Understanding functions of LTR retrotransposon activation in preimplantation development

Understanding the functions of LTR retrotransposon activation in preimplantation development Abstract Accumulating evidence indicates that retrotransposons, especially long terminal repeats (LTRs), can act as cis- regulatory elements, such as promoters and enhancers, to regulate host gene expression. After fertilization, the mammalian zygote activates its own genome in a process called zygotic gene activation (ZGA). Hundreds of retrotransposon subfamilies—which are repetitive elements that constitute ~40% of our genomes—become activated as well. This wave of retrotransposon activation is considered critical for ZGA and early embryogenesis, with its disruption leading to embryonic arrest. Similarly, in human in vitro fertilization, ~40% of embryos arrest during ZGA, and this is associated with mis-expression of retrotransposons. Thus, understanding the functions of retrotransposon activation during ZGA has important implications for both basic biology and reproductive medicine, but how specific retrotransposon subfamilies and single copies regulate ZGA, and preimplantation development is not known. The overall goal of this proposal is to understand to what extent LTR retrotransposon activation regulates ZGA and early embryogenesis. Genetic manipulation of retrotransposons is technically challenging because each retrotransposon subfamily has hundreds to thousands of copies interspersed throughout the mouse genome. Indeed, only a single LTR-derived promoter has been characterized in mouse preimplantation embryos. To overcome this barrier to progress, our lab recently developed a CRISPR interference (CRISPRi) method in mouse embryos that can systematically evaluate cis-regulatory functions of retrotransposons at the entire subfamily level (i.e., thousands of copies simultaneously). Using this method, we determined cis-regulatory activities of ~3500 copies of the MT2 subfamily, a ZGA and totipotency marker, at the 2-cell stage (when ZGA occurs). By integrating functional perturbation and multi-omics analyses, we identified hundreds of MT2 copies that function as promoters and/or enhancers during ZGA. Building upon this work, we aim to identify MT2-derived promoters and enhancers that are important for ZGA and preimplantation development and systematically evaluate to what extent ZGA depends on LTR retrotransposon activation. These goals will be achieved through integrative approaches such as epigenetic editing and base editing in zygotes, genetically engineered mouse models, and ultra-sensitive epigenome profiling. Completion of the proposed study will identify retrotransposon subfamilies and single copies that are important for early embryogenesis and provide novel insights on ZGA regulation in preimplantation embryos. Our findings will set the stage for understanding how retrotransposons participate in gene regulation in human embryogenesis. Collectively, these studies will advance our fundamental understanding of mammalian early development, with broader implications for improving reproductive technologies.