Project Summary Retrotransposons, mainly LINEs, SINEs, and endogenous retroviruses, occupy 40% of the mammalian genome. Retrotransposons have an enormous capacity to metastasize throughout the genome using a “copy and paste” mechanism involving reverse transcription. While retrotransposons play an important role in genome evolution, their mobilization can be detrimental to genome integrity. Indeed, more than 60 human genetic diseases are caused by transposon insertion. Retrotransposons exploit the host cellular machinery to proliferate. In response, the host has evolved multiple mechanisms to suppress retrotransposons to protect genome integrity, particularly within the germline. The piRNA pathway is a major evolutionarily conserved small non-coding RNA-based silencing mechanism for retrotransposons in germ cells. In the previous funding period, we demonstrated that MOV10L1, a germ cell-specific RNA helicase, is a master regulator of biogenesis of all piRNAs in mouse. MOV10L1 interacts with all Piwi proteins and binds to piRNA precursors to initiate piRNA biogenesis. Deficiency of Mov10l1 leads to upregulation of retrotransposons, a block in meiosis, and male sterility. Upregulation of retrotransposon transcripts does not necessarily lead to a proportionate increase in new retrotransposition, suggesting that additional host factors block retrotransposition. While previous studies have made tremendous progress delineating mechanisms responsible for transcriptional and post- transcriptional silencing of retrotransposons, host restriction factors that prevent genomic integration of retrotransposons in vivo have not yet been identified. Using our unique mouse models, we plan to 1) investigate the molecular mechanism underlying the essential role of MOV10L1 in piRNA biogenesis during spermatogenesis;; 2) elucidate the critical role of a host restriction factor in inhibition of retrotransposition in the mouse germline;; 3) interrogate the multi-generational impact of retrotransposon-driven genome expansion on genome stability, reproduction, and diseases. Completion of this project will have strong impacts on our understanding of retrotransposon silencing, genome expansion, and etiology of human diseases including male infertility, pregnancy loss, and birth defects.