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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72932完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 朱雪萍(Hsueh-Ping Chu) | |
| dc.contributor.author | Sheng-Ning Chan | en |
| dc.contributor.author | 詹勝甯 | zh_TW |
| dc.date.accessioned | 2021-06-17T07:10:48Z | - |
| dc.date.available | 2019-07-24 | |
| dc.date.copyright | 2019-07-24 | |
| dc.date.issued | 2019 | |
| dc.date.submitted | 2019-07-22 | |
| dc.identifier.citation | 1. Azzalin, C. M., Reichenbach, P., Khoriauli, L., Giulotto, E., & Lingner, J. (2007). Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science, 318(5851), 798-801. doi:10.1126/science.1147182
2. Celli, G. B., & de Lange, T. (2005). DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion. Nat Cell Biol, 7(7), 712-718. doi:10.1038/ncb1275 3. Chen, L. Y., Redon, S., & Lingner, J. (2012). The human CST complex is a terminator of telomerase activity. Nature, 488(7412), 540-544. doi:10.1038/nature11269 4. Chu, H. P., Cifuentes-Rojas, C., Kesner, B., Aeby, E., Lee, H. G., Wei, C., . . . Lee, J. T. (2017). TERRA RNA Antagonizes ATRX and Protects Telomeres. Cell, 170(1), 86-101 e116. doi:10.1016/j.cell.2017.06.017 5. Damle, R. N., Batliwalla, F. M., Ghiotto, F., Valetto, A., Albesiano, E., Sison, C., . . . Chiorazzi, N. (2004). Telomere length and telomerase activity delineate distinctive replicative features of the B-CLL subgroups defined by immunoglobulin V gene mutations. Blood, 103(2), 375-382. doi:10.1182/blood-2003-04-1345 6. de Lange, T. (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev, 19(18), 2100-2110. doi:10.1101/gad.1346005 7. Denchi, E. L., & de Lange, T. (2007). Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature, 448(7157), 1068-1071. doi:10.1038/nature06065 8. Doksani, Y., Wu, J. Y., de Lange, T., & Zhuang, X. (2013). Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation. Cell, 155(2), 345-356. doi:10.1016/j.cell.2013.09.048 9. Feng, X., Hsu, S. J., Bhattacharjee, A., Wang, Y., Diao, J., & Price, C. M. (2018). CTC1-STN1 terminates telomerase while STN1-TEN1 enables C-strand synthesis during telomere replication in colon cancer cells. Nat Commun, 9(1), 2827. doi:10.1038/s41467-018-05154-z 10. Graf, M., Bonetti, D., Lockhart, A., Serhal, K., Kellner, V., Maicher, A., . . . Luke, B. (2017). Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle. Cell, 170(1), 72-+. doi:10.1016/j.cell.2017.06.006 11. Greider, C. W., & Blackburn, E. H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell, 43(2 Pt 1), 405-413. 12. Harley, C. B., Futcher, A. B., & Greider, C. W. (1990). Telomeres shorten during ageing of human fibroblasts. Nature, 345(6274), 458-460. doi:10.1038/345458a0 13. Montero, J. J., Lopez-Silanes, I., Megias, D., M, F. F., Castells-Garcia, A., & Blasco, M. A. (2018). TERRA recruitment of polycomb to telomeres is essential for histone trymethylation marks at telomeric heterochromatin. Nat Commun, 9(1), 1548. doi:10.1038/s41467-018-03916-3 14. Palm, W., & de Lange, T. (2008). How shelterin protects mammalian telomeres. Annu Rev Genet, 42, 301-334. doi:10.1146/annurev.genet.41.110306.130350 15. Porro, A., Feuerhahn, S., Reichenbach, P., & Lingner, J. (2010). Molecular dissection of telomeric repeat-containing RNA biogenesis unveils the presence of distinct and multiple regulatory pathways. Mol Cell Biol, 30(20), 4808-4817. doi:10.1128/MCB.00460-10 16. Rice, C., & Skordalakes, E. (2016). Structure and function of the telomeric CST complex. Comput Struct Biotechnol J, 14, 161-167. doi:10.1016/j.csbj.2016.04.002 17. Roberts, B., Haupt, A., Tucker, A., Grancharova, T., Arakaki, J., Fuqua, M. A., . . . Gunawardane, R. N. (2017). Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization. Mol Biol Cell, 28(21), 2854-2874. doi:10.1091/mbc.E17-03-0209 18. Saretzki, G. (2018). Telomeres, Telomerase and Ageing. Subcell Biochem, 90, 221-308. doi:10.1007/978-981-13-2835-0_9 19. Schmidt, J. C., Zaug, A. J., & Cech, T. R. (2016). Live Cell Imaging Reveals the Dynamics of Telomerase Recruitment to Telomeres. Cell, 166(5), 1188-1197 e1189. doi:10.1016/j.cell.2016.07.033 20. Schoeftner, S., & Blasco, M. A. (2008). Developmentally regulated transcription of mammalian telomeres by DNA-dependent RNA polymerase II. Nat Cell Biol, 10(2), 228-236. doi:10.1038/ncb1685 21. Schonenberger, F., Deutzmann, A., Ferrando-May, E., & Merhof, D. (2015). Discrimination of cell cycle phases in PCNA-immunolabeled cells. BMC Bioinformatics, 16, 180. doi:10.1186/s12859-015-0618-9 22. Takasawa, K., Arai, Y., Yamazaki-Inoue, M., Toyoda, M., Akutsu, H., Umezawa, A., & Nishino, K. (2018). DNA hypermethylation enhanced telomerase reverse transcriptase expression in human-induced pluripotent stem cells. Hum Cell, 31(1), 78-86. doi:10.1007/s13577-017-0190-x 23. Wright, W. E., Tesmer, V. M., Liao, M. L., & Shay, J. W. (1999). Normal human telomeres are not late replicating. Exp Cell Res, 251(2), 492-499. doi:10.1006/excr.1999.4602 24. Yamada, T., Yoshimura, H., Shimada, R., Hattori, M., Eguchi, M., Fujiwara, T. K., . . . Ozawa, T. (2016). Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres. Sci Rep, 6, 38910. doi:10.1038/srep38910 25. Zhang, X., Mar, V., Zhou, W., Harrington, L., & Robinson, M. O. (1999). Telomere shortening and apoptosis in telomerase-inhibited human tumor cells. Genes Dev, 13(18), 2388-2399. doi:10.1101/gad.13.18.2388 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72932 | - |
| dc.description.abstract | 端粒為真核生物染色體末段的特殊線性構造，能夠保護染色體末端的完整性。而端粒的組成包含:重複性序列(TTAGGG)n 與端粒結合蛋白，可以阻止DNA損傷反應的發生、核酸酶降解、以及染色體的融合。在經過每一次循環的DNA複製後，端粒將會變得越來越短，導致「染色體末端複製問題」。端粒酶能夠藉由本身的RNA模板和反轉錄酶(TERT)進行延長端粒區域重複性序列的反應。近期的研究顯示端粒附近的區域能夠轉錄出一段long non-coding RNA (lncRNA)，稱為Telomeric repeat- containing RNA (TERRA)，攜帶著(UUAGGG)n重複性序列在自身的3’-end。
我們的實驗發現到TERRA會與端粒結合蛋白交互作用並且能夠調控端粒的完整性。為了進一步了研究TERRA是如何調控端粒，我們利用antisense-oligo (ASO)的方式將老鼠胚胎幹細胞中的TERRA去除。藉由Immuno-DNA-FISH，我觀察到了在TERRA去除之後，端粒酶在端粒上的比例會下降，間接說明了TERRA可能會幫助端粒酶徵招到染色體尾端的作用。相反的，在ChIP-qPCR的資料中顯示POT1與CTC1在端粒的位置上比率在TERRA去除之後會有所提升，這說明了TERRA會阻止端粒結合蛋白與CST complex結合至端粒上。結合先前的研究發現到CST complex會終止端粒酶在端粒上延長的作用，這個結果推測TERRA幫助端粒酶結合到DNA上可能是藉由抑制CST complex到端粒上的徵招。我們在另外一項實驗中發現到TERRA與端粒酶在細胞週期中的S時期的中期時回提升，並且在細胞週期到G2時期時會下降。此項結果與端粒酶與端粒ＤＮＡ 結合的時間相吻合。總括上述，我們的研究發現TERRA可以調控端粒酶與端粒DNA的結合，並影響端粒蛋白招募到端粒ＤＮＡ上，進而維持端粒的完整性。 | zh_TW |
| dc.description.abstract | Telomeres are nucleoprotein complex composed of repetitive sequence ”TTAGGG” and shelterin proteins (TRF1/TRF2/POT1/TPP1/TIN2). Telomerase (TERT) extends telomeres through binding its RNA template to G-rich strand of telomeres. Recent studies have shown that telomeres transcribe a long non-coding RNA, TERRA, which carries “UUAGGG” repeats at 3’-end.
