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研究生:楊楚光
研究生(外文):Chu-Kuang Yang
論文名稱:Tet1和5-hydroxymethylcytosine在淋巴生發中心的形成和功能的角色
論文名稱(外文):The role of Tet1 and 5-hydroxymethylcytosine in the formation and function of germinal centers
指導教授:鄭永銘鄭永銘引用關係
指導教授(外文):Yung-Ming Jeng
口試委員:葉秀慧張以承劉兆蓮
口試委員(外文):Shiou-Hwei YehYi-Cheng ChangChao-Lien Liu
口試日期:2015-06-30
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:病理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:60
中文關鍵詞:淋巴生發中心TET1Bcl2Kaiso
外文關鍵詞:germinal centerTET1Bcl2Kaiso
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B細胞活化和淋巴生發中心的形成對於適應型免疫系統以及產生高專一性的抗體是非常重要的。這些正常生理過程受到許多轉錄因子的調控。 Ten eleven translocation (TET) 蛋白是一群染色質重塑因子,主要參與在DNA去甲基化的過程中。在本篇研究中,我們觀察到5-hydroxymethylcytosine在淋巴生發中心有明顯減少的情形,而且我們也經實驗發現TET1的表現量最受到抑制。在SKW6.4細胞株的實驗中,我們也發現在IL6所誘導的漿細胞分化過程中,有Tet1再表現的現象,且過度表現TET1能誘導此細胞株分化為漿細胞。利用即時聚合酶鍊鎖反應,我們發現Bcl2和Kaiso是TET1可能的下游目標基因。我們進一步利用染色質免疫沉澱實驗和亞硫酸置換結合定序的實驗中,驗證這些候選目標基因在活化過程中,其促進子甲基化狀態的改變。我們也製作轉殖基因小鼠來探討TET1在淋巴生發中心形成過程所扮演的角色。我們的結果顯示TET1的負調控對於B細胞經過生發中心的過程是重要的,而且可能藉此機制調控Bcl2和Kaiso的表現量。

B cell activation and germinal center (GC) formation are essential for the adaptive immune system and the generation of high-affinity protective antibodies against pathogens. These important biological processes are strictly regulated by multiple transcription factors. The ten eleven translocation (TET) family of proteins served as chromatin remodeling factors and involved in DNA demethylation process. In this study, we observed loss of 5-hydroxymethylcytosine in germinal centers. Of the three TET proteins, we found that TET1 is the most significantly downregulated TET protein during B cell activation. Using SKW6.4 cell line, we found that Tet1 re-expressed in IL6-induced plasma cell differentiation model. Overexpression of TET1 induced plasma cell differentiation. Using real-time PCR analysis, we found that Bcl2 and Kaiso are the candidate downstream targets of TET1. Chromatin immunoprecipitation and bisulfite PCR combined sequencing confirmed change of the methylation status of promoters in these candidate genes during B cell activation. We also generated a transgenic mice model to study the roles of TET1 in GC formation. Our study suggests that downregulation of TET1 is critical for GC transition and the regulation of Bcl2 and Kaiso expressions is probably the mechanism.

口試委員會審定書………………………………………………………...I
謝辭………………………………………………………………………..II
中文摘要………………………………………………………………….III
Abstract…………………..……………………………………………….IV
Content……………………………………………………………….……V

1. Introduction 1
1.1 Immune systems……………………………………………………….……….........1
1.2 B cell development………………………………………………………………….1
1.3 GC responses and B cell transcription factors……………………………………….2
1.4 B cell development and epigenetic regulation……………………………………….3
1.5 TET protein family and TET1…………………………………………………….…4
1.6 Aims of this study…………………………………………………………...........….5
2. Materials and Methods 6
2.1 Materials………………………………………………………………..…………6
2.2 Cell culture…………………………………………………………….……………6
2.3 Plasmids and electroporation…………………………………………………...……6
2.4 Murine splenocytes isolation and activation………………………………..........….7
2.5 B-cell fractionation………………………………………………………...………..8
2.6 RNA isolation……………………………………………………………...………..8
2.7 Real-time RT-PCR…………………………………………………………..………9
2.8 Genomic DNA isolation and dot blot assay……………………………………….9
2.9 Western blot………………………………………………………………………….9
2.10 IgM ELISA assay……………………………………………………………….10
2.11 Chromatin immunoprecipitation assay (ChIP)……………………………………11
2.12 Immunohistochemistry……………………………………………………….…12
2.13 Immunofluorescence………………………………………………….…………13
2.14 NP-KLH immunization…………………………………………………………13
2.15 Cell cycle analysis………………………………………………………………13
2.16 Apoptosis assay…………………………………………………………………14
2.17 Bisulfite PCR combined sequencing……………………………………………14
3. Results 16
3.1 Loss of 5-hmC in lymphoid germinal centers…………………………………….16
3.2 Down-regulation of TET1 in murine B220+ B cell activation……………………16
3.3 tet1 gene expression in naïve and GC B cells……………………………………17
3.4 IL6-induced B cell differentiation increase tet1 gene expression and 5-hmC levels in SKW6.4 GC-B cell line………………………………………………………………...17
3.5 pcDNA3β-tet1, EμVh-tet1 and pCEP4 vector constructs…………………………18
3.6 TET1 induces plasma cell differentiation and results in cell cycle arrest and apoptosis………………………………………………………………………………..18
3.7 Identification of the genes regulated by methylation during B cell activation…………………………………………………………………….…………19
3.8 Validation of TET1 target genes in IL6-induced differentiation model and SKW6.4 cells with TET1 stable expression by real-time PCR analysis…………………………20
3.9 KAISO and BCL2 are downstream targets of TET1……………….…………….…20
3.10 Bisulfite sequencing to confirm methylaion changes in KAISO and BCL2…….21
4. Discussion 22
4.1 Germinal center formation is manipulated by epigenetic regulation…………….22
4.2 Loss of 5-hmC and TET1 expression are involved in B cell activation…………....22
4.3 TET1 re-expression induces plasma cell differentiation…………………………...23
4.4 Kaiso and Bcl2 are downstream targets of TET1…………………………………..24
4.5 TET1 plays a master role and controls GC transition……………………………25
5. Figures and Tables 26
Figure 1. Loss of 5-hmC in lymphoid germinal centers……………………...……….26
Figure 2. Down-regulation of TET1 in murine B220+ B cell activation………...…...28
Figure 3. Tet1 gene expression in naïve and germinal center B cells…………….……30
Figure 4. IL6-induced B cell differentiation increase tet1 gene expression and 5-hmC levels in SKW6.4 GC-B cell line……………………………………………………….33
Figure 5. TET1 induces plasma cell differentiation and results in cell cycle arrest and apoptosis………………………………………………………………………………..36
Figure 6. pcDNA3β-tet1, EμVh-tet1 and pCEP4 vector construct……………………...37
Figure7.Identification of the genes regulated by methylation during B cell activation….……………………………………………………………………………40
Figure 8. Validation of TET1 target genes in IL6-induced differentiation model and SKW6.4 cells with TET1 stable expression by real-time PCR analysis…………….....42
Figure 9. KAISO and BCL2 are downstream targets of TET1……………………….44
Figure 10. Confirmation of methylaion changes in KAISO and BCL2 promoter by bisulfite sequencing…………………………………………………………………….46
6. Reference 53

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