請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78989
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 周子賓 | |
dc.contributor.author | Chih-Yi Yang | en |
dc.contributor.author | 楊芝宜 | zh_TW |
dc.date.accessioned | 2021-07-11T15:34:37Z | - |
dc.date.available | 2023-08-21 | |
dc.date.copyright | 2018-08-21 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-15 | |
dc.identifier.citation | Aapola, U., Kawasaki, K., Scott, H.S., Ollila, J., Vihinen, M., Heino, M., Shintani, A., Kawasaki, K., Minoshima, S., Krohn, K., et al. (2000). Isolation and initial characterization of a novel zinc finger gene, DNMT3L, on 21q22.3, related to the cytosine-5-methyltransferase 3 gene family. Genomics 65, 293-298.
Aapola, U., Liiv, I., and Peterson, P. (2002). Imprinting regulator DNMT3L is a transcriptional repressor associated with histone deacetylase activity. Nucleic Acids Res 30, 3602-3608. Aapola, U., Lyle, R., Krohn, K., Antonarakis, S.E., and Peterson, P. (2001). Isolation and initial characterization of the mouse Dnmt3l gene. Cytogenet Cell Genet 92, 122-126. Atlasi, Y., and Stunnenberg, H.G. (2017). The interplay of epigenetic marks during stem cell differentiation and development. Nat Rev Genet 18, 643-658. Bender, C.M., Gonzalgo, M.L., Gonzales, F.A., Nguyen, C.T., Robertson, K.D., and Jones, P.A. (1999). Roles of cell division and gene transcription in the methylation of CpG islands. Mol Cell Biol 19, 6690-6698. Bergman, R.J., Gazit, D., Kahn, A.J., Gruber, H., McDougall, S., and Hahn, T.J. (1996). Age-related changes in osteogenic stem cells in mice. J Bone Miner Res 11, 568-577. Bethel, M., Chitteti, B.R., Srour, E.F., and Kacena, M.A. (2013). The changing balance between osteoblastogenesis and adipogenesis in aging and its impact on hematopoiesis. Curr Osteoporos Rep 11, 99-106. Bloushtain-Qimron, N., Yao, J., Shipitsin, M., Maruyama, R., and Polyak, K. (2009). Epigenetic patterns of embryonic and adult stem cells. Cell Cycle 8, 809-817. Bourc'his, D., and Bestor, T.H. (2004). Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 431, 96-99. Bourc'his, D., Xu, G.L., Lin, C.S., Bollman, B., and Bestor, T.H. (2001). Dnmt3L and the establishment of maternal genomic imprints. Science 294, 2536-2539. Campisi, J., and d'Adda di Fagagna, F. (2007). Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol 8, 729-740. Caplan, A.I. (1991). Mesenchymal stem cells. J Orthop Res 9, 641-650. Chedin, F., Lieber, M.R., and Hsieh, C.L. (2002). The DNA methyltransferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. Proc Natl Acad Sci U S A 99, 16916-16921. Chen, H., Zhou, X., Fujita, H., Onozuka, M., and Kubo, K.Y. (2013). Age-related changes in trabecular and cortical bone microstructure. Int J Endocrinol 2013, 213234. Chen, Z.X., Mann, J.R., Hsieh, C.L., Riggs, A.D., and Chedin, F. (2005). Physical and functional interactions between the human DNMT3L protein and members of the de novo methyltransferase family. J Cell Biochem 95, 902-917. Cheng, C.C., Lee, Y.H., Lin, S.P., Huangfu, W.C., and Liu, I.H. (2014). Cell-autonomous heparanase modulates