Our previous study has shown that TERRA interacts with shelterin proteins and regulates telomere integrity (Chu et al., 2017). To investigate how TERRA regulates telomere maintenance, I depleted TERRA using anti-sense oligos in mouse embryonic stem cells (mESCs). By immuno-DNA FISH staining, I observed that the number of TERT foci at telomeres was decreased upon TERRA depletion, implying that TERRA promotes the recruitment of telomerase to telomere ends. In contrast, ChIP-qPCR showed that POT1 and CTC1 occupancy at telomeres were elevated upon TERRA depletion, indicating that TERRA prevents CTC1 and shelterin complex from binding to telomere ends. Combined with the previous study showing that CTC1 terminates telomerase, these data suggest that TERRA may assist telomerase loading to DNA by expelling CTC1 from binding to telomeres. In agreement, the levels of both TERRA and telomerase at telomeres are high in Mid-S phase and low in G2 phase in mESCs. Together, this study provides a mechanism in which TERRA regulates the telomere maintenance by balancing the recruitment of telomerase and telomeric DNA binding proteins to telomeres. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T07:10:48Z (GMT). No. of bitstreams: 1 ntu-108-R06b43031-1.pdf: 5366580 bytes, checksum: c6fecb73531ff65c3142df18566f8da1 (MD5) Previous issue date: 2019 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………………………………………………………I
Contents………………………………………………………………………….. …II-III 誌謝…………………………………………………………………………………….IV 中文摘要………………………………………………………………………………..V Abstract………………………………………………………………………….....VI-VII Figure list………………………………………………………………………....VIII-X Table list…………………………………………………………………………………XI Chapter 1. Introduction…………………………………………………………………1 1.-1 Telomere ………………………………………………………………………1 1-2 Shelterin complex …………………………………………………………...1-2 1-3 Telomerase …………………………………………………………………….2 1-4 CST complex …………………………………………………………………..2 1-5 TERRA ………………………………………………………………………...3 Chapter 2. Materials and Methods……………………………………………………..4 2-1 Cell culture…………………………………………………………………..4 2-2 FISH…………………………………………………………………………4 2-3 Immuno- FISH…………………………………………………………….4-6 2-4 TERRA depletion……………………………………………………………6 2-5 Quantitative RT-PCR………………………………………………………...6 2-6 Western blotting…………………………………………………………...6-7 2-7 Chromatin-IP...………………………………………….…………………7-8 2-9 Flow cytometry………………………………………………………………8 2-8 Quantitative and statistical analysis………………………………….……8-9 Acknowledgements………………………………………………………………...…..10 Chapter 3. Results…………………………………………………………………..11-17 Chapter 4. Discussion ……………………………………………………………..18-20 Chapter 5. References…………………………………………………………50-54 Abbreviations………….……………………………………………………………55-58 | |
| dc.language.iso | en | |
| dc.title | TERRA如何在哺乳類動物細胞中調控端粒的完整性 | zh_TW |
| dc.title | TERRA regulates telomeres maintenance in mammalian cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 107-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 冀宏源(Hung-Yuan Chi),陳律佑(Chen, Liuh-Yow) | |
| dc.subject.keyword | 端粒,CST complex,TERRA,DNA damage response, | zh_TW |
| dc.subject.keyword | telomere,telomerase,TERRA,DNA damage response,CTC1, POT1,shelterin complex,long non-coding RNA, | en |
| dc.relation.page | 58 | |
| dc.identifier.doi | 10.6342/NTU201901548 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2019-07-22 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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