Project Summary/Abstract Embryonic stem cells (ESCs) hold great promise for medicine because they can be propagated to virtually unlimited numbers and can generate any disease relevant cell type. ESCs have three unique cell biological features that make them distinct from somatic cell lineages: (i) A pluripotency transcriptional network that promotes its own activity; (ii) An atypically rapid cell cycle with short G1 phase that lacks a typical restriction point; (iii) An atypical response to Mitogen Activated Protein Kinase (MAPK) activity1–3. While a lot of attention has focused on the maintenance of ESC pluripotency by a transcriptional network, research on the ESC cell cycle network has been largely descriptive, and the mechanistic links between the two networks have yet to be explored. In addition, a critical but poorly understood process is how ESCs use MAPK pathway to control the exit from pluripotency. My long-term career goal is to discover the molecular mechanisms that allows ESCs to choose between two conflicting fates, i.e, self-renewal vs. fate commitment. The driving hypothesis of this proposal is that the ESC-specific cell cycle is functionally linked with the transcriptional pluripotency network by mutual, positive feedback that is regulated by upstream activity of MAPK signaling. To test this hypothesis, we propose to investigate the function of phosphorylation sites on the pluripotency factors using both genetic and biochemical methods (Aim1). We will employ single cell quantitative imaging to measure dynamics of cell cycle in ESCs expressing reporters of pluripotency and cell cycle. To test if pluripotency factors directly promote cell cycle progression, we are proposing to develop a novel method by repurposing the CRISPR/Cas9 technology to examine the function of specific transcription factor binding sites in vivo (Aim2). To determine how MAPK signaling regulates exit from pluripotency, we will combine protein engineering and quantitative phosphoproteomics to uncover novel targets of this pathway (Aim3). Completion of these aims will reveal the mechanisms by which ESCs choose between opposing fates, i.e. self-renewal vs. fate commitment. During the training phase of this award (K99), I plan to leverage quantitative insight of Skotheim’s lab to advance an interdisciplinary research plan to study cell fate in pluripotent cells in my lab. To this end, I have established collaboration with Wernig’s lab (Stanford), Qi’s lab(Stanford) and Macek’s lab(Tubingen University, Germany) that will greatly facilitate the progress of my research project. To prepare for transition to an independent investigator, I will take part in career training courses such as The Future Faculty Series that are offered by Stanford University. In addition to representing an important advance in basic biological sciences, our mechanistic insight may facilitate propagation and lineage differentiation of ESCs for regenerative medicine.