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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 | 蔣依恬 | zh_TW |
| dc.contributor.author | I-Tien Chiang | en |
| dc.date.accessioned | 2023-09-22T16:39:30Z | - |
| dc.date.available | 2023-11-09 | - |
| dc.date.copyright | 2023-09-22 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-08-09 | - |
| dc.identifier.citation | 1. Chakravarti, D., K.A. LaBella, and R.A. DePinho, Telomeres: history, health, and hallmarks of aging. Cell, 2021. 184(2): p. 306-322.
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Bates, P.J., et al., G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms. Biochimica et Biophysica Acta (BBA) - General Subjects, 2017. 1861(5, Part B): p. 1414-1428. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89914 | - |
| dc.description.abstract | G-四聯體DNA是由富含鳥嘌呤的序列形成的非典型的二級結構，在基因組中廣泛存在。 G4結構被認為與轉錄、翻譯和基因組穩定性有關。長鏈非編碼端粒RNA (TERRA)，是由次端粒區域轉錄至端粒末端的一種非編碼RNA。 TERRA含有UUAGGG重複序列，使其能夠形成G-四聯體結構並與G-四聯體蛋白相互作用（例如ATRX，RTEL1和BLM DNA解旋酶），進而抑制G4形成，維持基因組穩定性。在本研究中，我們表明了TERRA透過其富含G序列調控G-四聯體蛋白並維持DNA G-四聯體結構形成。
我們先前的G4 ChIP-seq顯示TERRA降解後，減少轉錄起始位點（TSS）的G-四聯體形成，並伴隨著ATRX覆蓋率的增加。抑制ATRX緩解了TERRA knockdown所造成細胞中基因表達的變化，表示ATRX介導了TERRA knockdown所引起的基因表達失調。為了研究TERRA調控BLM在小鼠基因組中的分佈和其與DNA G-四聯體結構形成的關聯性，我們對BLM進行了染色質免疫沉澱測序。TERRA knockdown增加了BLM在端粒、染色質間端粒重複序列和一些特殊重複序列中的富集程度。此外， BLM富集程度的增加與G-四聯體結構形成減少相關。免疫染色結果證實TERRA knockdown導致BLM和RTEL1在染色質上的覆蓋率增加，並減少DNA G-四聯體結構的形成。然而，將富含G序列的DNA寡核苷酸送入細胞後可以減緩這些反應。此外，引入小片段的TERRA RNA可以降低染色質上BLM和RTEL1的覆蓋率並增加DNA G4結構的形成。基於這些發現，我們提出TERRA RNA藉由其富含G序列阻止G-四聯體蛋白到染色質上，防止G-四聯體結構被解開。總結來說，我們的研究揭示了TERRA具有阻止G4解旋酶募集到基因組上以維持G4結構形成的角色。 | zh_TW |
| dc.description.abstract | G-quadruplex (G4) DNA, a non-B form secondary structure formed with Guanine-rich sequences, widely exists in the genome. G4 structures are thought to be involved in the regulation of transcription, translation and genome stability. Telomeric repeat containing RNA (TERRA), a long non-coding RNA, is transcribed from subtelomeric region toward telomeric end. TERRA containing UUAGGG repetitive sequences enables folding to G-quadruplex and interacts with G4 regulators, including ATRX, RTEL1 and BLM DNA helicases that function in suppressing G4 formation to maintain genome stability. Here, we show that TERRA regulates G4 binding proteins and maintains G4 formation through its G-rich sequences.
Our previous G4 ChIP-seq analysis revealed a decrease in G4 formation at transcription start sites (TSS) after TERRA depletion, accompanied by elevated ATRX occupancy. Here I found that ATRX is increased in the chromatin bound fraction upon TERRA depletion. Silencing ATRX mitigates the effect of altered gene expression in TERRA knockdown cells, suggesting that ATRX mediates the dysregulation of gene expression caused by TERRA depletion. To investigate whether TERRA mediates the BLM occupancy in the genome, BLM chromatin immunoprecipitation (ChIP) sequencing was performed. TERRA depletion increased BLM abundance at telomeres, interstitial telomeric repeats, and some repetitive sequences. Additionally, such increased BLM occupancy was associated with G4 reduction. Immunostaining results support that TERRA depletion results in elevated abundance of BLM and RTEL1 on chromatin and a reduction of G4 formation, whereas introducing G-rich DNA oligos counteracts these effects. Moreover, the delivery of TERRA RNA oligos reduces BLM and RTEL1 occupancies on chromatin and increases DNA G4 structures. Based on these findings, we propose that TERRA RNA sequesters G4 binding proteins through its G-rich sequences, thereby preventing DNA G4 from unwinding. Overall, our findings uncover the role of TERRA in regulating the occupancies of G4 binding proteins and preserving G4 formation on the genome. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-09-22T16:39:30Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2023-09-22T16:39:30Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | Contents
