In vivo biology of mammalian L1 retrotransposition - supplement

哺乳动物 L1 逆转录转座的体内生物学 - 补充

• 批准号: 10578990
• 负责人: Jeffrey S Han
• 金额: $ 24.99万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-24 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10578990
• 关键词: Aging; Alleles; Animal Model; Animals; Automobile Driving; Biochemical; Biogenesis; Biological; Biology; Cell Line; Complex; DNA Damage; Defect; Disease; Elements; Family; Future; Genes; Genetic Screening; Genome; Germ Cells; Germ Lines; Human; Infertility; Integration Host Factors; Knock-out; Knowledge; Light; Male Infertility; Male Sterility; Malignant Neoplasms; Mammals; Meiotic Prophase I; Mus; Mutate; Mutation; Nuclear; Pathway interactions; Phenotype; Play; Regulation; Retrotransposition; Role; Small RNA; Sorting - Cell Movement; Wild Type Mouse; Work; Yeasts; cell immortalization; genome integrity; human male; human tissue; in vivo; insight; male; mammalian genome; mouse genetics; mouse model; mutant; nervous system disorder; overexpression; piRNA; tissue culture; trafficking; transposon/insertion element

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.

项目摘要 长时间散布的核元素（线）是一类可逆转的元素，它们不断突变 基因组，包括哺乳动物基因组。目前活跃的哺乳动物系列称为LINE-1 （L1）。哺乳动物基因组中有成千上万的L1副本，未经检查的表达 人类和/或模型生物中的这个元素与各种异常状态（例如癌症）有关 不育，衰老和神经疾病。我们不知道L1在这些疾病中是否起因作用， 部分是因为我们对体内L1生物学的了解是基本的，这限制了我们的实验能力 以特定方式操纵内源性L1活性。尽管许多可以改变的细胞宿主因素 L1逆转录置位已从永生细胞中的生化下拉和遗传筛选中鉴定出来 这些因素在进化相关的生殖细胞中的体内生物学相关性尚不清楚。 以前我们的实验室发现，运输所需的内体分类复合物（ESCRT）扮演关键 酵母和人组织培养中生产线逆转录的作用。该建议将使用鼠标 遗传学检查是否将ESCRT用于L1雄性的细胞内运输和逆转录。 种系，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线缺陷 不育模型。为此，我们将同时利用野生型小鼠和小鼠，其中L1的已知增加 在种系中的表达。在大漩涡中删除的小鼠（mael - / - ）的小鼠对L1的过度表达巨大， 减数分裂预言I，是雄性无菌的。漩涡参与小rNA的生物发生 当相关的piRNA生物发生时，可见生殖系，称为PIRNA和类似的转座子/不育缺陷 在小鼠中敲出基因（例如MOV10L1）。目前尚不清楚L1是否是不育的驱动因素 在这些老鼠中。为了减少L1活动，我们将引入ALIX敲除等位基因（Alix是 ESCRT复合体）。在AIM 1中，我们将评估生殖系L1 RNP定位，调节和逆转录的定位 当L1/ESCRT相互作用中断时。在AIM 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提供宝贵的见解 贩运细菌。拟议的工作还将阐明是否过多的L1转载词是 驱动因子负责PIRNA途径突变体中的不育症。因为L1和PIRNA途径是 在人类中保守的，PIRNA途径基因中的突变与人类不育症有关，我们 期望这项工作将成为未来研究转座监管与某些关系之间关系的基础 人类男性不育症的案例。