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???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
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dc.contributor.advisor | 林劭品(Shau-Ping Lin) | |
dc.contributor.author | Hsien-Hen Lin | en |
dc.contributor.author | 林咸亨 | zh_TW |
dc.date.accessioned | 2021-06-17T02:41:14Z | - |
dc.date.available | 2020-08-31 | |
dc.date.copyright | 2017-08-31 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-16 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68903 | - |
dc.description.abstract | 生殖細胞在基因延續與物種演化中扮演重要的角色。因此,性腺與生殖幹細胞能適當且正確地維持與分化極為重要。跳躍子 (transposon) 能在基因間移動的特性,可能影響生殖幹細胞的基因穩定度而造成缺失。為了調控跳躍子的表現,生殖細胞發展出轉錄與後轉錄機制,分別於不同層面調節跳躍子。然而部分報導指出,兩個機制可能合作並協同調控跳躍子的表現。類第三型 DNA 甲基化酶 (DNMT3L) 是轉錄調控機制中參與 DNA 甲基化的重要蛋白,而研究顯示 DNMT3L 可能會參與後轉錄調控機制 PIWI-interacting RNA pathway,於成熟 piRNA 進入細胞核調控表觀基因體時,協助建立DNA 甲基化以調節跳躍子的表現。近期,我們實驗室的初步研究顯示 DNMT3L 極可能不只於 piRNA pathway 下游間接透過轉錄機制調控跳躍子,還可能參與 piRNA pathway 其他層面。於不同發育階段的小鼠生殖細胞研究結果顯示,剔除 DNMT3L 會造成 8 天大之小鼠睪丸中 piRNA pathway 相關蛋白的 RNA 表現量改變。其中一個相關蛋白 MAEL 於細胞內的表現量及位置,會因生殖細胞發育階段之不同而有不同程度之變異。此外,DNMT3L 的缺失也會影響 piRNA 的族群結構。儘管種種證據指出 DNMT3L 不只在 piRNA pathway 的下游作用,目前仍不清楚 DNMT3L 是如何對 piRNA pathway 造成影響。本研究利用生物資訊工具進行正常小鼠及 Dnmt3l 基因剔除小鼠 piRNA 序列分析,探討在 0 天小鼠生殖細胞與 8 天小鼠精原母細胞 (spermatogonial stem cell THY1+ enriched population) 中 DNMT3L 可能參與 piRNA pathway 的層面為何。結果顯示,microRNA 及 piRNA 的相對比例並沒有因為 DNMT3L 剔除出現明顯變化。然而,在 DNMT3L 缺失的 8 天小鼠精原母細胞中,piRNA 生成的位置與組成有極大的變化,且位於基因上的位置相較於野生型明顯不同。進一步研究 piRNA前驅物 (precursor) 發現,DNMT3L 缺失基因型生成的 piRNA 前驅物有極高比例帶有 ZBTB12、STAT3 或 FOXP1 蛋白的結合位。本研究顯示 DNMT3L 除了 piRNA pathway下游外,還會影響 piRNA 前驅物的生成位置並導致 piRNA 組成改變。然而 DNMT3L 是否會參與更多層面並影響 piRNA pathway 仍須更深入的研究。 | zh_TW |
dc.description.abstract | It is crucial to preserve appropriate germline development, differentiation and maintenance, as germ cells are responsible for genetic inheritance, reproduction and evolution. Transposons may cause genome instability and lead to germ cell defects and infertility due to their dysregulation and derepression. To combat with transposons, transcriptional as well as post-transcriptional regulations have been developed in germline, and these two pathways have been indicated to cooperate. In mice, DNA methyltransferase 3-like protein (DNMT3L), an important factor participating in de novo DNA methylation for transcriptional regulation. It is also suggested that DNMT3L may act downstream of PIWI-interacting RNA (piRNA) pathway, which is mainly responsible for post-transcriptional regulation. Mature piRNA-PIWI complex may also guide sequence specific histone modification and subsequent de novo DNA methylation potentially facilitated partly by DNMT3L. However, according to our following preliminary data, DNMT3L seems to act not only downstream but also participate in other level of piRNA pathway. As the result of studying mice spermatogonial stem cells (SSCs) from different developmental stage, in 8 dpp Dnmt3l-deficiency samples, RNA expression of several piRNA pathway components are down regulated; MAEL, one of the piRNA pathway components, is mislocalized in E18.5 dpc germ cells from Dnmt3l KO embryos, and its expression level varies in the mutant spertmatogonia cells of different developmental stages compared to those from wild type littermates; besides, Dnmt3l-deficiency results in distinct piRNA composition. Though evidence from different developmental stages above suggests the noncanonical role of DNMT3L, it is still unclear how DNMT3L participates in and makes influence on piRNA pathway. In this study, we apply bioinformatics tools to analyze small RNA sequencing datasets to investigate the potential role of DNMT3L in 0 dpp germ cells and 8 dpp spermatogonial stem cell (SSC) enriched THY1+ population. Results indicated that relative proportion of microRNA and piRNA were similar between WT and Dnmt3l KO mice. However, piRNA origin and composition significantly differed in 8 dpp Dnmt3l-deficiency samples, and genomic location of predicted precursors remarkably shifted as well. Further analysis revealed that the binding sites of ZBTB12, STAT3 