Improving Bovine Cloning Efficiency by Enhancing Reprogramming during Embryonic Genome Activation (EGA)

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This F32 proposal outlines research objectives to investigate the early gene expression regulation of bovine somatic cell nuclear transfer (SCNT) cloned embryos, and improve their developmental potential by harnessing our knowledge of IVF bovine preimplantation development. Based on several published reports, including my own work, my hypothesis is that failed embryonic genome activation (EGA) is the rate-limiting step negatively impacting the robust development of cloned animals. Because many EGA activated genes are involved in epigenetic reprogramming embryonic chromatin, failure to appropriately turn on these genes leads to developmental dysregulation and failure, often manifested much later in gestation. Building upon both our published work, showing that DUX transcription factors (TF) are the master regulators of gene expression at the egg-to-embryo transition, and utilizing my extensive unpublished preliminary data laying the ground work for our hypothesis in the bovine system, I will first address this problem using cutting-edge genomic, epigenomic, and embryological methods. First, I will test whether the putative enhancer regions I identified in IVF bovine embryos show stage-specific transcriptional activity in vivo. I will also dissect which TFs the putative enhancer regions are responsive to using a cellular system. Then I will identify open chromatin regions in bovine SCNT embryos and compare them to the high-quality IVF embryo open chromatin maps I have previously generated. This will help me understand how much of the SCNT developmental phenotype is caused by failure to activate EGA chromatin or failure to decommission somatic cell open chromatin. Finally, I will use our data that cow DUXC is a major activator of bovine EGA to test our hypothesis that DUXC deficiency can be overcome by ectopically expressing DUXC in SCNT embryos and that this will increase the efficiency of SCNT. With significance for human assisted reproductive technologies, development of large animal models of human embryogenesis, agricultural significance of cloning, and ecological impacts of endangered species preservation through SCNT, this work integrates innovative strategies to impact multiple fields.