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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 朱雪萍 | zh_TW |
| dc.contributor.advisor | Hsueh-Ping Chu | en |
| dc.contributor.author | 李姿瑢 | zh_TW |
| dc.contributor.author | Tzu-Rong Lee | en |
| dc.date.accessioned | 2025-08-20T16:14:11Z | - |
| dc.date.available | 2025-08-21 | - |
| dc.date.copyright | 2025-08-20 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-08-13 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/98906 | - |
| dc.description.abstract | G-四聯體是由富含鳥糞嘌玲的核酸序列摺疊形成的非典型二級結構，可透過去氧核醣核酸（DNA）或核醣核酸（RNA）構成。DNA G-四聯體廣泛分布於基因組中，並參與DNA複製、轉錄和基因組穩定性的調控。TERRA是一類由RNA聚合酶Ⅱ從次端粒和端粒區域轉錄而成的長片段非編碼RNA，含有不等數量的UUAGGG重複序列，可摺疊形成RNA G-四聯體結構。之前研究顯示TERRA可與G4解旋酶BLM結合。在小鼠胚胎幹細胞中進行TERRA下調處理後，BLM在染色質的結合量上升，且DNA G-四聯體的形成下降。
為了確認TERRA能否透過其富含鳥糞嘌呤的重複序列調控DNA G-四聯體形成，我們將含有四個UUAGGG重複序列的RNA（TERRA RNA）轉染至HeLa細胞中。免疫螢光染色結果顯示，BLM在染色質的結合下降，而核內DNA G-四聯體訊號上升，進一步支持TERRA具備促進DNA G-四聯體形成的想法。 為了進一步探討TERRA的表觀遺傳功能，之前研究整合ChIP-seq與RNA-seq資料，發現TERRA下調會導致部分基因轉錄起始點（TSS）附近的DNA G-四聯體訊號下降，而且這些基因與TERRA下調後表現量降低的基因有關。近期有其他研究指出DNA G-四聯體可能影響CTCF的DNA結合調控。CTCF是一種高度保守的染色質結構蛋白，參與染色質圈構（chromatin looping）的形成，並影響基因表現。為了確認TERRA能否藉由DNA G-四聯體的調控進一步影響CTCF與染色質的結合，我們在TERRA下調的小鼠胚胎幹細胞中進行CTCF ChIP-seq實驗，觀察到大多數CTCF在結合位點都有訊號上升的趨勢，而且在G4訊號下降的位置，CTCF訊號上升程度更為顯著。然而，這些位點附近的G-四聯體訊號變化不一，顯示CTCF結合的改變可能並非單純由DNA G-四聯體調控所致。此外，CTCF與cohesin共結合的區域訊號上升更為顯著，暗示TERRA可能參與染色質三維結構的調控。 綜上所述，本研究透過TERRA RNA轉染系統與TERRA KD系統，分別確認TERRA會影響染色質上的BLM結合與DNA G-四聯體的形成，並進一步指出TERRA可能透過對CTCF的影響，揭示潛在的表觀遺傳調控機制。 | zh_TW |
| dc.description.abstract | G-quadruplexes (G4) are noncanonical secondary structures formed by guanine-rich sequences in DNA and RNA. DNA G4s are widely distributed throughout the genome and play roles in regulating replication, transcription, and genome stability. TERRA, a long non-coding RNA transcribed by RNA polymeraseⅡ, transcribed from subtelomeric regions to telomeres, with varying numbers of UUAGGG repeats capable of forming RNA G4 structures. TERRA has been shown to interact with BLM, an ATP-dependent helicase known to unwind G4 structure, and TERRA knockdown in mESCs leads to increased BLM occupancy and reduced DNA G4 levels.
