Project Summary Most mammalian retrotransposons are strictly silenced in development and physiology, yet induction of some retrotransposons can be observed during specific developmental processes. Interestingly, a portion of the reactivated retrotransposons, particularly LTR retrotransposons, confer a gene regulatory role, at least in part, by acting as alternative promoters to drive chimeric transcripts with proximal protein-coding genes. Such retrotransposon promoters frequently alter gene structure and/or gene expression, yet their functional importance remains largely unclear. Mammalian preimplantation embryos are an excellent experimental system to probe the functional importance of retrotransposons. Dynamic induction of retrotransposons in preimplantation embryos have been observed in all 8 mammalian species examined, and the global retrotransposon expression profiles across mammalian species are similar. Using published single-cell RNA-seq data from mouse, human, primate and livestock preimplantation embryos, we discovered hundreds of retrotransposon promoters, which drive preimplantation-specific, proximal gene isoforms. Interestingly, most retrotransposon sequences and integrations are species specific, yet many retrotransposon promoters yield gene isoforms that encode evolutionarily conserved proteins. Hence, these data suggest that retrotransposon promoters can regulate conserved protein sequences and bestow them with species-specific gene regulation. One of such evolutionarily conserved retrotransposon driven gene isoform, Cdk2ap1N(MT2B2), encodes an N-terminally truncated isoform for Cdk2ap1, a negative regulator for cell proliferation by repressing Cdk2. Cdk2ap1N(MT2B2) is generated by an MT2B2 promoter, whose deletion in mice yield reduced cell proliferation, impaired implantation and embryonic lethality. This is among the first study demonstrating an essential function of a retrotransposon element in development. Here, we hypothesize that retrotransposon-mediate gene regulation play an essential role in mammalian preimplantation development. Using bioinformatics prediction combined with experimental validation, we propose to comprehensively and accurately categorize retrotransposon-promoters in mouse, primate and livestock preimplantation embryos, and elucidate the diverse molecular mechanisms for retrotransposon-mediated gene regulation. Additionally, we will employ a highly efficient CRISPR technology, CRISPR-EZ, to generate mouse deletion mutants for selected retrotransposon promoters or for the corresponding canonical gene isoforms. We will compare the roles of retrotransposon-dependent gene isoform and the canonical gene isoform, elucidate the molecular mechanisms of their action and explore the evolutionary significance of such regulation. Taken together, these proposed studies will generate a comprehensive atlas of retrotransposon-dependent gene regulation during preimplantation development, and provide a new paradigm to ...