Previous groundbreaking discoveries have made it possible to grow pluripotent embryonic stem (ES) cells in vitro and to reprogram adult cells back to a pluripotent state. These advances have opened new opportunities for regenerative medicine by providing the cellular starting point from which we can derive adult cell types with physiological properties to study and treat human disease. Attaining cell types suitable for direct therapeutic application or as cellular models of human disease for drug development both require a substantial level of similarly to the in vivo cellular counterpart. Most often, investigaors attempt to attain the best in vivo mimics by replicating key aspects of a cell's developmental progression through embryogenesis. Given the appreciation that epigenetic effects are cumulative and progressive through embryogenesis, a central hypothesis of this proposal is that the initial steps in lineage specification are critical, and epigenetic mistakes made during exit from pluripotency can have undesirable effects even after cells have reached the targeted differentiated state. To address this problem, we aim to elucidate the mechanisms that eliminate or decommission ES cell- specific gene regulatory elements (i.e. enhancers) from lineage specified cell types. We discovered that a sequence-specific DNA-binding transcription factor, Tcf7l1, binds specifically to enhancers in ES cells that are targeted for decommissioning. Given our previous findings that showed Tcf7l1 stimulated differentiation of ES cells, we propose testing the hypothesis that Tcf7l1 is necessary to decommission ES specific enhancers. The proposal will further determine how this effect of Tcf7l1 changes the responses of cells to extracellular signals, and measure how important it is for genuine lineage specification using established in vitro differentiation protocols. The immediate outcome of completing the proposed research will be a better understanding of how cells exit the pluripotent state, and a demonstration of how defects during early steps can diminish the efficiency of subsequent stages of differentiation. Finally, given the oncogenic potential of accessible ES- specific enhancers in adult cells, the importance of the proposed work for increasing the safety of cell-based therapies should be appreciated.