In vivo biology of mammalian L1 retrotransposition

项目总结 长散布核素(线)是一类不断发生突变的逆转座子 基因组，包括哺乳动物的基因组。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，并且不受控制地表达L1 在人类和/或模型生物中，这种元素与各种异常状态有关，例如癌症， 不孕不育、衰老和神经系统疾病。我们不知道L1是否在这些疾病中起到了致病作用， 部分原因是我们对体内L1生物学的知识还很初级，这限制了我们进行实验的能力 以特定的方式操纵内源性L1活性。尽管许多蜂窝宿主因素可以改变 在永生化细胞中已经从生化下拉和遗传筛选中鉴定出L1逆转录转座 这些因子在进化上相关的生殖细胞中的体内生物学相关性尚不清楚。 此前，我们的实验室发现，运输所需的内体分选复合体(ESCRT)在 生产线逆转座在酵母和人类组织培养中的作用。这项提案将使用鼠标 遗传学检查ESCRT是否用于男性L1细胞内转运和逆转位 生殖系，以及ESCRT/L1相互作用的中断是否可以减轻在小鼠中发现的生殖系缺陷 不孕不育模型。为此，我们将利用野生型小鼠和L1已知增加的小鼠 在生殖系中的表达。大漩涡基因(MAEL-/-)缺失的小鼠有大量的L1过表达， 停滞于减数分裂前期I，且均为雄性不育。Maelstrom参与了小RNA的生物发生 当相关的piRNA生物发生时，被称为piRNAs的生殖系和类似的转座子/不育缺陷被看到 基因(例如MOV10L1)在小鼠体内被敲除。目前尚不清楚L1是否是不孕不育的驱动因素 在这些老鼠身上。为了减少L1活性，我们将引入Alix基因敲除等位基因(Alix是 ESCRT复合体)。在目标1中，我们将评估生殖系L1 RNP的定位、调控和逆转录转座 当L1/ESCRT相互作用中断时。在目标2中，我们将确定L1过表达对 小鼠的生殖细胞表型。该项目将为ESCRT是否启用L1 RNP提供有价值的见解 贩卖种子线。这项拟议的工作还将阐明过量的L1逆转位是否是一种 在piRNA途径突变体中导致不孕的驱动因素。因为L1和piRNA途径是 在人类中是保守的，piRNA途径基因的突变与人类不孕症有关，我们 期望这项工作将成为未来研究转座子调控和一些 人类男性不育病例。

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.