DNA甲基化抑制病毒與內源性轉位子之探討及應用

2009-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/703185摘要：上位遺傳/後生遺傳(Epigenetics)乃一正常之生理現象，其在不影響DNA 序列的前提下，經由DNA 甲基化、組蛋白(Histone)修飾及功能性RNA 之作用途徑，影響特定基因之表現。此等上位遺傳對於基因體之修飾作用即為幹細胞之所以有別於已分化體細胞之差異所在。上位遺傳於各類細胞間不同之印記是具有可逆性的。這種可逆性是謂幹細胞及再生醫學研究之關鍵課題。配子生成過程即為上位遺傳印記再程序化(Epigenetic Reprogramming)之關鍵時刻。此計畫將有系統的探討雄性小鼠精子生成(spermatogenesis) 中之上位遺傳調控，以及分析DNA 甲基化對於病毒及轉位子相關序列 (transposon-related sequences)的抑制作用。目前為止，唯一已知對於有害之內源性轉錄子有著專一性的抑制作用之調控因子乃配子及胚幹細胞專一性表現之類DNA 甲基化脢3L(DNA metyltransferase 3-like; Dnmt3L)。本試驗將利用病毒來感染胚幹細胞或已被剔除Dnmt3L 的胚幹細胞，並偵測病毒序列被甲基化的表現情形，以證明Dnmt3L 可以將病毒序列重新甲基化。此外，我們並將找出參與此離體系統中甲基化病毒序列之其他作用因子。男性生殖細胞之發育及再程序化過程中已知的上位遺傳(Eepigenetic)調控機制包括 DNA 甲基化及微型RNA 之作用途徑，前者會去抑制男性原始生殖細胞中轉位因子相關序列(transposon-related sequences)，使得配子生成過程可以順利進行。我們將配合由孔祥智博士所領導的中央研究院基因突變鼠核心實驗室建立之突變鼠表現型的篩選方法，首先進行突變鼠睪丸小於正常百分之五十之篩選，進而找出具上位遺傳缺陷之突變鼠家族，以進行突變基因之定性分析。成熟體細胞並不會表現Dnmt3L 蛋白質，我們將利用基因轉殖技術建立Dnmt3L（及其他於本計畫篩選出之共同作用基因）可調控之基因轉殖動物(conditional Dnmt3L transgenic animals)，嘗試抑制病毒序列的表現。本計畫所得之成果更可大幅提昇吾等對上位遺傳機制誘發之基因抑制作用的瞭解，是謂深入探討生殖細胞(germ cell)之分化、幹細胞之上位遺傳之調控及提供再生醫學研究之基礎。<br> Abstract: Epigenetics is a physiological process that uses DNA methylation, histone protein modifications and non-coding RNAs to control gene expression without causing any changes in DNA sequences itself. This process marked the differences between Stem cells and differentiated cells and is crucial for normal development and metabolism, including X-inactivation, silencing of endogenous retrotransposons and genomic imprinting. The reversible nature of these Epigenetic marks is the reason that somatic nuclear transfer in mammals is possible and it sets the foundation of regenerative medical research. However, we are still in a primitive stage in understanding these epigenetic regulations. In this current project, we will carry out a systematic and stepwise approach to study DNA methylation silencing of virus-derived sequences. The germ cell and embryonic stem cell specifically expressed DNA methyltransferase 3-Like (Dnmt3L) protein is the only trans-acting regulator identified so far that is specifically responsible for silencing endogenous retrotransposons during spermatogenesis. It is very logical to deduce that it also has the capacity to silence newly infected viral sequences. This has not been proven before, but will be clarified in this project by testing the methylation of newly infected viral sequence in embryonic stem (ES) cells with and without Dnmt3L expression. If so, we will identify the Dnmt3L interacting factors while this de novo methylation process takes place, in a Dnmt3Ltag/tag ES cell lines we established. In addition, the possibility of introducing Dnmt3L (and the newly identified non-ubiquitously expressed interacting factors from this project) to silence newly infected viruses will be tested in somatic cell lines, which do not usually express Dnmt3L and conditional Dnmt3L transgenic animals. In addition to the in vitro system described above, we will also study the regulators that are involved in silencing endogenous retrotransposons during spermatogenesis. Taking advantage of an effective, unbiased ENU (N-ethyl-N-nitrosourea)-mutagenesis based screen, we will be able to identify all regulators responsible for silencing retrotransposons. In the absence of these epigenetic regulators, reactivation of the retrotransponsons occurs and results in serious meiotic defects and azospermia pheonotypes. These epigenetic deregulated mice have the hallmark of significantly smaller testis associated with azospermia. We can therefore easily incorporate a primary screen for ENU mutant lines having significantly smaller testis with the existing ENU screen at the Mouse Mutagenesis Program Core Facility (MMP) at the Academia Sinica headed by Dr. John Kung. There are many groups involved in the forward genetic screening based on altered blood composition phenotype. We can make the best use of the G3 adult mice generated from other ongoing screens by checking the size of the ovaries and testes when they are to be sacrificed. This can reduce a great amount of cost. The in vitro and in vivo studies of DNA methylation mediated gene silencing described in this project serve as a perfect model system to explain epigenetic mechanism in general, which can easily be adapted to Stem cell and Regenerative medical research. We also have the opportunity in developing therapeutic protocol to prevent and cure retroviral infections.DNA 甲基化病毒胚幹細胞&#64029子生成上位遺傳DNA MethylationRetrovirusRetrotransposonsEpigeneticsSpermatogenesis,DNA甲基化抑制病毒與內源性轉位子之探討及應用