誌謝 i 中文摘要 ii Abstract iii Contents v Content of figures viii Content of supplementary figures x Content of tables xi Abbreviations xii Chapter 1 Introduction 1 1.1 Telomere 1 1.2 Telomeric repeat-containing RNA (TERRA) 2 1.3 G-quadruplex (G4) 3 1.4 ATRX 5 1.5 G-quadruplex helicase - BLM 6 1.6 G-quadruplex helicase – RTEL1 7 Chapter 2 Materials and Methods 9 2.1 Mouse embryonic stem cell culture 9 2.2 Nucleofection 10 2.3 siRNA knockdown (ATRX, BLM) 11 2.4 Double knockdown of ATRX and TERRA 12 2.5 RNA extraction 12 2.6 cDNA synthesis 13 2.7 Real-time quantitative PCR (qPCR) 14 2.8 Northern blot 14 2.9 Preparing samples for ChIP 16 2.10 Chromatin immunoprecipitation (ChIP) 17 2.11 Sample library preparation for sequencing 20 2.12 Immunofluorescence 23 2.13 Nuclear cell fractionation 24 2.14 Western blot 25 Chapter 3 Results 28 3.1 ATRX was increased in the chromatin fraction after TERRA depletion. 28 3.2 ATRX inhibits DNA G4 formation and contributes to gene repression upon TERRA depletion 29 3.3 TERRA depletion compromised the effect of elevated G4 formation in ATRX knockdown cells 32 3.4 BLM deficiency significantly increases G4 intensity in nuclei 33 3.5 TERRA regulates BLM binding to chromatin 33 3.6 BLM binds to G-rich motif 34 3.7 TERRA prevents the recruitment of BLM to unique G-rich repetitive sequences 35 3.8 TERRA prevents BLM recruitment to interstitial telomeric repeats (ITS) and telomere 36 3.9 Introducing TERRA RNA oligos elevates DNA G4 abundance and reduces BLM occupancy 38 3.10 The delivery of G4 DNA oligos reverses the effects of increased BLM occupancy and decreased DNA G4 formation 39 Chapter 4 Discussion 76 Chapter 5 Supplementary information 79 Chapter 6 References 89 Content of figures Figure 1. ATRX was increased in the chromatin fraction after TERRA depletion. 42 Figure 2. ATRX mediates gene repression caused by TERRA depletion. 44 Figure 3. TERRA depletion alleviates the effect of increased G4 formation in ATRX knockdown mES cells. 47 Figure 4. BLM depletion increases DNA G4 formation in mouse ES cells. 48 Figure 5. BLM was elevated in the chromatin fraction after TERRA depletion. 49 Figure 6. BLM binds to G-rich motif. 51 Figure 7. BLM abundance increases at (TCTCTG)n repeats displaying G4 reduction upon TERRA depletion. 53 Figure 8. TERRA depletion elevates BLM occupancies at (CAGAG)n repeats containing G4 peaks. 55 Figure 9. BLM is highly enriched at (CTCTG)n repeats containing G4 peaks. 57 Figure 10. TERRA depletion elevates BLM occupancy and reduces DNA G4 formation at telomeres. 59 Figure 11. TERRA depletion increases BLM occupancy and reduces DNA G4 formation at interstitial telomeric repeats (ITS). 61 Figure 12. Transfecting TERRA RNA oligos elevates DNA G4 abundance and reduces BLM and RTEL1 occupancy. 63 Figure 13. The delivery of G4 DNA oligos reverses the effects of increased BLM occupancy and decreased DNA G4 formation. 65 Figure 14. Models of TERRA modulating G4 binding proteins occupancy and G4 formation through its G-rich sequences. 67 Content of supplementary figures Figure.S1 Increased ATRX occupancy around the transcription start sites is associated with gene regulation after TERRA depletion. 68 Figure S2. TERRA promotes DNA G4 formation in mouse ES cells. 70 Figure S3. Reduced G4 formation near transcription start sites is associated with DEGs after TERRA depletion. 72 Figure S4. ATRX suppresses DNA G4 formation in mouse ES cells. 73 Figure S5. TERRA depletion increases BLM and RTEL1 loading on chromatin. 75 Content of tables Table 1. DNA and RNA oligo nucleotides used in nucleofection 79 Table 2. siRNA used in transfection 79 Table 3. Reagent for cDNA synthesis 80 Table 4. Primers and probes used in qPCR and Northern blot 81 Table 5. Antibodies used in ChIP, IF and WB 82 Table 6. NGS-code list 83 Table 7. Reagents and chemicals 84 | - |
| dc.language.iso | en | - |
| dc.subject | TERRA | zh_TW |
| dc.subject | G-四聯體 | zh_TW |
| dc.subject | BLM | zh_TW |
| dc.subject | G-四聯體解旋酶 | zh_TW |
| dc.subject | ATRX | zh_TW |
| dc.subject | RTEL1 | zh_TW |
| dc.subject | RTEL1 | en |
| dc.subject | ATRX | en |
| dc.subject | BLM | en |
| dc.subject | G-quadruplex | en |
| dc.subject | TERRA | en |
| dc.subject | G-quadruplex helicases | en |
| dc.title | TERRA作為一個表觀遺傳學的調控者維持全基因組中DNA G-四聯體的形成 | zh_TW |
| dc.title | TERRA acts as an epigenetic regulator to maintain DNA G-quadruplex formation in the genome | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 高承福;林敬哲 | zh_TW |
| dc.contributor.oralexamcommittee | Cheng-Fu Kao;Jing-Jer Lin | en |
| dc.subject.keyword | TERRA,G-四聯體,G-四聯體解旋酶,ATRX,BLM,RTEL1, | zh_TW |
| dc.subject.keyword | TERRA,G-quadruplex,G-quadruplex helicases,ATRX,BLM,RTEL1, | en |
| dc.relation.page | 92 | - |
| dc.identifier.doi | 10.6342/NTU202303458 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2023-08-11 | - |
| 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 | 2028-08-07 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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