self-renewal and migration in bone marrow-derived mesenchymal stem cells. J Biomed Sci 21, 21. Cheng, C.C., Lian, W.S., Hsiao, F.S., Liu, I.H., Lin, S.P., Lee, Y.H., Chang, C.C., Xiao, G.Y., Huang, H.Y., Cheng, C.F., et al. (2012). Isolation and characterization of novel murine epiphysis derived mesenchymal stem cells. PLoS One 7, e36085. Ciccone, D.N., Su, H., Hevi, S., Gay, F., Lei, H., Bajko, J., Xu, G., Li, E., and Chen, T. (2009). KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature 461, 415-418. Collins, C.A., Olsen, I., Zammit, P.S., Heslop, L., Petrie, A., Partridge, T.A., and Morgan, J.E. (2005). Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122, 289-301. Deplus, R., Brenner, C., Burgers, W.A., Putmans, P., Kouzarides, T., de Launoit, Y., and Fuks, F. (2002). Dnmt3L is a transcriptional repressor that recruits histone deacetylase. Nucleic Acids Res 30, 3831-3838. Feng, J., Liu, T., Qin, B., Zhang, Y., and Liu, X.S. (2012). Identifying ChIP-seq enrichment using MACS. Nat Protoc 7, 1728-1740. Friedenstein, A.J., Deriglasova, U.F., Kulagina, N.N., Panasuk, A.F., Rudakowa, S.F., Luria, E.A., and Ruadkow, I.A. (1974). Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 2, 83-92. Friedenstein, A.J., Petrakova, K.V., Kurolesova, A.I., and Frolova, G.P. (1968). Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6, 230-247. Garcia-Prat, L., and Munoz-Canoves, P. (2017). Aging, metabolism and stem cells: Spotlight on muscle stem cells. Mol Cell Endocrinol 445, 109-117. Gowher, H., Liebert, K., Hermann, A., Xu, G., and Jeltsch, A. (2005). Mechanism of stimulation of catalytic activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L. J Biol Chem 280, 13341-13348. Guenatri, M., Duffie, R., Iranzo, J., Fauque, P., and Bourc'his, D. (2013). Plasticity in Dnmt3L-dependent and -independent modes of de novo methylation in the developing mouse embryo. Development 140, 562-572. Hajkova, P., Erhardt, S., Lane, N., Haaf, T., El-Maarri, O., Reik, W., Walter, J., and Surani, M.A. (2002). Epigenetic reprogramming in mouse primordial germ cells. Mech Dev 117, 15-23. Hashimoto, H., Vertino, P.M., and Cheng, X. (2010). Molecular coupling of DNA methylation and histone methylation. Epigenomics 2, 657-669. Hata, K., Okano, M., Lei, H., and Li, E. (2002). Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 129, 1983-1993. Hirokawa, N. (1996). Organelle transport along microtubules - the role of KIFs. Trends Cell Biol 6, 135-141. Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279, 519-526. Hirokawa, N., Noda, Y., and Okada, Y. (1998). Kinesin and dynein superfamily proteins in organelle transport and cell division. Curr Opin Cell Biol 10, 60-73. Holz-Schietinger, C., Matje, D.M., Harrison, M.F., and Reich, N.O. (2011). Oligomerization of DNMT3A controls the mechanism of de novo DNA methylation. J Biol Chem 286, 41479-41488. Hsiao, F.S., Cheng, C.C., Peng, S.Y., Huang, H.Y., Lian, W.S., Jan, M.L., Fang, Y.T., Cheng, E.C., Lee, K.H., Cheng, W.T., et al. (2010). Isolation of therapeutically functional mouse bone marrow mesenchymal stem