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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 周子賓(Tze-Bin Chou) | |
| dc.contributor.author | Hung-Chun Tung | en |
| dc.contributor.author | 董虹君 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:33:28Z | - |
| dc.date.available | 2020-08-24 | |
| dc.date.copyright | 2017-08-24 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-17 | |
| dc.identifier.citation | Aravin, A., Gaidatzis, D., Pfeffer, S., Lagos-Quintana, M., Landgraf, P., Iovino, N., Morris, P., Brownstein, M.J., Kuramochi-Miyagawa, S., Nakano, T., et al. (2006). A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442, 203-207.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68748 | - |
| dc.description.abstract | 組織恆定及再生有賴於幹細胞的維持。幹細胞為一群具有自我更新及分化能力的細胞，能夠不斷分化出子細胞。而生殖幹細胞能夠生成配子，並將其遺傳和表觀遺傳訊息傳遞給下一代，對物種延續及子代健康極具重要性。而在生殖細胞發育過程中，有DNA甲基化去除及再建立的過程以建立生殖細胞特化之基因模式，後者有賴於3號DNA甲基化酶(DNMT3s, DNA methyltransferases 3)的幫助。由於甲基化的去除會造成反轉錄轉位子(retrotransposons)的活化，而此舉恐會使轉位子有能力在基因中任意跳躍而影響基因體的完整性，甚至造成無法產生配子。生殖細胞因此演化出特化小片段RNA (piRNA, PIWI-interacting RNA)路徑得以抑制轉位子的跳躍。
類3號DNA甲基化酶(DNMT3L, DNA methyltransferase 3-like)雖不具酵素功能，卻得以輔助3號A型DNA甲基化酶(DNMT3A, DNA methyltransferase 3A)催化的甲基化再建立，以抑制轉位子的跳躍，並影響piRNA的組成；且本實驗室發現其在精原細胞之細胞質表現，有可能與轉錄後調節相關。本篇論文著重於探討8天大小鼠精原細胞在缺少類3號DNA甲基化酶情況下生殖細胞維持分子和piRNA生成相關因子之表現量、細胞內座落位置及相互關係是否受到影響。本論文第一部分證實在缺少DNMT3L的情況下，會劇烈影響精原細胞之自我更新、分化，以及piRNA路徑相關基因的表現量，且反轉錄轉位子無法被抑制而大量活化。早幼粒細胞白血病鋅指蛋白(PLZF, Promyelocytic Leukemia Zinc Finger)為幹細胞與前軀細胞自我更新，及表觀基因體調節相關因子，而由於DNMT3L與PLZF的相互關係已被本實驗室證實，且MILI及PLZF此二蛋白在文獻中都指出另參與在轉錄後或轉譯層面的調控，因此第二部分實驗著重於此二蛋白。藉由蛋白質免疫定量及細胞免疫螢光染色確立Dnmt3l突變鼠之精原細胞中，PLZF的表現量顯著減少，且幾乎完全喪失位於細胞質之PLZF。而DNMT3L對MILI的影響則局限於表現量。進一步利用免疫共沉澱法證實MILI與PLZF相互結合關係，並觀察到TDRD2 (Tudor Domain-Containing Protein 2)及MILI的相互結合關係是會受到DNMT3L所影響的。 總結，本論文結果顯示Dnmt3l存在與否直接影響精原細胞中piRNA生成相關因子之表現量，因此極可能對piRNA生成產生嚴重影響。並推斷DNMT3L在轉錄及後轉錄層面影響MILI及PLZF的座落位置及表現量，以及MILI-PLZF共同參與抑制反轉錄轉位子的角色的可能性。更由於MILI為piRNA路徑的重要角色之一，故建議進一步研究PLZF參與調節piRNA路徑的可能性。 | zh_TW |
| dc.description.abstract | Tissue homeostasis and regeneration relies on the maintenance of stem cells. Stem cells are a group of cells with self-renewal and differentiation ability and can continue to differentiate into daughter cells. Germline stem cells can produce gametes, and transfer its genetic and epigenetic information to next generation, that is crucial for producing viable and healthy offspring. During germ cell development, DNA sequences undergo demethylation and re-establishment of methylation pattern to regulate germ cell specific gene pattern, and the latter depends on DNA methyltransferases 3 (DNMT3s). The removal of methylation can cause the re-activation of retrotransposons. Activated retrotransposons have the ability to randomly jump in the genome and affect the integrity of the genome, and even cause infertility. For this reason, germ cells have developed specialized piRNA (PIWI-interacting RNA) pathway to suppress the transposition of the transposons.
