21.Grigoryan, G., A.W. Reinke, and A.E. Keating. 2009. Design of protein-interaction specificity gives selective bZIP-binding peptides. Nature 458:859-864.
22.Kerppola, T.K., and T. Curran. 1994. A conserved region adjacent to the basic domain is required for recognition of an extended DNA binding site by Maf/Nrl family proteins. Oncogene 9:3149-3158.
23.Kataoka, K., M. Noda, and M. Nishizawa. 1994. Maf nuclear oncoprotein recognizes sequences related to an AP-1 site and forms heterodimers with both Fos and Jun. Mol Cell Biol 14:700-712.
24.Nakamura, M., M. Hamada, K. Hasegawa, M. Kusakabe, H. Suzuki, D.R. Greaves, T. Moriguchi, T. Kudo, and S. Takahashi. 2009. c-Maf is essential for the F4/80 expression in macrophages in vivo. Gene 445:66-72.
25.Yoshida, T., T. Ohkumo, S. Ishibashi, and K. Yasuda. 2005. The 5''-AT-rich half-site of Maf recognition element: a functional target for bZIP transcription factor Maf. Nucleic Acids Res 33:3465-3478.
26.Kusunoki, H., H. Motohashi, F. Katsuoka, A. Morohashi, M. Yamamoto, and T. Tanaka. 2002. Solution structure of the DNA-binding domain of MafG. Nat Struct Biol 9:252-256.
27.Kerppola, T.K., and T. Curran. 1994. Maf and Nrl can bind to AP-1 sites and form heterodimers with Fos and Jun. Oncogene 9:675-684.
28.Ring, B.Z., S.P. Cordes, P.A. Overbeek, and G.S. Barsh. 2000. Regulation of mouse lens fiber cell development and differentiation by the Maf gene. Development 127:307-317.
29.Rajaram, N., and T.K. Kerppola. 2004. Synergistic transcription activation by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract. Mol Cell Biol 24:5694-5709.
30.Huang, W., N. Lu, H. Eberspaecher, and B. De Crombrugghe. 2002. A new long form of c-Maf cooperates with Sox9 to activate the type II collagen gene. J Biol Chem 277:50668-50675.
31.Kataoka, K., S. Shioda, K. Ando, K. Sakagami, H. Handa, and K. Yasuda. 2004. Differentially expressed Maf family transcription factors, c-Maf and MafA, activate glucagon and insulin gene expression in pancreatic islet alpha- and beta-cells. J Mol Endocrinol 32:9-20.
32.Cao, S., J. Liu, L. Song, and X. Ma. 2005. The protooncogene c-Maf is an essential transcription factor for IL-10 gene expression in macrophages. J Immunol 174:3484-3492.
33.Kim, J.I., I.C. Ho, M.J. Grusby, and L.H. Glimcher. 1999. The transcription factor c-Maf controls the production of interleukin-4 but not other Th2 cytokines. Immunity 10:745-751.
34.Hiramatsu, Y., A. Suto, D. Kashiwakuma, H. Kanari, S.I. Kagami, K. Ikeda, K. Hirose, N. Watanabe, M.J. Grusby, I. Iwamoto, and H. Nakajima. 2010. c-Maf activates the promoter and enhancer of the IL-21 gene, and TGF-{beta} inhibits c-Maf-induced IL-21 production in CD4+ T cells. J Leukoc Biol
35.Bauquet, A.T., H. Jin, A.M. Paterson, M. Mitsdoerffer, I.C. Ho, A.H. Sharpe, and V.K. Kuchroo. 2009. The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells. Nat Immunol 10:167-175.
36.Pot, C., H. Jin, A. Awasthi, S.M. Liu, C.Y. Lai, R. Madan, A.H. Sharpe, C.L. Karp, S.C. Miaw, I.C. Ho, and V.K. Kuchroo. 2009. Cutting edge: IL-27 induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells. J Immunol 183:797-801.
37.Kurata, H., H.J. Lee, A. O''Garra, and N. Arai. 1999. Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription factors in developing Th1 cells. Immunity 11:677-688.
38.Yang, Y., J. Ochando, A. Yopp, J.S. Bromberg, and Y. Ding. 2005. IL-6 plays a unique role in initiating c-Maf expression during early stage of CD4 T cell activation. J Immunol 174:2720-2729.
