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標題: | 以hTERT基因探討G-quadruplex與DNA甲基化之交互影響機制 Investigate the biological effect of DNA methylation on G-quadruplex of hTERT gene |
作者: | Yen-Min Kuan 管彥民 |
指導教授: | 陳進庭(Chin-Tin Chen) |
關鍵字: | DNA甲基化,G-quadruplex,hTERT,CTCF, DNA methylation,G-quadruplex,hTERT,CTCF, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | DNA甲基化修飾與DNA二級結構G-quadruplex皆被認為具有抑制基因表現的可能。但兩者之交互作用是否會調控基因表現,卻尚未有文獻證實此一現象。由於與癌症發展有極高關聯性的hTERT基因,其啟動子區域受DNA甲基化調控機制已被廣為探討,並且該區域也存在具有G-quadruplex結構等特性,故合適做為探討G-quadruplex和DNA甲基化兩者可能的交互作用是否影響轉錄因子結合調控基因表現,進而改變細胞生理功能,並深具進一步的探討其作用機制的重要性。本研究首先以QGRS Mapper (Quadruplex forming G-Rich Sequences)進行分析後hTERT基因後,發現主要受到甲基化調控的區域CTCF (CCCTC-binding factor) 結合位序列會形成G-quadruplex結構,隨後以核磁共振(Nuclear Magnetic Resonance, NMR)確認此一事實。我們建構了含hTERT 啟動子區域且包含不同甲基化修飾之報導基因質體,從報導基因表現結果證實位於CTCF結合位之G-quadruplex結構,會和hTERT甲基化調控有交互影響而改變基因表現。我們也利用ChIP assay確認CTCF結合能力會因為此交互作用影響而改變。本研究證明了G-quadruplex結構和甲基化之間的互相影響會透過影響轉錄因子CTCF的結合,改變hTERT基因表現。未來也將透過相同模式探討其他已知有G-quadruplex結構且同樣會受到甲基化調控的基因,確認此現象為普遍存在並深入探討。 It has been shown that gene expression will be affected by DNA methylation and secondary structures in the promoter region. However, the gene expression profile has not been addressed when the DNA methylation occurs at the G-quadruplex secondary structures. DNA methylation and G-quadruplex structures has been found in the promoter region of hTERT (human telomerase reverse transcriptase) gene. In this study, we investigated whether the methylation of G-quadruplex will occur at the promoter region of hTERT and further modulate its gene expression level. Using QGRS Mapper (Quadruplex forming G-Rich Sequences), a G-quadruplex structure was identified on the CTCF (CCCTC-binding factor) binding site of hTERT gene. NMR (nuclear magnetic resonance) analysis further confirmed this possible G-quadruplex structure. Reporter plasmids were constructed under the control of different hTERT promoter region. Here we further showed that the expression of hTERT was regulated by the G-quadruplex at the CTCF binding site and DNA methylation at the G-quadruplex further modulate the gene expression. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56922 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
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