Though DNMT3L (DNA methyltransferase 3 like) has no catalytic ability, but is critical to facilitate DNMT3A (DNA methyltransferase 3A) mediated DNA methylation re-establishment to repress retrotransposons. DNMT3L also affects the compositions of piRNA and the localization of DNMT3L in cytoplasm at postnatal stage, implies its influence on post-transcriptional regulation. This thesis focus on studying whether the absence of Dnmt3l would affect the expression, subcellular localization and interactions among germ cell surviving factors and piRNA biogenesis related factors in spermatogonia of 8 dpp mouse testes. In the first part of this thesis, I confirmed that in the absence of Dnmt3l, most of the tested self-renewal factors, differentiation factors, and piRNA pathway components are seriously down-regulated and also cause the failure of retrotransposon repression. Our lab previously observed the interaction between DNMT3L and PLZF (Promyelocytic Leukemia Zinc Finger) which is a self-renewal factor involving in epigenetic modulation. In addition, since both MILI and PLZF proteins are shown to be involved in post-transcriptional and translational regulation of retrotransposons, I focused on these two proteins in the second part of this thesis. Western blotting and immunofluorescent staining analyses confirmed that the expression and localization of PLZF were affected in Dnmt3l KO testes, while the effect on MILI was only restricted in the expression level. Furthermore, I confirmed the interaction between MILI and PLZF in 8 dpp testes by co-immunoprecipitation. I also observed a DNMT3L-dependent interaction between TDRD2 (Tudor Domain-Containing Protein 2) and MILI. In conclusion, the presence of Dnmt3l affects the expression level of piRNA biogenesis related factors that shows great importance for the production of piRNAs. My data also indicated that DNMT3L affected the expression and localization of PLZF. The observed MILI-PLZF interaction provided further implications on their potential collaborative function in retrotransposon silencing at transcriptional or post-transcriptional level. Moreover, as MILI is the most important player in piRNA pathway, the interaction of MILI and PLZF also suggested the potential influence of PLZF in the piRNA pathway. | en |
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| dc.description.tableofcontents | 致謝 I
中文摘要 II ABSTRACT IV CONTENTS VII LIST OF TABLES IX LIST OF FIGURES X ABBREVIATIONS XI CHAPTER 1 INTRODUCTION 1 1.1 MOUSE MALE GERMLINE DEVELOPMENT AND MAINTENANCE 1 1.1.1 STEM CELLS, THE FOUNDATION OF TISSUE HOMEOSTASIS 1 1.1.2 PRECURSORS OF UNDIFFERENTIATED SPERMATOGONIA 3 1.1.3 SPERMATOGENESIS 5 1.1.4 TRANSPOSONS 7 1.2 IMPORTANT FACTORS FOR TRANSPOSON SILENCING 8 1.2.1 DNMT3L (DNA METHYLATRANSFERASE 3-LIKE) 8 1.2.2 PIRNA PATHWAY: THE GUARD OF GENOME INTEGRITY 9 1.2.3 MILI: A KEY COMPONENT IN PIRNA PATHWAY FOR FACILITATING DE NOVO METHYLATION 10 1.2.4 PLZF IN GERMLINE MAINTENANCE AND RETROTRANSPOSON SILENCING 10 1.3 SIGNIFICANCE 12 1.4 HYPOTHESIS 12 1.5 SPECIFIC AIMS 13 CHAPTER 2 MATERIALS AND METHODS 14 2.1 BREEDING OF DNMT3L MUTANT MOUSE 14 2.2 GENOTYPING 14 2.3 H&E STAINING 15 2.4 REVERSE TRANSCRIPTION QUANTITATIVE PCR (RT-QPCR) 16 2.5 PREPARATION OF SINGLE CELL SUSPENSION FROM GERM CELL ENRICHED SEMINIFEROUS TUBULE CONTENTS 18 2.6 IMMUNOCYTOCHEMISTRY 20 2.7 IMMUNOHISTOCHEMISTRY 21 2.8 WESTERN BLOTTING 23 2.9 IMMUNOPRECIPITATION 25 2.10 STATISTICAL ANALYSIS 27 2.11 RNA SEQUENCING ANALYSIS 27 CHAPTER 3 RESULTS 28 3.1 GRADUAL DECLINE OF GERM CELLS IN DNMT3L KO TESTES AT POSTNATAL STAGE 28 3.2 CRUCIAL FUNCTIONS OF DNMT3L IN EXPRESSIONS OF GERMLINE MAINTENANCE FACTORS AND RETROTRANSPOSON SILENCING 29 3.3 EXPRESSION PATTERNS AND LOCALIZATION OF PLZF IN DNMT3L KO GERM CELLS AT 8 DPP. 31 3.4 DNMT3L CONTRIBUTES TO MILI-PLZF AND MILI-TDRD2 INTERACTIONS IN 8 DPP TESTES 32 CHAPTER 4 DISCUSSION 33 4.1 ESSENTIAL ROLE OF DNMT3L IN EXPRESSIONS OF GERMLINE MAINTENANCE FACTORS 33 4.2 THE ISOFORM OF MILI 34 4.3 POTENTIAL NOVEL ROLES OF DNMT3L IN POSTNATAL SPERMATOGONIA THROUGH INFLUENCING MILI-PLZF AND MILI-TDRD2 INTERACTIONS 35 4.4 PERSPECTIVE 37 CHAPTER 5 REFERENCES 59 | |
| dc.language.iso | en | |
| dc.title | 類3號DNA甲基化酶維持初生小鼠精原細胞恆定之機制探討 | zh_TW |
| dc.title | DNMT3L Mediated Germline Maintenance in Postnatal
Spermatogonia | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 林劭品(Shau-Ping Lin) | |
| dc.contributor.oralexamcommittee | 靖永皓(Yun-Hao Ching),曾大千(Ta-Chien Tseng),洪瑞鴻(Jui-Hung Hung) | |
| dc.subject.keyword | piRNA 路徑,PLZF,類 3 號 DNA 甲基化?,精原細胞, | zh_TW |
| dc.subject.keyword | DNMT3L,piRNA pathway,PLZF,spermatogonia, | en |
| dc.relation.page | 64 | |
| dc.identifier.doi | 10.6342/NTU201702966 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-18 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-106-1.pdf 目前未授權公開取用 | 2.95 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。