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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳進庭(Chin-Tin Chen) | |
dc.contributor.author | Yen-Min Kuan | en |
dc.contributor.author | 管彥民 | zh_TW |
dc.date.accessioned | 2021-06-16T06:31:18Z | - |
dc.date.available | 2017-08-08 | |
dc.date.copyright | 2014-08-08 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-06 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56922 | - |
dc.description.abstract | 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。 | zh_TW |
dc.description.abstract | 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. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T06:31:18Z (GMT). No. of bitstreams: 1 ntu-103-R01B22026-1.pdf: 2701561 bytes, checksum: ceb340865893caf4183ce0cd644efbde (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 目錄
摘要 I Abstract II 第一章 緒論 1 1.1 DNA二級結構 – G-quadruplex 1 1.1.1 G-quadruplex基本介紹 1 1.1.2 G-quadruplex對啟動子活性的影響 2 1.2 DNA甲基化 4 1.2.1 DNA甲基化與轉錄調控 4 1.2.2 DNA甲基化相關酵素 5 1.2.3 藥物對於DNA甲基化調控的影響 7 1.3 hTERT基因表現 8 1.3.1 端粒與端粒酶 8 1.3.2 hTERT轉錄調控 8 1.4轉錄因子CTCF(CCCTC-binding factor) 10 1.5研究動機與目的 12 第二章 材料與方法 13 2.1藥品與儀器 13 2.1.1藥品 13 2.1.2細胞培養試劑 15 2.1.3儀器 15 2.2細胞株 16 2.2.1 細胞繼代培養 17 2.2.2 細胞冷凍與解凍 17 2.2.3 細胞計數 17 2.3 核磁共振光譜 NMR spectra 18 2.4 甲基化調控 18 2.4.1 ALA光動力處理 18 2.4.2 Trichostatin A (TSA) 處理 19 2.5 mRNA定量分析 19 2.5.1 RNA 萃取 (RNA extraction) 19 2.5.2 反轉錄 (Reverse Transcription, RT) 19 2.5.3 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 20 2.5.4 洋菜膠體電泳分析 21 2.6 Methylation cassette assay 21 2.6.1 以CpG-free vector建構hTERT promoter質體 21 2.6.1.1 目標基因序列的PCR放大 21 2.6.1.2 TA cloning 22 2.6.1.3 質體建構 23 2.6.2 定點突變 (site-directed mutagenesis) 24 2.6.3 甲基化 25 2.6.4 建構partial-methylation質體 25 2.6.5 細胞轉染 26 2.7 亞硫酸鹽定序 (Bisulfite sequencing) 26 2.8 染色質免疫沉澱 (chromatin immunoprecipitation) 28 2.9 螢光素酶報導基因分析 (Luciferase reporter assay) 29 2.10 統計分析 30 第三章 結果 31 3.1 分析CTCF結合位序列是否形成G-quadruplex 31 3.2 確認hTERT表現量和甲基化相關酵素之間的影響 32 3.2.1 TSA處理細胞後DNMT1/hTERT表現量的改變 33 3.2.2 ALA-PDT處理細胞後DNMT1 / Gadd45α / hTERT表現量的改變 34 3.2.3 ALA-PDT處理對hTERT啟動子甲基化程度之影響 35 3.3 探討甲基化和G-quadruplex的交互作用是否影響hTERT基因表現 35 3.3.1 hTERT啟動子質體之報導基因表現 36 3.3.2 TSA處理對於報導基因影響 37 3.3.3 ALA-PDT處理對於報導基因影響 38 3.4 CTCF結合能力差異 39 3.5 G-quadruplex影響hTERT基因表現 40 第四章 討論 43 4.1 為何選擇hTERT之CTCF結合位作為主要研究對象 43 4.2 G-quadruplex分析方法 44 4.3 定點突變影響 45 4.4 ALA-PDT所引發hTERT可能調控機制 45 4.5 RT-PCR實驗結果與報導基因實驗結果之差異 47 4.5.1 TSA處理 47 4.5.2 ALA-PDT處理 49 4.6 G-quadruplex影響hTERT基因甲基化程度 50 第五章 結論與未來工作 52 圖 53 附錄 67 參考文獻 75 | |
dc.language.iso | zh-TW | |
dc.title | 以hTERT基因探討G-quadruplex與DNA甲基化之交互影響機制 | zh_TW |
dc.title | Investigate the biological effect of DNA methylation on G-quadruplex of hTERT gene | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張大釗(Ta-Chau Chang),張麗冠(Li-Kwan Chang),林晉玄(Ching-Hsuan Lin) | |
dc.subject.keyword | DNA甲基化,G-quadruplex,hTERT,CTCF, | zh_TW |
dc.subject.keyword | DNA methylation,G-quadruplex,hTERT,CTCF, | en |
dc.relation.page | 86 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-08-07 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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