To examine whether TERRA regulates DNA G4 through its G-rich repetitive sequences, we introduced synthetic TERRA RNA oligos containing four tandem UUAGGG repeats into HeLa cells. Immunofluorescence staining revealed that TERRA RNA reduced BLM occupancy and increased nuclear DNA G4 signals, suggesting that TERRA promotes G4 formation. To further investigate the epigenetic role of TERRA, integrated analyses of G4 ChIP-seq and RNA-seq showed that TERRA knockdown led to G4 signal reduction near the transcription start sites (TSSs), particularly in genes downregulated upon TERRA depletion. Given that studies from other labs revealed that G4 formation may influence CTCF binding, we tested whether TERRA regulates CTCF binding to chromatin. CTCF ChIP-seq analysis revealed a genome-wide increase in CTCF occupancy in TERRA knockdown mESCs. Notably, G4 reduction correlates with the greatest increase in CTCF binding. However, changes in G4 signal near CTCF binding sites were not positively correlated with changes in CTCF occupancy in TERRA knockdown cells, suggesting that G4 modulation alone may not fully account for the increased in CTCF occupancy upon TERRA depletion. Moreover, the increase in CTCF binding was more pronounced at sites co-occupied with cohesin complexes, implicating a potential role for TERRA in modulating chromatin looping via the CTCF-cohesin axis. Taken together, our findings suggest a previously unrecognized epigenetic mechanism by which TERRA influences chromatin architecture and regulates gene expression. | en |
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| dc.description.provenance | Made available in DSpace on 2025-08-20T16:14:11Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 審定書 i
致謝 ii 中文摘要 iii Abstract v Contents vii Contents of Figure xi Contents of Table xiii Contents of Supplementary Figure xiv Contents of Supplementary Table xv Abbreviations xvi Chapter 1 Introduction 1 1.1 G-quadruplex (G4) 1 1.2 Telomeric Repeat-Containing RNA (TERRA) 2 1.3 G4C2 repeats in C9orf72 gene 4 1.4 BLM (Bloom’s syndrome helicase) 5 1.5 CCCTC-binding factor (CTCF) 7 1.6 Hypothesis and aim in this study 9 Chapter 2 Materials and Methods 11 2.1 HeLa cell culture 11 2.2 Small RNA transfection 11 2.3 Slide preparation for staining 12 2.4 Immunofluorescence (IF) 12 2.5 Microscopy data quantification 13 2.6 Mouse embryonic stem (ES) cell culture 13 2.7 TERRA knockdown 14 2.8 RNA extraction 15 2.9 cDNA synthesis 16 2.10 qPCR (Real-time Quantitative Polymerase Chain Reaction) 16 2.11 Chromatin immunoprecipitation for CTCF 17 2.12 Library preparation for sequencing 21 2.13 NGS analysis for CTCF ChIP-seq 24 2.14 NGS analysis for DNA G4 signal nearby CTCF binding sites 27 2.15 NGS analysis of CTCF signal at CTCF binding sites with or without cohesin co-binding 29 2.16 Cell fractionation 30 2.17 Western blotting 31 2.18 Quantification for western blotting 33 2.19 NGS analysis for CTCF CLIP-seq 33 2.20 NGS analysis for TERRA KD RNA-seq 34 Chapter 3 Results 35 3.1 RNA G4 promotes DNA G4 formation and reduce BLM occupancy. 35 3.2 Investigation of CTCF occupancy upon TERRA depletion in mESCs. 37 3.3 CTCF occupancy increases upon TERRA depletion. 39 3.4 CTCF binding increases significantly in regions with G4 reduction. 41 3.5 Increased CTCF occupancy after TERRA KD is more pronounced at cohesin co-binding sites 43 3.6 TERRA KD leads to a modest increase in CTCF chromatin occupancy 44 Chapter 4 Discussion 45 Chapter 5 Figures and Tables 54 Chapter 6 Supplementary Figures and Tables 84 Chapter 7 References 101 | - |
| dc.language.iso | en | - |
| dc.subject | TERRA | zh_TW |
| dc.subject | G-四聯體 | zh_TW |
| dc.subject | DNA G-四聯體 | zh_TW |
| dc.subject | BLM | zh_TW |
| dc.subject | CTCF | zh_TW |
| dc.subject | TERRA | en |
| dc.subject | CTCF | en |
| dc.subject | DNA G-quadruplex | en |
| dc.subject | BLM | en |
| dc.subject | G-quadruplex | en |
| dc.title | TERRA RNA對基因組中G4與CTCF的影響 | zh_TW |
| dc.title | TERRA RNA impacts G-quadruplexes and CTCF occupancy across the genome | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 林敬哲;陳世淯 | zh_TW |
| dc.contributor.oralexamcommittee | Jing-Jer Lin;Shih-Yu Chen | en |
| dc.subject.keyword | TERRA,G-四聯體,DNA G-四聯體,BLM,CTCF, | zh_TW |
| dc.subject.keyword | TERRA,G-quadruplex,DNA G-quadruplex,BLM,CTCF, | en |
| dc.relation.page | 107 | - |
| dc.identifier.doi | 10.6342/NTU202504256 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-08-14 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | - |
| dc.date.embargo-lift | 2025-08-21 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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|---|---|---|---|
| ntu-113-2.pdf | 6.33 MB | Adobe PDF | 檢視/開啟 |
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