PROJECT SUMMARY Totipotency, a remarkable cellular plasticity of a single cell in generating a complete organism with both embryonic and extraembryonic contribution, is essential for multicellularity and development. In mice, fully totipotent cells exist transiently in the zygote and cleavage-stage blastomeres, although totipotent 2-cell (2C)- like cells (2CLCs) can also arise sporadically or induced genetically in cultured embryonic stem cell (ESCs) that are pluripotent and can only contribute to embryonic lineages. Recently, expanded potential stem cells and extended pluripotent stem cells, collectively known as EPSCs, with totipotency features were derived and stably cultured in vitro using two distinct sets of small molecule inhibitors. However, 2CLCs and the two EPSC lines differ in their culture conditions, transcriptomes, and expression of core pluripotency factors, suggestive of alternative totipotent states. 2CLCs and EPSCs represent easily accessible cell source and valuable in vitro models for understanding totipotency, the knowledge of which will be critical to capture and stabilize the totipotent 2CLC state, which is not currently possible. Defining molecular pathways underlying these various totipotent cells will facilitate unraveling the complex regulatory mechanisms of totipotency and achieving the capture/stabilization of totipotent 2CLCs. The totipotent 2CLCs were reported to reactivate transcription of endogenous retroviruses (ERVs), in particular MERVL, whose activation has now been found to be causative for totipotency in our preliminary studies. ZSCAN4 is also sharply expressed in 2C-stage embryo and marks those sporadic ESCs exiting from the ESC state towards 2CLCs. By characterizing MERVL/ZSCAN4 double reporter positive (DR+/+) ESCs, we have begun to dissect the molecular pathways underlying 2CLC totipotency and discovered miR-344 and its direct target ZMYM2 as novel positive and negative regulators, respectively, for MERVL activation and 2CLC totipotency. The objective of this application is to extend our knowledge of the divergent cellular potency states in ESC culture, and define the MERVL contribution to totipotency and explore transcriptional and post-transcriptional mechanisms underlying totipotency. We hypothesize that 2CLCs represent an alternative totipotent state that is regulated by a novel molecular axis encompassing the transcriptional and post-transcriptional mechanisms. We propose the following studies to test our hypothesis. 1) Define a molecular roadmap for alternative totipotent states in EPSCs and 2CLCs. 2) Establish the functional contribution of MERVL elements to 2CLC totipotency. We will define and distinguish functional roles of individual MERVL elements in contributing to 2CLC totipotency. 3) Dissect the molecular mechanism underlying 2CLC totipotency by establishing miR-344 as a new paradigm for understanding mammalian totipotency. Understanding the molecular mechanisms that drive and induce totipo...