Tet1和5-hydroxymethylcytosine在淋巴生發中心的形成和功能的角色

The role of Tet1 and 5-hydroxymethylcytosine in the formation and function of germinal centers

楊楚光(Chu-Kuang Yang)

指導教授 ： 鄭永銘

國立臺灣大學/醫學院/病理學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.00076

摘要

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。

關鍵字

淋巴生發中心 ； TET1 ； Bcl2 ； Kaiso

並列摘要

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.

並列關鍵字

germinal center ； TET1 ； Bcl2 ； Kaiso

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