In vivo biology of mammalian L1 retrotransposition

哺乳动物 L1 逆转录转座的体内生物学

• 批准号: 10367362
• 负责人: Jeffrey S Han
• 金额: $ 40.39万
• 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: 2022-01-12 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10367362
• 关键词: Affect; Aging; Alleles; Amino Acid Motifs; Animal Model; Animals; Attenuated; Automobile Driving; Biochemical; Biogenesis; Biological; Biology; Breeding; Cell Line; Complex; Cultured Cells; DNA Damage; Defect; Disease; Elements; Enterobacteria phage P1 Cre recombinase; Event; Family; Frequencies; Future; Genes; Genetic Polymorphism; Genetic Recombination; Genetic Screening; Genome; Genomics; Germ Cells; Germ Lines; Human; Infertility; Integration Host Factors; Knock-in Mouse; Knock-out; Knockout Mice; Knowledge; Leptotene Stage; Light; Male Infertility; Male Sterility; Malignant Neoplasms; Mammals; Meiosis; Meiotic Prophase I; Modeling; Mus; Mutate; Mutation; Nuclear; Pachytene Stage; Pathway interactions; Phenotype; Play; Prophase; Regulation; Retrotransposition; Ribonucleoproteins; Role; Small RNA; Sorting - Cell Movement; Spermatocytes; Transgenes; Wild Type Mouse; Work; Yeasts; Zygonema; cell immortalization; genome integrity; human male; human tissue; in vivo; insight; male; mammalian genome; mouse genetics; mouse model; mutant; nervous system disorder; overexpression; particle; piRNA; promoter; 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.

项目概要 长散布核元件 (LINE) 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。目前哺乳动物中活跃的 LINE 家族称为 LINE-1 （L1）。哺乳动物基因组中有数十万个 L1 拷贝，且未受控制的表达 人类和/或模型生物体内的这种元素与各种异常状态有关，例如癌症、 不孕症、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， 部分原因是我们对体内 L1 生物学的了解还很初级，这限制了我们进行实验的能力 以特定方式操纵内源性 L1 活性。尽管许多细胞宿主因素可以改变 L1 逆转录转座已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物 (ESCRT) 起着至关重要的作用。 生产性 LINE 逆转录转座在酵母和人体组织培养中的作用。本提案将使用鼠标 遗传学检查 ESCRT 是否用于男性的 L1 细胞内运输和逆转录转座 种系，以及破坏 ESCRT/L1 相互作用是否可以减轻小鼠中发现的种系缺陷 不孕不育模型。为此，我们将利用野生型小鼠和已知 L1 增加的小鼠 在种系中表达。 Maelstrom (Mael-/-) 基因缺失的小鼠 L1 大量过度表达， 停滞于减数分裂前期 I，并且是雄性不育的。 Maelstrom 参与小 RNA 的生物合成 种系，称为 piRNA，当相关 piRNA 生物发生时，会看到类似的转座子/不育缺陷 小鼠体内的基因（例如 Mov10l1）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 敲除等位基因（ALIX 是 ESCRT 复合体）。在目标 1 中，我们将评估种系 L1 RNP 定位、调节和逆转录转座 当 L1/ESCRT 相互作用被破坏时。在目标 2 中，我们将确定 L1 过度表达对 小鼠生殖细胞表型。该项目将为 ESCRT 是否支持 L1 RNP 提供有价值的见解 贩运种系。拟议的工作还将阐明过量的 L1 逆转录转座是否是一种 piRNA 途径突变体中导致不孕的驱动因素。因为 L1 和 piRNA 途径是 piRNA 通路基因在人类中保守，并且 piRNA 通路基因的突变与人类不孕不育有关，我们 期望这项工作能够为未来研究转座子调控与某些基因之间的关系奠定基础。 人类男性不育症案例。