Nearly half of both human and mouse genomes originate from ancient retroviral integrations. While silenced in nearly all cells, retrotransposon reactivation is a recognized phenomenon occurring in preimplantation embryos. In a handful of reports, disruption of retrotransposon family expression resulted in embryonic lethality, suggesting essential functions, but the cause of this is completely unknown. The majority of retrotransposons have been inactivated through mutation. Still, many retain regulatory and structural features of intact elements, with rare reports of retrotransposon influence of nearby genes. The highly repetitive nature of retrotransposons has made studying their individual functions difficult, however re-analysis of single cell pre-implantation mouse embryos revealed striking levels of dynamically expressed retrotransposon families. Most retrotransposons are only active during defined windows of time, sometimes spanning a single cell division. Interestingly, a subset of these loci are spliced with nearby protein coding genes, generating ?chimeric transcripts? that form hundreds of novel embryonic specific promoters, exons and polyadenylation sites. As a proof-of-principle, a highly efficient electroporation based CRISPR embryo editing method developed in the lab was used to generate retrotransposon deletions of two chimeric promoters. The deletion of the MT2B1 promoter driving Rpl41 results in delayed global translation, causing stress induced arrest. The second, deletion of the MT2C_Mm promoter of the cell cycle regulator Cdk2ap1, results in small litters, physical abnormalities, embryo spacing, crowding and implantation into unsuitable uterine sites, reminiscent of the human pregnancy complications placenta previa, accreta and potentially pre-eclampsia. Thus, the applicant hypothesizes that retrotransposon reactivation and transcript chimerism in preimplantation is essential for early embryonic development and implantation. During the K99 phase, the applicant will train in three cutting edge technologies: automated live cell spinning disk confocal imaging, single cell/embryo Western Blot with Dr. Amy Herr and ultra-low input Ribosome Profiling of Embryos with Dr. Nicholas Ingolia. These collaborations and additional coursework will help the applicant master the techniques needed for long term academic and career goals. To this end, Aim1 will serve to understand the cellular cause of the implantation defects of the M2TC_Mm:CKD2ap1 deletion, offering insight into novel explanations for related human pregnancy issues.
Aim2 will work to elucidate the role of M2TB1:Rpl41 in global translation as well as to gain insight into the oxidative and metabolic needs of the developing embryo.
Aims 1 and 2 will be completed during the K99 phase. During the R00 phase, Aim 3 will work to determine the extent of retrotransposon influence on the preimplantation embryo. Together with Dr. Davide Risso, parallel analysis of the embryo ribosome profiling and matched RNA-SEQ data will help to unravel and further test the complex re- wiring of the preimplantation embryo by retrotransposon reactivation. As these reactivations are not unique to the mouse, this work will establish a method to investigate this overlooked but important novel regulatory network and offer additional explanations to human developmental phenotypes that thus far have no clear genetic cause.
Nearly 50% of infertility and recurrent pregnancy loss cases are currently without a genetic explanation. Disruption of retrotransposon activity in the mouse preimplantation embryo leads to altered global protein synthesis, improper uterine implantation that resembles human pregnancy complications and embryonic lethality. This project will adapt powerful proteomics, genetics, bioinformatics and CRISPR/Cas9 genome editing technologies to generate a meaningful understanding of the role of retrotransposon activity in development and disease that will reveal an overlooked and essential form of regulation present in early embryonic development.