cells within 3 h by an effective single-step plastic-adherent method. Cell Prolif 43, 235-248. Hu, J.L., Zhou, B.O., Zhang, R.R., Zhang, K.L., Zhou, J.Q., and Xu, G.L. (2009). The N-terminus of histone H3 is required for de novo DNA methylation in chromatin. Proc Natl Acad Sci U S A 106, 22187-22192. Hu, Y.G., Hirasawa, R., Hu, J.L., Hata, K., Li, C.L., Jin, Y., Chen, T., Li, E., Rigolet, M., Viegas-Pequignot, E., et al. (2008). Regulation of DNA methylation activity through Dnmt3L promoter methylation by Dnmt3 enzymes in embryonic development. Hum Mol Genet 17, 2654-2664. In 't Anker, P.S., Scherjon, S.A., Kleijburg-van der Keur, C., Noort, W.A., Claas, F.H., Willemze, R., Fibbe, W.E., and Kanhai, H.H. (2003). Amniotic fluid as a novel source of mesenchymal stem cells for therapeutic transplantation. Blood 102, 1548-1549. Jackson, M., Krassowska, A., Gilbert, N., Chevassut, T., Forrester, L., Ansell, J., and Ramsahoye, B. (2004). Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells. Mol Cell Biol 24, 8862-8871. Jaiswal, N., Haynesworth, S.E., Caplan, A.I., and Bruder, S.P. (1997). Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64, 295-312. Jia, D., Jurkowska, R.Z., Zhang, X., Jeltsch, A., and Cheng, X. (2007). Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature 449, 248-251. Jilka, R.L., Weinstein, R.S., Takahashi, K., Parfitt, A.M., and Manolagas, S.C. (1996). Linkage of decreased bone mass with impaired osteoblastogenesis in a murine model of accelerated senescence. J Clin Invest 97, 1732-1740. Jurkowska, R.Z., Rajavelu, A., Anspach, N., Urbanke, C., Jankevicius, G., Ragozin, S., Nellen, W., and Jeltsch, A. (2011). Oligomerization and binding of the Dnmt3a DNA methyltransferase to parallel DNA molecules: heterochromatic localization and role of Dnmt3L. J Biol Chem 286, 24200-24207. Kao, T.H., Liao, H.F., Wolf, D., Tai, K.Y., Chuang, C.Y., Lee, H.S., Kuo, H.C., Hata, K., Zhang, X., Cheng, X., et al. (2014). Ectopic DNMT3L triggers assembly of a repressive complex for retroviral silencing in somatic cells. J Virol 88, 10680-10695. Kareta, M.S., Botello, Z.M., Ennis, J.J., Chou, C., and Chedin, F. (2006). Reconstitution and mechanism of the stimulation of de novo methylation by human DNMT3L. J Biol Chem 281, 25893-25902. Kilian, K.A., Bugarija, B., Lahn, B.T., and Mrksich, M. (2010). Geometric cues for directing the differentiation of mesenchymal stem cells. Proc Natl Acad Sci U S A 107, 4872-4877. Kim, D., Pertea, G., Trapnell, C., Pimentel, H., Kelley, R., and Salzberg, S.L. (2013). TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14, R36. Kim, M., Kim, C., Choi, Y.S., Kim, M., Park, C., and Suh, Y. (2012). Age-related alterations in mesenchymal stem cells related to shift in differentiation from osteogenic to adipogenic potential: implication to age-associated bone diseases and defects. Mech Ageing Dev 133, 215-225. Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357-359. Li, C., Zhen, G., Chai, Y., Xie, L., Crane, J.L., Farber, E., Farber, C.R., Luo, X., Gao, P., Cao, X., et al. (2016). RhoA determines lineage fate of mesenchymal stem cells by modulating CTGF-VEGF complex in extracellular matrix. Nat Commun 7, 11455. Liao, H.F., Chen, W.S., Chen, Y.H., Kao, T.H., Tseng, Y.T., Lee, C.Y., Chiu, Y.C., Lee, P.L., Lin, Q.J., Ching, Y.H., et al. (2014). DNMT3L promotes quiescence in postnatal spermatogonial progenitor cells. Development 141, 2402-2413. Liao, H.F., Mo, C.F., Wu, S.C., Cheng, D.H., Yu, C.Y., Chang, K.W., Kao, T.H., Lu, C.W., Pinskaya, M., Morillon, A., et al. (2015). Dnmt3l-knockout donor cells improve somatic cell nuclear transfer reprogramming efficiency. Reproduction 150, 245-256. Liao, H.F., Tai, K.Y., Chen, W.S., Cheng, L.C., Ho, H.N., and Lin, S.P. (2012). Functions of DNA methyltransferase 3-like in germ cells and beyond. Biol Cell 104, 571-587. Lopez-Otin, C., Blasco, M.A., Partridge, L., Serrano, M., and Kroemer, G. (2013). The hallmarks of aging. Cell 153, 1194-1217. Lunyak, V.V., and Rosenfeld, M.G. (2008). Epigenetic regulation of stem cell fate. Hum Mol Genet 17, R28-36. Matsumura, Y., Nakaki, R., Inagaki, T., Yoshida, A., Kano, Y., Kimura, H., Tanaka, T., Tsutsumi, S., Nakao, M., Doi, T., et al. (2015). H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation. Mol Cell 60, 584-596. Meunier, P., Aaron, J., Edouard, C., and Vignon, G. (1971). Osteoporosis and the replacement of cell populations of the marrow by adipose tissue. A quantitative study of 84 iliac bone biopsies. Clin Orthop Relat Res 80, 147-154. Molaro, A., Falciatori, I., Hodges, E., Aravin, A.A., Marran, K., Rafii, S., McCombie, W.R., Smith, A.D., and Hannon, G.J. (2014). Two waves of de novo methylation during mouse germ cell development. Genes Dev 28, 1544-1549. Mueller, S.M., and Glowacki, J. (2001). Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges. J Cell Biochem 82, 583-590. Neri, F., Krepelova, A., Incarnato, D., Maldotti, M., Parlato, C., Galvagni, F., Matarese, F., Stunnenberg, H.G., and Oliviero, S. (2013). Dnmt3L antagonizes DNA methylation at bivalent promoters and favors DNA methylation at gene bodies in ESCs. Cell 155, 121-134. Niyibizi, C., Wang, S., Mi, Z., and Robbins, P.D. (2004). The fate of mesenchymal stem cells transplanted into immunocompetent neonatal mice: implications for skeletal gene therapy via stem cells. Mol Ther 9, 955-963. Ooi, S.K., Qiu, C., Bernstein, E., Li, K., Jia, D., Yang, Z., Erdjument-Bromage, H., Tempst, P., Lin, S.P., Allis, C.D., et al. (2007). DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 448, 714-717. Otani, J., Nankumo, T., Arita, K., Inamoto, S., Ariyoshi, M., and Shirakawa, M. (2009). Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain. EMBO Rep 10, 1235-1241. Park, D., Spencer, J.A., Koh, B.I., Kobayashi, T., Fujisaki, J., Clemens, T.L., Lin, C.P., Kronenberg, H.M., and Scadden, D.T. (2012). Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration. Cell Stem Cell 10, 259-272. Phinney, D.G., Kopen, G., Isaacson, R.L., and Prockop, D.J. (1999). Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation. J Cell Biochem 72, 570-585. