LINE-1 Retrotransposition in Human Embryonic Stem Cells

描述(申请人提供)：长穿插元件-1(LINE-1或L1)是一种丰富的反转录转座子，约占人类DNA的17%。绝大多数L1不能再移动(即后转位)。然而，人类基因组平均含有约80-100个逆转座子能力L1(RC-L1)，它们在生殖系和体细胞中的迁移性导致了多种遗传疾病，包括血友病A、肌肉营养不良和结肠癌。RC-L1s编码的蛋白质还可能负责Alu元件、某些短散布元件(Sine)的动员和加工假基因的形成，这些元件加起来占人类DNA的13%。因此，无论是直接还是通过细胞RNA的混杂动员，L1介导的逆转录转座事件都负责人类DNA中至少10亿个碱基，并对人类基因组的进化产生了巨大的影响。最近的研究表明，L1逆转座事件可以发生在胚胎发育的早期和神经前体细胞中。我们假设HES细胞代表了一个与发育相关的体外模型系统，以研究L1介导的逆转录转座事件在人类发育的不同阶段影响基因组完整性的机制。了解影响HES细胞基因组完整性的基本分子过程很重要，因为这些细胞最终可能被用于治疗广泛的疾病。这项建议的具体目的是：1.确定L1逆转录转座事件在HES细胞中的影响。2.确定在HES细胞中动员的内源性L1的星座。3.研究L1逆转位对神经元分化的影响。 通俗叙述：LINE-1元件是DNA序列，它可以通过一种称为逆转座的过程从染色体的一个区域“跳”到另一个区域。它们约占人类DNA的17%，在基因组进化中发挥了重要作用。绝大多数LINE-1分子发生突变，不能再跳，但仍有约100个LINE-1分子能够动员。有害的LINE-1基因插入导致了血友病A、肌肉营养不良等疾病，也与肿瘤的发生有关。由LINE-1元件编码的蛋白质还负责Alu元件、某些短散布元件(Sine)的动员和加工假基因的形成，这些元件加在一起至少占人类DNA的13%。因此，无论是直接还是通过细胞RNA的混杂动员，L1介导的逆转录转座事件都负责人类DNA中至少10亿个碱基，并对人类基因组的进化产生了巨大的影响。尽管有这些发现，但仍不清楚在体内L1逆转录转座的频率、细胞类型以及在发育过程中的时间。最近的研究表明，L1逆转座事件可以发生在胚胎发育的早期和神经前体细胞中。我们假设HES细胞代表了一个与发育相关的体外模型系统，以研究L1介导的逆转录转座事件在人类发育的不同阶段影响基因组完整性的机制。了解影响HES细胞基因组完整性的基本分子过程很重要，因为这些细胞最终可能被用于治疗广泛的疾病。

DESCRIPTION (provided by applicant): Long Interspersed Element-1 (LINE-1 or L1) is an abundant retrotransposon that comprises ~17% of human DNA. The overwhelming majority of L1s can no longer move (i.e., retrotranspose). However, the average human genome contains ~80-100 retrotransposition-competent L1s (RC-L1s) and their mobility, in both germ line and somatic cells, has resulted in a variety of genetic disorders, including Hemophilia A, muscular dystrophy, and colon cancer. The proteins encoded by RC-L1s also likely are responsible for the mobilization of Alu elements, certain Short Interspersed Elements (SINEs) and the formation of processed pseudogenes, which together comprise ~13% of human DNA. Thus, either directly or by the promiscuous mobilization of cellular RNAs, L1-mediated retrotransposition events are responsible for at least one billion bases in human DNA and have had a tremendous impact on human genome evolution. Recent studies indicate that L1 retrotransposition events can occur during early embryogenesis and in neural progenitor cells. We hypothesize that hES cells represent a developmentally relevant in vitro model system to study the mechanisms by which L1- mediated retrotransposition events impact genome integrity at various stages of human development. An understanding of the basic molecular processes that impact genome integrity in hES cells is important to evaluate since these cells ultimately may be used therapeutically to treat a wide-range of diseases. The specific aims of this proposal are: 1. To determine the impact of L1 retrotransposition events in hES cells. 2. To determine the constellation of endogenous L1s that are mobilized in hES cells. 3. To determine how L1 retrotransposition impacts neuronal differentiation. Lay Narrative: LINE-1 elements are DNA sequences that can "jump" from one area of a chromosome into another by a process called retrotransposition. They comprise ~17% of the human DNA and have played a major role in genome evolution. The vast majorities of LINE-1 elements are mutated and can no longer jump; however, ~100 LINE-1 elements are still able to mobilize. Deleterious LINE-1 insertions into genes have caused diseases such as Hemophilia A, muscular dystrophy, and also are implicated in tumorigenesis. The proteins encoded by LINE-1 elements also are responsible for the mobilization of Alu elements, certain Short Interspersed Elements (SINEs) and the formation of processed pseudogenes, which together comprise at least 13% of human DNA. Thus, either directly or by the promiscuous mobilization of cellular RNAs, L1-mediated retrotransposition events are responsible for at least one billion bases in human DNA and have had a tremendous impact on human genome evolution. Despite these findings, it still is unclear how often, in what cell types, and when during development L1 retrotransposes in vivo. Recent studies indicate that L1 retrotransposition events can occur during early embryogenesis and in neural progenitor cells. We hypothesize that hES cells represent a developmentally relevant in vitro model system to study the mechanisms by which L1-mediated retrotransposition events impact genome integrity at various stages of human development. An understanding of the basic molecular processes that impact genome integrity in hES cells is important to evaluate since these cells ultimately may be used therapeutically to treat a wide-range of diseases.