39.Parrish-Novak, J., S.R. Dillon, A. Nelson, A. Hammond, C. Sprecher, J.A. Gross, J. Johnston, K. Madden, W. Xu, J. West, S. Schrader, S. Burkhead, M. Heipel, C. Brandt, J.L. Kuijper, J. Kramer, D. Conklin, S.R. Presnell, J. Berry, F. Shiota, S. Bort, K. Hambly, S. Mudri, C. Clegg, M. Moore, F.J. Grant, C. Lofton-Day, T. Gilbert, F. Rayond, A. Ching, L. Yao, D. Smith, P. Webster, T. Whitmore, M. Maurer, K. Kaushansky, R.D. Holly, and D. Foster. 2000. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 408:57-63.
40.Sondergaard, H., and K. Skak. 2009. IL-21: roles in immunopathology and cancer therapy. Tissue Antigens 74:467-479.
41.Spolski, R., and W.J. Leonard. 2009. IL-21 and T follicular helper cells. Int Immunol 22:7-12.
42.Huber, M., A. Brustle, K. Reinhard, A. Guralnik, G. Walter, A. Mahiny, E. von Low, and M. Lohoff. 2008. IRF4 is essential for IL-21-mediated induction, amplification, and stabilization of the Th17 phenotype. Proc Natl Acad Sci U S A 105:20846-20851.
43.Korn, T., E. Bettelli, W. Gao, A. Awasthi, A. Jager, T.B. Strom, M. Oukka, and V.K. Kuchroo. 2007. IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells. Nature 448:484-487.
44.Vogelzang, A., H.M. McGuire, D. Yu, J. Sprent, C.R. Mackay, and C. King. 2008. A fundamental role for interleukin-21 in the generation of T follicular helper cells. Immunity 29:127-137.
45.Kurokawa, H., H. Motohashi, S. Sueno, M. Kimura, H. Takagawa, Y. Kanno, M. Yamamoto, and T. Tanaka. 2009. Structural basis of alternative DNA recognition by Maf transcription factors. Mol Cell Biol 29:6232-6244.
46.Hansen, L., H. Eiberg, and T. Rosenberg. 2007. Novel MAF mutation in a family with congenital cataract-microcornea syndrome. Mol Vis 13:2019-2022.
47.Vanita, V., D. Singh, P.N. Robinson, K. Sperling, and J.R. Singh. 2006. A novel mutation in the DNA-binding domain of MAF at 16q23.1 associated with autosomal dominant "cerulean cataract" in an Indian family. Am J Med Genet A 140:558-566.
48.Perveen, R., J. Favor, R.V. Jamieson, D.W. Ray, and G.C. Black. 2007. A heterozygous c-Maf transactivation domain mutation causes congenital cataract and enhances target gene activation. Hum Mol Genet 16:1030-1038.
49.Lyon, M.F., R.V. Jamieson, R. Perveen, P.H. Glenister, R. Griffiths, Y. Boyd, L.H. Glimcher, J. Favor, F.L. Munier, and G.C. Black. 2003. A dominant mutation within the DNA-binding domain of the bZIP transcription factor Maf causes murine cataract and results in selective alteration in DNA binding. Hum Mol Genet 12:585-594.
50.Betz, B.C., K.L. Jordan-Williams, C. Wang, S.G. Kang, J. Liao, M.R. Logan, C.H. Kim, and E.J. Taparowsky. 2010. Batf coordinates multiple aspects of B and T cell function required for normal antibody responses. J Exp Med 207:933-942.
51.Schraml, B.U., K. Hildner, W. Ise, W.L. Lee, W.A. Smith, B. Solomon, G. Sahota, J. Sim, R. Mukasa, S. Cemerski, R.D. Hatton, G.D. Stormo, C.T. Weaver, J.H. Russell, T.L. Murphy, and K.M. Murphy. 2009. The AP-1 transcription factor Batf controls T(H)17 differentiation. Nature 460:405-409.
52.Xu, J., Y. Yang, G. Qiu, G. Lal, Z. Wu, D.E. Levy, J.C. Ochando, J.S. Bromberg, and Y. Ding. 2009. c-Maf regulates IL-10 expression during Th17 polarization. J Immunol 182:6226-6236.
53.Lin, B.S., P.Y. Tsai, W.Y. Hsieh, H.W. Tsao, M.W. Liu, R. Grenningloh, L.F. Wang, I.C. Ho, and S.C. Miaw. 2010. SUMOylation attenuates c-Maf-dependent IL-4 expression. Eur J Immunol 40:1174-1184.
54.Leavenworth, J.W., X. Ma, Y.Y. Mo, and M.E. Pauza. 2009. SUMO conjugation contributes to immune deviation in nonobese diabetic mice by suppressing c-Maf transactivation of IL-4. J Immunol 183:1110-1119.