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147. Polioudaki, H., Kastrinaki, M.C., Papadaki, H.A., and Theodoropoulos, P.A. (2009). Microtubule-interacting drugs induce moderate and reversible damage to human bone marrow mesenchymal stem cells. Cell Prolif 42, 434-447. Reik, W. (2007). Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425-432. Rodriguez, J.P., Gonzalez, M., Rios, S., and Cambiazo, V. (2004). Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem 93, 721-731. Rosen, C.J., and Bouxsein, M.L. (2006). Mechanisms of disease: is osteoporosis the obesity of bone? Nat Clin Pract Rheumatol 2, 35-43. Ross, S.E., Hemati, N., Longo, K.A., Bennett, C.N., Lucas, P.C., Erickson, R.L., and MacDougald, O.A. (2000). Inhibition of adipogenesis by Wnt signaling. Science 289, 950-953. Sasaki, H., and Matsui, Y. (2008). Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nat Rev Genet 9, 129-140. Seisenberger, S., Andrews, S., Krueger, F., Arand, J., Walter, J., Santos, F., Popp, C., Thienpont, B., Dean, W., and Reik, W. (2012). The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol Cell 48, 849-862. Sethe, S., Scutt, A., and Stolzing, A. (2006). Aging of mesenchymal stem cells. Ageing Res Rev 5, 91-116. Sharp, D.J., Rogers, G.C., and Scholey, J.M. (2000). Microtubule motors in mitosis. Nature 407, 41-47. Suetake, I., Shinozaki, F., Miyagawa, J., Takeshima, H., and Tajima, S. (2004). DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction. J Biol Chem 279, 27816-27823. Surani, M.A., Barton, S.C., and Norris, M.L. (1984). Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308, 548-550. Trapnell, C., Hendrickson, D.G., Sauvageau, M., Goff, L., Rinn, J.L., and Pachter, L. (2013). Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31, 46-53. Vacanti, V., Kong, E., Suzuki, G., Sato, K., Canty, J.M., and Lee, T. (2005). Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture. J Cell Physiol 205, 194-201. Vasiliauskaite, L., Berrens, R.V., Ivanova, I., Carrieri, C., Reik, W., Enright, A.J., and O'Carroll, D. (2018). Defective germline reprogramming rewires the spermatogonial transcriptome. Nat Struct Mol Biol 25, 394-404. Wegmeyer, H., Broske, A.M., Leddin, M., Kuentzer, K., Nisslbeck, A.K., Hupfeld, J., Wiechmann, K., Kuhlen, J., von Schwerin, C., Stein, C., et al. (2013). Mesenchymal stromal cell characteristics vary depending on their origin. Stem Cells Dev 22, 2606-2618. Young, H.E., Steele, T.A., Bray, R.A., Hudson, J., Floyd, J.A., Hawkins, K., Thomas, K., Austin, T., Edwards, C., Cuzzourt, J., et al. (2001). Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors. Anat Rec 264, 51-62. Zamudio, N., Barau, J., Teissandier, A., Walter, M., Borsos, M., Servant, N., and Bourc'his, D. (2015). DNA methylation restrains transposons from adopting a chromatin signature permissive for meiotic recombination. Genes Dev 29, 1256-1270. Zhang, Y., Jurkowska, R., Soeroes, S., Rajavelu, A., Dhayalan, A., Bock, I., Rathert, P., Brandt, O., Reinhardt, R., Fischle, W., et al. (2010). Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail. Nucleic Acids Res 38, 4246-4253. Zhang, Z.M., Jiang, L.S., Jiang, S.D., and Dai, L.Y. (2009). Osteogenic potential and responsiveness to leptin of mesenchymal stem cells between postmenopausal women with osteoarthritis and osteoporosis. J Orthop Res 27, 1067-1073. Zuk, P.A., Zhu, M., Ashjian, P., De Ugarte, D.A., Huang, J.I., Mizuno, H., Alfonso, Z.C., Fraser, J.K., Benhaim, P., and Hedrick, M.H. (2002). Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13, 4279-4295. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78989 | - |
dc.description.abstract | 類3號DNA甲基化酶(DNA methyltransferase 3-lke, DNMT3L)為主要表現於胚胎幹細胞和生殖細胞的表觀遺傳因子,其雖不具酵素功能,但可透過與DNA甲基轉移酶3A和3B作用以催化DNA甲基化之建立。先前實驗室觀察到一歲以上缺少Dnmt3l的小鼠和野生型相比,體型明顯瘦小,且擁有較少量的白色脂肪組織,以及高於3的骨骼結構模型指數(Structure model index, SMI); 此指數數值是在較年長者中可觀察到的趨勢,並且暗示缺少Dnmt3l的小鼠可能有提早衰老現象。除此之外,我們也證實可分化成脂肪以及硬骨的前驅細胞-間葉幹細胞(Mesenchymal stem/stromal cells, MSCs),從缺乏Dnmt3l的小鼠骨髓中分離進行體外培養後,成骨能力明顯下降。
雖然使用通用的蛋白質表現量偵測技術,例如西方墨點法,DNMT3L在一般體細胞譜系中檢測不到,包括間葉幹細胞也未偵測到DNMT3L的表現。然而本實驗室過去針對完全沒有DNMT3L表現之小鼠胚胎纖維母細胞的研究顯示,相較於野生型同胎胚胎取得之纖維母細胞,來自Dnmt3l缺失小鼠胚胎的胚胎纖維母細胞,體外培養後觀察到提早衰老和相對鬆散的染色質結構,說明在胚胎幹細胞(Embryonic stem cells, ESCs)及某些前驅細胞中短暫表達的DNMT3L,不只影響胚胎幹細胞當下的表觀遺傳標記與基因表現,經由部分表觀基因體標記之保留性,於幹細胞分化後,DNMT3L不再表現時,仍得以影響體細胞的染色質結構以及細胞功能。因此,本論文著重於探討在胚胎幹細胞時DNMT3L存在與否所影響的表觀基因體標示,其效應是否會延續至間葉幹細胞,而對間葉幹細胞及其衍生細胞系的基因表達造成影響,使得從缺乏Dnmt3l的小鼠骨髓中分離之間葉幹細胞,分化成骨骼系細胞的潛能下降。 於本論文中,我們首先利用細胞表面抗原表現概觀從野生型以及缺乏Dnmt3l小鼠所分離的間葉幹細胞細胞族群組成相似,另外,除了間葉幹細胞硬骨分化能力受到基因缺乏Dnmt3l影響外,根據細胞在低密度環境下形成群落的效率測試中發現,來自缺乏Dnmt3l的間葉幹細胞形成聚落的效率較低。然而細胞生長速率在較前期(P4)的細胞代數中並無明顯差異,但過去實驗室結果顯示從骨骺分離的間葉幹細胞族群中,缺乏Dnmt3l小鼠的間葉幹細胞在相對後期的細胞代數(P6)有較低的生長速率。綜合自我更新以及生長速率測試結果暗示缺乏Dnmt3l可能造成小鼠的間葉幹細胞提早衰老,造成幹細胞耗竭。 另外,我們藉由RNA定序結果中剖析來自野生型和缺乏Dnmt3l小鼠的間葉幹細胞在誘導硬骨分化前以及分化第3天的基因表達。結果顯示在硬骨分化前,野生型和缺乏Dnmt3l小鼠來源的間葉幹細胞之間差異表達基因(Differentially expressed genes, DEGs)與骨頭型態的相關途徑相關 ; 而誘導硬骨分化三天的差異表達基因則顯示和微管(microtubule)的動態活性有關。這些基因表現分析暗示缺乏Dnmt3l小鼠來源的間葉幹細胞不正常的基因表達可能是失去幹細胞特性和硬骨分化能力有關。 接著我們試圖了解DNMT3L在胚胎幹細胞時期大量表現對於表觀基因組和轉錄組的影響,且探究此影響是否持續至間葉幹細胞並和維持幹細胞特性以及硬骨分化潛能有關。我們下載並重新分析三個已發表的資料集,包含DNMT3L直接結合位點(使用 ChIP-seq)、Dnmt3l knockdown的胚胎幹細胞以及控制組的基因表達 (Microarray) 和DNA甲基化程度 (MeDIP-seq)。並將上述的資料集個別和野生型以及缺失Dnmt3l間葉幹細胞之間,於硬骨分化前、以及誘導硬骨分化第三天,差異表現的基因進行聯集,以找出DNMT3L於胚胎幹細胞之存在與否所造成的基因表現以及DNA甲基化程度不同,和兩種基因型之間,間葉幹細胞的轉錄體組成有無直接的關聯性。我們發現在Dnmt3l knockdown的胚胎幹細胞中,總體啟動子和基因本體上的甲基化水平較高。此外,我們發現於兩種基因型間葉幹細胞中,差異表現之基因,有較高比例亦屬於胚胎幹細胞中,因DNMT3L存在與否而造成基因本體上甲基化的差異所導致。 總論,我們發現胚胎幹細胞中DNMT3L的表現,其效應可持續至間葉幹細胞並影響幹細胞特性以及硬骨分化。 | zh_TW |
dc.description.abstract | DNA methyltransferase 3-like (DNMT3L) is an epigenetic modulating factor, mainly expressed in embryonic stem cells and germ cells. It’s a well-known factor for transcriptional silencing and facilitating de novo DNA methylation with DNMT3A and DNMT3B. Previously, we observed that Dnmt3l knockout (KO) mice over one year old display lower body weight and reduced white adipose tissue associated with a higher structural model index, which is a trend observed in older animals and human, insinuating the pre-mature aging phenomenon. Moreover, we demonstrated that Dnmt3l KO mice derived mesenchymal stem/stromal cells (MSCs), the progenitor cells of adipocytes and osteoblasts, have seriously impaired osteogenesis ability in vitro.