and FOXP1 highly enriched among predicted precursors with high piRNA production in 8 dpp Dnmt3l KO samples. This report suggests that DNMT3L should act not only downstream of piRNA pathway, especially affecting piRNA precursor production and thus resulting in distinct piRNA composition. However, whether DNMT3L participates in other levels of piRNA pathway needs more investigation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:41:14Z (GMT). No. of bitstreams: 1 ntu-106-R04642001-1.pdf: 3873075 bytes, checksum: e5dfb363ccabd379a36b2d18f88a7862 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 致謝 ii 中文摘要 iii ABSTRACT iv CONTENTS vi LIST OF FIGURES ix LIST OF TABLES xi Chapter 1 Introduction 1 1.1 Germline development 1 1.1.1 The importance of germline research 1 1.1.2 The development stages of germ cells 1 1.1.3 Two waves of demethylation during preimplantation embryo and germline development 2 1.1.4 Proper epigenomic regulations maintain adequate germline functions 3 1.2 Transposon: a potential threat to genome stability 3 1.2.1 Two types of transposons 3 1.2.2 The mechanism of transposons participating in and interfering with genome stability and development 4 1.2.3 The regulation systems against transposons 6 1.3 piRNA pathway: an evolutionarily conserved regulatory system against transposons 6 1.3.1 The structure of piRNA and PIWI proteins 6 1.3.2 The piRNA clusters 7 1.3.3 The biogenesis of piRNA, including primary piRNAs and secondary piRNAs 8 1.3.4 The pathway of piRNA modulating transposons 10 1.4 DNMT3L: an important regulators of gene expression 11 1.4.1 DNMT3L in facilitating de novo methylation 11 1.4.2 DNMT3L interacts with histone H3 and influences histone modification 12 1.4.3 The relationship between DNMT3L and piRNA pathway 13 Chapter 2 Specific aim 15 Chapter 3 Material and Methods 17 3.1 Breeding of Dnmt3l mouse and sample preparation 17 3.2 Small RNA-sequencing and bioinformatics filtering for piRNA candidates 18 3.3 RNA-sequencing and analysis 21 3.4 piRNA precursors prediction 21 3.5 MeDIP sequencing and analysis 22 3.6 Transcription factor binding site enrichment analysis 22 3.7 Prediction and GO term analysis of piRNA Targets 23 Chapter 4 Results 24 4.1 Small RNA production machineries still remain functional in Dnmt3l knockout germ cells 24 4.2 piRNA biogenesis was still active in Dnmt3l knockout mice 28 4.3 piRNA compositions were distinct between wildtype and Dnmt3l knockout pups 31 4.4 The increased reads in repeat region were mainly generated from DNMT3L-modulated transposon 35 4.5 Standard piRNA precursors were defined via merging predicted piRNA precursors and RNA transcripts 37 4.6 Several transcription factor binding sites were enriched in candidate precursors in wildtype and Dnmt3l knockout 43 4.7 piRNAs produced from candidate precursors may target specific genes related to nervous system 47 Chapter 5 Discussion 52 5.1 Our bioinformatics pipeline could appropriately isolate piRNA candidate 52 5.2 Is piRNA biogenesis really uninfluenced after Dnmt3l KO? 55 5.3 Validation of predicted piRNA precursors 56 5.4 Function of the transcription factors that bound to the top three enriched TFBS 57 5.5 Is piRNA targeting to genes related to nervous system just an artifact? 60 Chapter 6 Conclusion 61 REFERENCE 63 | |
dc.language.iso | en | |
dc.title | 利用生物資訊工具探討類第三型 DNA 甲基化酶 DNMT3L 於piRNA 生成過程扮演之角色 | zh_TW |
dc.title | Tackling the Roles of DNMT3L in piRNA Production with Bioinformatic Analysis | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蔡孟勳(Mong-Hsun Tsai),林詩舜(Shih-Shun Lin),洪瑞鴻(Jui-Hung Hung),陳柏仰(Pao-Yang Chen),靖永皓(Yung-Hao Ching) | |
dc.subject.keyword | 生殖細胞,精原母細胞,跳躍子,piRNA,RNA 定序, | zh_TW |
dc.subject.keyword | germ cell,spermatogonial stem cell,transposon,piRNA,RNA-seq, | en |
dc.relation.page | 93 | |
dc.identifier.doi | 10.6342/NTU201702732 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-16 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 生物科技研究所 | zh_TW |
Appears in Collections: | 生物科技研究所 |
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