Genetic and Molecular Analyses of Human LINE-1 Retrotransposition

摘要 长散布的Element-1(Line-1或L1)转座元件衍生序列包括 大约17%的人类基因组DNA。尽管绝大多数行-1已呈现为 随着进化时间的推移，突变过程不会移动，一个平均的人类基因组包含大约 100个活性LINE-1，可以通过RNA中间体通过称为 逆转位。在胚系中、在早期发育过程中或在选择过程中的活性品系-1s的逆转座 体细胞继续产生个体间和个体内的遗传变异，并可导致零星病例 人类遗传性疾病，包括血友病A，血友病B，杜氏肌营养不良，以及某些 癌症。我们假设，对LINE-1生物学的机械论理解对于理解 对人类疾病、人类基因变异和人类进化做出贡献。这项提案是第三个 GM060518的竞争性续展申请，并寻求持续支持，以继续我们对1号线的研究 生物学。我们将以我们在移动遗传元素领域的经验和知识为基础，利用洞察力 从研究进化相关的反转录转座子的移动机制中获得的，并使用了一条直线- 1培养细胞逆转录转座试验与综合策略相结合， 回答前沿问题的分子生物学、生化、多组学和计算方法 在生物学的第一行。这项建议包括两个目标，这两个目标建立在我们广泛的试剂工具箱的基础上， 之前的发现和初步数据。在目标1中，我们将阐明1号线的机械方面 逆转位。在目标2中，我们将确定宿主蛋白如何促进或限制LINE-1 逆转位。该项目将受益于莫兰和莫兰之间长期成功的合作 密歇根大学的KIDD实验室和我们杰出的研究团队。圆满完成 以上的具体目标将增加我们对LINE-1s如何移动到新的基因组位置，如何 寄主已经进化出防止LINE-1逆转录转座的机制，以及LINE-1是如何利用 帮助其逆转录转座的细胞蛋白质。这一知识还将提供有关1号线如何 逆转录转座有助于人类疾病、人类遗传变异和人类基因组进化。

Abstract Long INterspersed Element-1 (LINE-1 or L1) transposable element derived sequences comprise approximately 17% of human genomic DNA. Although the overwhelming majority of LINE-1s have been rendered immobile by mutational processes over evolutionary time, an average human genome contains approximately 100 active LINE-1s that can mobilize to new genomic locations via an RNA intermediate by a process known as retrotransposition. The retrotransposition of active LINE-1s in the germline, during early development, or in select somatic cells continues to generate inter- and intra-individual genetic variation and can lead to sporadic cases of human genetic diseases, including hemophilia A, hemophilia B, Duchenne muscular dystrophy, and certain cancers. We posit that a mechanistic understanding of LINE-1 biology is essential to understand the forces that contribute to human disease, human genetic variation, and human evolution. This proposal represents the third competing renewal application of GM060518, and seeks ongoing support to continue our studies of LINE-1 biology. We will build upon our experience and knowledge in the field of mobile genetic elements, use insights gained from studies of the mobility mechanisms of evolutionarily related retrotransposons, and employ a LINE- 1 cultured cell retrotransposition assay in conjunction with an integrated strategy that combines genetic, molecular biological, biochemical, multi-omic, and computational approaches to answer cutting-edge questions in LINE-1 biology. This proposal comprises two Aims, which build upon our extensive toolbox of reagents, previous findings, and preliminary data. In Aim 1, we will elucidate mechanistic aspects of LINE-1 retrotransposition. In Aim 2, we will determine how host proteins act to facilitate or restrict LINE-1 retrotransposition. This project will benefit from a long-standing successful collaboration between the Moran and Kidd laboratories at the University of Michigan and our outstanding research teams. The successful completion of the above Specific Aims will increase our understanding how LINE-1s mobilize to new genomic locations, how the host has evolved mechanisms to prevent unabated LINE-1 retrotransposition, and how LINE-1 has exploited cellular proteins to aid in its retrotransposition. This knowledge also will provide insights into how LINE-1 retrotransposition contributes to human disease, human genetic variation, and human genome evolution.