Although we cannot exclude the lower DNMT3L expression in some subpopulation of MSCs, DNMT3L is certainly not expressed in mouse embryonic fibroblasts (MEFs). However, premature senescence and relatively relaxed chromatin structure are observed in cultured mouse embryonic fibroblast derived from Dnmt3l KO embryos compared to counterparts from wild type littermates. These results suggested that transiently expressed DNMT3L mediated epigenetic marks established in embryonic stem cells (ESCs) may have long-lasting effect on the chromatin structure and phenotype of differentiated cells, long after DNMT3L is silenced. Therefore, this thesis focuses on studying whether DNMT3L mediated epigenetic legacy in ESCs may affect their differentiation downstream gene expression and osteogenic potential in MSCs in vitro. First, we investigated the expressions of typical cell surface markers on mesenchymal stem cell populations isolated from WT and Dnmt3l-deficient mice, and observed that these cells exhibit similar cell surface markers expression. However, besides the osteogenic potential that was affected by the lack of DNMT3L, the lower colony forming efficiency in Dnmt3l KO mice-derived MSCs was observed. Although there was no significant difference in growth rate between WT and Dnmt3l KO mice-derived MSCs at the early passage (P4), our results showed that a decreased rate of cell growth at late-passage (P6) in the epiphysis-derived MSCs population from Dnmt3l KO mice. Furthermore, the results of colony forming efficiency assay and growth curve test implicated that MSCs from Dnmt3l KO mice have the lower self-renewal capacity and premature senescence phenotype. In the second part of the thesis, I investigated the gene expression pattern using RNA-sequencing analysis of MSCs derived from WT and Dnmt3l KO mice. I compared the transcriptome from the two genotypes for both undifferentiated MSCs and differentiating derivatives on day 3 of osteogenic induction, and found that differentially expressed genes (DEGs) between WT and Dnmt3l KO mice-derived MSCs are highlighted in various pathways related to bone morphology in undifferentiated group and associated with microtubule activity in 3 days of osteogenic induction. These gene ontology analyses implied that distinct gene expression on proliferation and differentiation may serve as the cue of compromised stemness in Dnmt3l KO mice-derived MSCs. In order to investigate the mechanisms of how DNMT3L in ESCs trigger a cascade of epigenomic and transcriptome changes that lead to long-term effect on the MSCs and modulate the differentiation potential of MSCs for osteogenesis. We downloaded and reanalyzed the published dataset including DNMT3L binding region (by ChIP-seq), gene expression profile (by Microarray) and DNA methylation level (by MeDIP-seq) in Dnmt3l knockdown and control ESCs. We compared these DNMT3L associated changes in ESCs with Dnmt3l genotype dependent gene expression profile of undifferentiated and differentiating MSCs. The global methylation level of promoter and gene body regions are both higher in Dnmt3l knockdown ESCs. In addition, we found that the obvious differentially expressed genes between WT and Dnmt3l KO MSCs are also genes associated with DNMT3L-dependent methylation on gene bodies in ESCs. Collectively, this thesis reveals that transient DNMT3L expression in ESCs has lasting effect in MSCs, including their self-renew and osteogenic differentiation. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:34:37Z (GMT). No. of bitstreams: 1 ntu-107-R05b43001-1.pdf: 2778034 bytes, checksum: 796b8b1441c9799c6b234ca7643956fc (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 致謝 i
