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 two unique cell biological features that make them distinct from somatic cell lineages: (i) they possess a pluripotency transcriptional network that promotes its own activity, and (ii) they have an atypically rapid and mitogen-independent cell cycle. While a lot of attention has focused on the ESC pluripotency transcriptional network, research on the ESC cell cycle network has been largely descriptive, and the potential links between the two networks are unexplored. Cell division is phenomenologically linked to the pluripotent state of embryonic stem cells. Here, we aim to discover the molecular mechanisms linking the cell division cycle with pluripotency. 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. In this model, the ESC pluripotency network drives division and the cell cycle control network promotes pluripotency. Previous results suggest that cell cycle kinases regulate pluripotency and differentiation. Our preliminary results suggest that central pluripotency transcription factors are phosphorylated cell cycle kinase, which would provide a mechanism directly connecting cell cycle control with pluripotency. To test this hypothesis, we propose to investigate the function of phosphorylation sites on the pluripotency factors using both genetic and biochemical methods. We will employ novel single cell quantitative imaging to measure and characterize cell division in ESCs expressing reporters of pluripotency and cell cycle. To test if pluripotency factor directly promotes cell cycle progression, we are proposing to develop a novel method repurposing the CRISPR/Cas9 technology to examine the function of specific transcription factor binding sites in vivo. In addition to representing an important advance in basic biological sciences, our mechanistic insight may facilitate propagation and lineage differentiation of ESCs in vitro for regenerative medicine.