指導教授：林劭品臺灣大學：生物科技研究所高資皓Kao, Tzu-HaoTzu-HaoKao2014-11-262018-06-292014-11-262018-06-292014http://ntur.lib.ntu.edu.tw//handle/246246/261700哺乳類動物的基因組序列中包含許多反轉錄跳躍子 (retrotransposable elements)，類3號DNA甲基化酶(DNMT3L)在精細胞發育過程中對抑制內源性的反轉錄跳躍子(retrotransposon)扮演重要角色，Dnmt3L也專一性表現在未分化的胚幹細胞中，但已分化的體細胞則無法偵測其表現，反轉錄病毒的活性在胚幹細胞中會受強烈地抑制，但體細胞卻無此抑制活性。DNMT3L的交互作用蛋白KAP1可招募上位遺傳調控蛋白HDAC1和SETDB1以抑制反轉錄跳躍子。本研究旨在闡明DNMT3L在胚幹細胞或體細胞中對新感染病毒是否具有抑制作用，是否可藉由DNMT3L在胚幹細胞反轉錄病毒的抑制效果，外源性表現DNMT3L在體細胞，作為抗病毒的應用。本研究以Mo-MuLV為反轉錄病毒材料感染細胞進而偵測病毒甲基化情形或表現活性，結果顯示DNMT3L和KAP1對於胚幹細胞中新嵌插病毒DNA序列的甲基化與抑制基因表現扮演重要功能。外源性大量表現DNMT3L在體細胞3T3與MEF (Mouse embryonic fibroblast)中，雖然無法使體細胞具有甲基化病毒序列的能力，然而卻可誘導體細胞抗反轉錄病毒活性，此活性已藉由點突變分析證實DNMT3L只有在能夠結合到組蛋白H3，或能與DNMT3A正確交互作用時才具有抗反轉錄病毒活性，且此活性依附KAP1抑制機制路徑，由此結果推論DNMT3L是藉由引起與KAP1交互作用的蛋白達成反轉錄病毒抑制。本研究之細胞染色結果顯示外源性表現DNMT3L於MEF中可誘使大部分表現在細胞質的HDAC1移至細胞核，蛋白免疫共沉澱分析結果顯示外源性表現DNMT3L於MEF中可促進KAP1 、HDAC1和SETDB1蛋白複合體的形成，染色質免疫沉澱分析發現外源性表現DNMT3L於MEF中可誘導KAP1 、HDAC1和SETDB1蛋白結合到新嵌插的病毒DNA序列以進行靜默修飾作用。外源性表現DNMT3L可使MEF細胞持續繼代40次以上，經由長時間培養後發現MEF細胞型態發生轉型且可偵測到Oct4表現且伴隨強烈的反轉錄病毒抑制活性。本研究結果顯示DNMT3L具有體細胞抗反轉錄病毒應用的潛力，未來將有利於體細胞抗病毒相關研究之進展。Mammalian genomes are replete with retrotranspoable elements including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L), an epigenetic regulator highly expressed in germ cells and embryonic stem (ES) cells, is crucial for the long-term suppression of endogenous retrotransposons. Here we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methylatransfearse 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28)/ KRAB-associated protein 1 (KAP1) in ES cells, and orchestrates retroviral-silencing activity with TRIM28 by mechanisms including but not limited to facilitating de novo DNA methylation. Gain of function studies by the introduction of ectopic DNMT3L into somatic cells demonstrate its DNA-methylation independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to the newly infected Moloney-Murine Leukemia Virus (Mo-MuLV). Concurrent with this recruitment, we also observe the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma, as well as reduced H3K9 and H3K27 acetylation at newly integrated Mo-MuLV proviral sequences. Ectopic DNMT3L also re-distributes cytoplasmically localized HDAC1 to the nucleus in late passage mouse embryonic fibroblasts (MEFs) and 3T3 cell line. Formation of this epigenetic modifying complex formation requires Dnmt3L interaction with DNMT3A as well as with histone H3 tail. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding between TRIM28, DNMT3A, SETDB1, and HDAC1. We have therefore uncovered a potential histone-level epigenetic silencing activity of DNMT3L, and this activity is beyond the known scope of DNMT3L in facilitating de novo DNA methylation and interpretation of histone modifications and chromatin context. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks on endogenous retrotransposons and imprinting control regions.口試委員會審定書 i 誌謝 ii 中文摘要 iii ABSTRACT iv CONTENTS vi LIST OF FIGURES xiii LIST OF TABLES xv General Introduction 1 Chapter 1 DNMT3L and TRIM28 are required for Mo-MuLV proviral silencing in ES cells 12 1.1 INTRODUCTION 12 1.2 MATERIAL AND METHODS 13 1.2.1 Ethics Statement 13 1.2.2 Cell culture and sample preparation 13 1.2.3 Construction of expression vectors containing wild-type and mutant Dnmt3l 14 1.2.4 Cell transfection 15 1.2.5 Retroviral preparation and infection 15 1.2.6 Luciferase activity assay 16 1.2.7 DNA Methylation analysis 17 1.2.8 Oligonucleotides for PCR amplification of bisulfite-treated DNA 18 1.2.9 Antibodies 18 1.2.10 Immunoprecipitation 19 1.2.11 Dnmt3l KO fetal testes preparation 19 1.3 RESULTS 21 1.3.1 DNMT3L and TRIM28 are required for efficient de