In vivo biology of mammalian L1 retrotransposition

项目摘要 长散布核元件（LINEs）是一类不断突变的逆转录转座元件 基因组，包括哺乳动物基因组。哺乳动物中目前活跃的LINE家族被称为LINE-1 （L1）。在哺乳动物基因组中有数十万个L1拷贝， 人类和/或模式生物体中的这种元素与各种异常状态，例如癌症， 不孕、衰老和神经系统疾病。我们不知道L1是否在这些疾病中起着致病作用， 部分原因是我们对L1生物学的了解还很初级，这限制了我们在实验上研究L1生物学的能力。 以特定方式操纵内源性L1活性。尽管一些细胞宿主因素可以改变 L1反转录转座已经从永生化细胞的生化下拉和遗传筛选中鉴定出来 线，这些因素在进化相关的生殖细胞的体内生物学相关性尚不清楚。 以前我们的实验室发现，运输所需的内体分选复合物（ESCRT）在运输中起着关键作用。 在酵母和人类组织培养中生产性LINE反转录转座的作用。这个建议将使用鼠标 遗传学，以检查ESCRT是否用于男性中的L1细胞内运输和逆转录转座 以及ESCRT/L1相互作用的破坏是否可以减轻小鼠中发现的生殖系缺陷 不孕症的模型。为此，我们将利用野生型小鼠和已知L1增加的小鼠 在生殖细胞系中表达。缺失Maelstrom基因（Mael-/-）的小鼠具有L1的大量过表达， 在减数分裂前期I停滞，并且是雄性不育的。Maelstrom参与小RNA的生物合成， 生殖系，称为piRNA，当相关的piRNA生物合成时， 基因（例如Mov10l1）在小鼠中被敲除。目前尚不清楚L1是否是不孕症的驱动因素 在这些老鼠身上。为了降低L1活性，我们将引入ALIX敲除等位基因（ALIX是L1基因的一个组成部分）。 ESCRT复合物）。在目标1中，我们将评估生殖系L1 RNP的定位、调控和反转录转座 当L1/ESCRT相互作用被破坏时。在目标2中，我们将确定L1过表达对 小鼠生殖细胞表型。该项目将为ESCRT是否能够实现L1 RNP提供有价值的见解 贩卖生殖细胞拟议的工作也将阐明过量的L1逆转录是否是一个潜在的问题。 导致piRNA途径突变体不育的驱动因子。因为L1和piRNA通路是 在人类中是保守的，piRNA途径基因的突变与人类不育有关，我们 我希望这项工作将成为未来研究转座子调控与某些生物学特性之间关系的基础。 人类男性不育的病例。

PROJECT SUMMARY Long Interspersed Nuclear Elements (LINEs) are a class of retrotransposable elements that continually mutate genomes, including mammalian genomes. The currently active family of LINEs in mammals is called LINE-1 (L1). There are hundreds of thousands of copies of L1 in mammalian genomes, and unchecked expression of this element in humans and/or model organisms is associated with various abnormal states, such as cancer, infertility, aging and neurologic disease. We do not know whether L1 plays a causative role in these disorders, partly because our knowledge of L1 biology in vivo is rudimentary, which has limited our ability to experimentally manipulate endogenous L1 activity in a specific manner. Although a number of cellular host factors that can alter L1 retrotransposition have been identified from biochemical pulldowns and genetic screens in immortalized cell lines, the in vivo biological relevance of these factors in the evolutionarily relevant germ cells is unclear. Previously our lab discovered that the endosomal sorting complex required for transport (ESCRT) plays a critical role for productive LINE retrotransposition in both yeast and human tissue culture. This proposal will use mouse genetics to examine whether ESCRT is used for L1 intracellular trafficking and retrotransposition in the male germ line, and whether disruption of the ESCRT/L1 interaction can alleviate germ line defects found in a mouse models of infertility. To this end, we will utilize both wild type mice and mice with known increases in L1 expression in the germline. Mice deleted in the gene for Maelstrom (Mael-/-) have massive overexpression of L1, arrest in meiotic prophase I, and are male sterile. Maelstrom is involved in the biogenesis of small RNAs in the germ line, called piRNAs, and similar transposon/infertility defects are seen when related piRNA biogenesis genes (e.g. Mov10l1) are knocked out in mice. It is currently unknown whether L1 is a driving factor of infertility in these mice. To reduce L1 activity, we will introduce an ALIX knockout allele (ALIX is a component of the ESCRT complex). In Aim 1 we will evaluate germ line L1 RNP localization, regulation, and retrotransposition when the L1/ESCRT interaction is disrupted. In Aim 2 we will determine the contribution of L1 overexpression to germ cell phenotypes in mice. This project will provide valuable insight into whether ESCRT enables L1 RNP trafficking in the germ line. The proposed work will also shed light on whether excess L1 retrotransposition is a driving factor responsible for infertility in piRNA pathway mutants. Because L1 and the piRNA pathway are conserved in humans, and mutations in piRNA pathway genes have been associated with human infertility, we expect that this work will form the basis for future study on the relation between transposon regulation and some cases of human male infertility.