Molecular feedback between cell division cycle and differentiation in pluripotent stem cells

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Project Summary A major long-term goal of my laboratory is to determine molecular feedback mechanisms responsible for coordination between the cell division cycle and differentiation of embryonic stem cells (ESCs). ESCs have great therapeutic potential for regenerative medicine, because they can differentiate into any cell type and have unlimited self-renewal potential. This remarkable biological potential, known as pluripotency, is associated with a complex transcription network, an ultrafast cell division cycle that lacks typical checkpoints, and an atypical response to activation of the Mitogen-Activated Protein Kinase (MAPK) pathway. While a lot of progress has been made in characterizing the pluripotency transcription network, less is known about the cell division cycle of ESCs and its molecular links to the pluripotency transcription network. In addition, a critical yet poorly understood process is how ESCs use the MAPK pathway to control their self-renewal and differentiation. Our driving hypothesis is that the cell division cycle and pluripotency transcription network are linked through a bidirectional molecular feedback loop that is regulated by the MAPK activity. To test our hypothesis, we will: I. Characterize novel regulators of the MAPK pathway identified in our CRISPR screen in ESCs II. Determine functional substrate network of the MAPK kinases, Mek and Erk, in ESCs using chemical-genetic kinase engineering and quantitative phosphoproteomics. III. Determine mechanisms of pluripotency maintenance by the G1 cell cycle kinase (Cdk2) using direct labeling of the substrates by chemical-genetic engineering of Cdk2. To successfully complete proposed experiments, I have established collaborations with Professor Stanley Qi laboratory (Stanford University), Professor Alice Ting laboratory (Stanford University), Professor Seth Rubin laboratory (University of California, Santa Cruz) and Professor Boris Macek laboratory (University of Tubingen). By gaining detailed insight into the molecular mechanisms linking the cell division cycle and differentiation of ESCs, the outcome of this R35 proposal will provide novel strategies to address a key challenge in the field of regenerative medicine that is efficient and reproducible differentiation of ESCs for therapeutic purposes.