Saturday, January 28, 2023
1/28/2023
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.
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