臺灣博碩士論文加值系統

反轉錄跳躍子 (retrotransposons)嵌入基因體的現象在演化上扮演著重要的角色，其大約佔據了哺乳動物一半的DNA組成。以生殖細胞為例。若沒有縝密的調控機制抑制這群「跳躍基因」，反轉錄跳躍子可能會影響並破壞正常基因的表現。 異常喪失反轉錄跳躍子的抑制功能已被證實和生殖細胞的發育缺陷有關。Promyelocytic leukemia zinc finger (PLZF) 於睪丸中絕大部分表現在精原幹細胞及前軀細胞，其調控細胞自我更新及分化以維持正常的生精作用以避免精原幹細胞 (spermatogonial stem cells) 耗損。 本研究使用Plzflu/lu突變鼠和同胞野生型小鼠探討PLZF對於調節睪丸反轉錄跳躍子的角色。藉由生物資訊學和RT-qPCR分析發現PLFZ會和重複序列 (repeat element) 結合，且PLZF缺失會造成反轉錄跳躍子、LINE1 和IAP表現異常，進而證明PLZF在睪丸調控反轉錄跳躍子扮演不可或缺的角色。本研究室曾以四周齡THY1抗體純化的精原幹細胞作為材料，發現PLZF和MILI 和MAEL有交互作用。然而我在出生後八日的睪丸細胞中，發現PLZF僅和MILI有作用。由於已知MILI會藉由piRNA路徑，在生殖細胞發育中參與抑制反轉錄跳躍子以維持生殖細胞基因體的完整性，本研究假設PLZF亦藉由piRNA路徑調控反轉錄跳躍子。然而，分析出生後八日的睪丸細胞小RNA序列，並未觀察到PLZF突變鼠之piRNA生成明顯異於野生型小鼠。
總而言之，PLZF在睪丸中或可藉由影響反轉錄跳躍子轉錄，mRNA的穩定度、和piRNA路徑重要的相關基因結合以於多個層次影響反轉錄跳躍子抑制。但在piRNA生成作用無明顯差異的情況下，可能暗示著PLZF調節MILI的作用無關於piRNA pathway，而是可能在生殖細胞中調節轉譯並補償其他路徑以抑制反轉錄跳躍子。

The integration of retrotransposons into genomes plays an important role in evolution and make up about half of mammalian genome. Without tight and finely tuned regulation to repress these “jumping genes”, retrotransposons may wreak havoc and disrupt normal gene expression, especially in germ cells. Aberrant derepression of retrotransposons is associated with germ cell developmental defects. Promyelocytic leukemia zinc finger (PLZF), expressed mainly in germ cells in testes, is known to have a crucial role in spermatogenesis through self-renewal and differentiation regulation to prevent the depletion of spermatogonial stem cells (SCCs). In this study, we used Plzflu/lu mutant and wild type mouse to explore the roles of PLZF in modulating retrotransposons in testes.
First, through bioinformatics and RT-qPCR analysis, I saw that PLZF binds to the DNA sequences of repeat elements in cultured SSCs and in 8 dpp testes of Plzflu/lu, retrotransposons, LINE1 and IAP, displayed a deregulation in expression when compared to that of wild type littermates, concluding that PLZF is necessary to regulate retrotransposon in testes. Moreover, based on the preliminary data from our lab, PLZF interacts with MILI and MAEL in 4 weeks SSC enriched THY1+ cells. However, in 8dpp whole testes, I was only able to confirm the interaction between PLZF and MILI. Since MILI plays an important role in piRNA pathways, which include the repression of retrotransposons after epigenetic reprogramming during germ cell development to maintain the integrity of germ cell genome, I hypothesize that PLZF partially modulates retrotransposons through piRNA pathways. However, my small RNA sequencing results from 8 dpp testes of Plzflu/lu and their counterpart wild type littermates did not reveal major differences on the piRNA production, characteristics and specific retrotransposon subfamily associated piRNA candidates.
All in all, we and others demonstrated that PLZF has many levels of modulation in testes, essential for germline maintenance and spermatogenesis, and may modulate transposable elements through transcriptional gene silencing and/or through post-transcriptional gene silencing by affecting retrotransposon mRNA stability. The lack of significant difference in piRNA biogenesis may imply that MILI-PLZF interaction modulates MILI’s function independent of piRNA pathway, beyond transposable element modulation which may further suggest PLZF’s function in translational control in germ cells to repress retrotransposons and potentially other transcripts.

致謝Acknowledgment i
中文摘要 iii
Abstract v
List of Tables x
List of Figures xi
Chapter 1. Introduction 1
1.1 Spermatogonial Stem Cell and Spermatogenesis 1
1.2 Promyelocytic Leukemia Zinc Finger 1
1.3 Role of PLZF in SSC 2
1.4 Retrotransposons 3
1.5 PiRNA and PIWI Protein 4
1.6 Role of MILI in SSC and Retrotransposon Repression 5
1.7 PLZF interacts with MILI and MAEL in 4 week THY1+ spermatogonial stem cells 6
1.8 Significance 7
Chapter 2. Specific Aims 8
Chapter 3. Materials and Methods 10
3.1. Animals 10
3.2. PLZF luxoid Mutant 10
3.3. PLZF Genotyping 11
3.4. Cryosectioning 12
3.5. Immunohistochemistry and Immunocytochemistry 13
3.6. Total RNA Extraction 14
3.7. Reverse Transcription PCR 16
3.8. RTqPCR 17
3.9. Strand Specific RTqPCR 17
3.10. Protein Isolation 18
3.11. Protein Quantification 18
3.12. Western Blot 19
3.11.1 SDS-Page Gel 19
3.11.2 Buffer Preparation 20
3.11.3 Protein Preparation & Loading 21
3.11.4 Electrophoresis 21
3.11.5 Transfer 22
3.11.6 Antibody 22
3.11.7 Chemiluminescent Blotting & Data Analysis 23
3.13. Co-Immunoprecipitation 24
3.12.1 Protein Isolation 24
3.12.2 Co-Immunoprecipitation 24
3.12.3 Western Blot 25
3.14. Small RNA Sequencing 26
3.15. Bioinformatics 27
3.16. Statistical Analysis 28
Chapter 4. Results 29
4.1 Derepression of retrotransposon in Plzflu/lu 29
4.2 PLZF binds to genes of some piRNA pathway components 30
4.3 Deregulation of some piRNA regulators in Plzflu/lu 31
4.4 PLZF interacts with MILI 32
4.5 Bioinformatics analysis of 8 dpp testes derived small RNA sequencing demonstrates similar patterns of retrotransposon derived piRNAs between Plzflu/lu mutant and wild type littermates 33
Chapter 5. Discussion 36
5.1. PLZF repressive function observed on retrotransposons 36
5.2. The potential function of PLZF in retrotransposon modulation associated factors 37
5.3. MILI piRNA pathway independent functions 37
5.4. PLZF – MILI interaction and MILI subcellular localization 38
5.5. Prospective Work 39
5.6. Summary 39
Tables 41
Figures 46
References 64

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