中文摘要 ii ABSTRACT v CONTENTS viii LIST OF FIGURES xii LIST OF TABLES xiv Chapter 1 Introduction 1 1.1 Epigenetic regulation in stem cells 1 1.2 DNA methyltransferase 3-like (DNMT3L): an important epigenetic modulator 2 1.2.1 DNMT3L in facilitating de novo DNA methylation 2 1.2.2 DNMT3L participates in histone modification 3 1.3 Dnmt3l KO mice display impaired mesenchymal lineages phenotype 5 1.4 Mesenchymal stem cells 6 1.4.1 History and property of MSCs 6 1.4.2 Senescence of MSCs 7 1.5 Dnmt3l KO mouse derived MSCs significantly decrease osteogenic potential 8 Chapter 2 Specific aims 11 Chapter 3 Material and Methods 12 3.1 Animals 12 3.2 Dnmt3l genotyping 12 3.3 Isolation and culture of mouse bone marrow-derived mesenchymal stem cells 13 3.4 Flow cytometry 15 3.5 Colony-forming Unit (CFU) assay 16 3.6 Cell growth assay 17 3.7 Osteogenic differentiation 17 3.8 Adipogenic differentiation 18 3.9 RNA library preparation and sequencing 19 3.10 RNA-sequencing analysis 19 3.11 Bioinformatic analysis on ChIP-seq and MeDIP-seq 20 3.12 Western blot 20 3.12.1 Protein extraction and BCA protein assay 20 3.12.2 Buffer preparation 21 3.12.3 Preparation of SDS-PAGE gel 22 3.12.4 Sample loading 22 3.12.5 Electrophoresis 22 3.12.6 Protein transfer 23 3.12.7 Antibody staining 23 Chapter 4 Results 25 4.1 Surface marker expression profile of MSCs isolated from Dnmt3l KO and wild-type littermates 25 4.2 Dnmt3l deficiency influences MSCs self-renew potential 26 4.3 The impaired properties in Dnmt3l KO mice-derived MSCs suggested by transcriptome analysis 27 4.4 The DNMT3L-mediated epigenetic hypothesis 29 4.5 Searching for potential DNMT3L-mediated epigenetic memory from ESCs 30 4.5.1 DNMT3L ChIP-sequencing analysis 30 4.5.2 DNMT3L dependent differentially expressed genes in ESCs and MSCs 31 4.5.3 DNA methylation analysis (MeDIP sequencing) in ESCs 32 Chapter 5 Discussion 33 5.1 Dnmt3l KO derived MSCs may have pre-mature aging characteristics 33 5.2 The imbalance of osteogenesis and adipogenesis in pre-senescent MSCs 33 5.3 Unusual bivalent H3K4/H3K9me3 modification of adipogenic related genes hypothesis 34 5.4 The potential epigenetic legacy from ESCs 36 Chapter 6 Figures 39 Chapter 7 Tables 52 Chapter 8 Appendix 55 REFERENCE 61 | |
dc.language.iso | en | |
dc.title | 類3號DNA甲基化酶對於骨髓間葉幹細胞之長期影響 | zh_TW |
dc.title | DNMT3L Mediated Legacy Has Long Term Effect on Bone Marrow-Derived Mesenchymal Stem/Stromal Cell | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 林劭品 | |
dc.contributor.oralexamcommittee | 劉逸軒,洪士杰,蕭士翔 | |
dc.subject.keyword | 表觀基因體,類3號DNA甲基化?,骨髓,間葉幹細胞,硬骨分化,提早老化, | zh_TW |
dc.subject.keyword | epigenetic,DNMT3L,bone-marrow,mesenchymal stem cells,osteogenesis,pre-senescence, | en |
dc.relation.page | 70 | |
dc.identifier.doi | 10.6342/NTU201803415 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-15 | |
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 |
dc.date.embargo-lift | 2023-08-21 | - |
顯示於系所單位: | 分子與細胞生物學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-107-R05b43001-1.pdf 目前未授權公開取用 | 2.71 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。