novo DNA methylation of newly-integrated Mo-MuLV proviral sequences in 129S4/SvJae ES cells 21 1.3.2 DNMT3L and TRIM28 are required for efficient de novo DNA methylation of newly-integrated Mo-MuLV proviral sequences in ES cells 24 1.3.3 DNMT3A and TRIM28 are required for efficient de novo DNA methylation of newly-integrated Mo-MuLV proviral sequences 30 1.3.4 DNMT3L is required for HDAC1/TRIM28/DNMT3A/SETDB1 protein complex formation in ES cells 33 1.3.5 DNMT3L-repressive chromatin modifier complex is present in fetal gonads 35 1.4 DISCUSSIONS 37 1.4.1 DNMT3L and TRIM28 synergistically silence proviral sequences in ES cells through multiple mechanisms 37 Chapter 2 DNMT3L induced viral silencing in somatic cells 39 2.1 INTRODUCTION 39 2.2 MATERIAL AND METHODS 40 2.2.1 Cell culture and sample preparation 40 2.2.2 Cell transfection 40 2.2.3 Retroviral preparation and infection 40 2.2.4 Luciferase activity assay 41 2.2.5 DNA Methylation analysis 42 2.2.6 Oligonucleotides for PCR amplification of bisulfite-treated DNA 43 2.2.7 Antibodies 43 2.2.8 Immunoprecipitation 44 2.2.9 Chromatin immunoprecipitation 44 2.3 RESULTS 46 2.3.1 Ectopic Dnmt3L induces modest retroviral silencing activity in later-passage MEFs 46 2.3.2 Dnmt3L can induce retroviral silencing activity in 3T3 51 2.3.3 Dnmt3L cannot induce retroviral silencing in RAT2 cell which lack of TRIM28-mediated silencing mechanism 54 2.3.4 Ectopic DNMT3L induces the formation of a repressive chromatin modifier complex to introduce repressive histone modifications at Mo-MuLV proviral DNA in DNMT3L-expressing MEFs 56 2.3.5 Ectopic DNMT3L induces the formation of a repressive chromatin modifier complex to introduce repressive histone modifications at ERVs in DNMT3L-expressing MEFs 58 2.3.6 Ectopic DNMT3L induces the formation of a repressive chromatin modifier complex in DNMT3L-expressing MEFs 61 2.3.7 DNMT3L induces HDAC1 translocation into the nucleus 64 2.4 DISCUSSION 70 2.4.1 Methylation-independent retroviral silencing by ectopic DNMT3L in somatic cells 70 2.4.2 DNMT3L-induced retroviral silencing does not rely on DNA methylation of proviruses in somatic cells 75 2.4.3 DNMT3L induces HDAC1 translocation into nucleus in later-passage MEFs 77 Chapter 3 DNMT3L overexpression induced cell fate changes in MEFs and 3T3 78 3.1 INTRODUCTION 78 3.2 MATERIAL AND METHODS 80 3.2.1 Cell culture of Dnmt3l-transfected MEFs 80 3.2.2 Immunocytochemistry 80 3.2.3 iTRAQ 81 3.3 RESULTS 83 3.3.1 DNMT3L induced accumulation effect of retroviral silencing activity in long-term cultured morphology changed MEFs 83 3.3.2 DNMT3L is sufficient for inducing strong retroviral silencing activity in long-term cultured MEFs 86 3.3.3 DNMT3L expression can induce morphological changes and mimic partial reprogramming in MEFs 90 3.3.4 Dnmt3L transfection can induce cell congregation in 3T3 cells 92 3.3.5 iTRAQ analysis of Dnmt3l-overexpression 3T3s 94 3.4 DISCUSSION 98 3.4.1 DNMT3L induces cell proliferation and pluripotent marks expression in later-passage MEFs 98 3.4.2 The roles of caveolin-1 in cancer initiation and progression 99 Chapter 4 General discussion 100 Methylation-independent retroviral silencing by DNMT3L 100 DNMT3L induces HDAC1 translocation into the nucleus 101 Host factors expression levels for retroviral silencing 102 DNMT3L-dependent chromatin-modifying activity may explain the necessity of DNMT3L activity in germ cell development 103 Conclusion …………………………………………………………………………106 References …………………………………………………………………………1083309727 bytesapplication/pdf論文使用權限：不同意授權上位遺傳甲基化反轉錄病毒抗病毒胚幹細胞thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261700/1/ntu-103-D95642003-1.pdf