Single-cell analysis of SOX2 protein level dynamics in pluripotency and neuroectodermal commitment
Strebinger D1 and Suter DM*1
1UPSUTER, The Institute of Bioengineering (IBI), School of Life Sciences, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland
Transcription factor (TF) networks govern the maintenance and changes of cell fates. In embryonic stem (ES) cells, SOX2 and OCT4 reside at the core of the pluripotency network and further play a role in germ layer specification. SOX2 levels are kept within a narrow range and we have recently shown that its expression at the M-G1 transition is important for pluripotency maintenance and neuroectodermal (NE) differentiation.  While earlier studies indicate that the fine-tuning of SOX2 protein levels regulate cell fate decisions , there is no quantitative live single-cell study correlating SOX2 expression dynamics with cell fate decisions.
Here we generated a knock-in mES cell line where both Sox2 endogenous alleles are tagged with NanoLuc, and one of the endogenous Sox1 alleles is fused to a P2A-Firefly Luciferase cassette. This allows using luminescence microscopy for highly sensitive, absolute quantification of endogenous SOX2 protein levels in single living cells, and the simultaneous monitoring of NE differentiation by SOX1 expression.
Analysis of absolute copy numbers and concentrations of SOX2 during pluripotency maintenance revealed a gradual increase over the cell cycle, proportional to the nuclear volume, resulting in near-constant concentrations. Cells expressing high or low SOX2 levels rapidly readjusted their expression towards the mean during the G1 phase. During differentiation, SOX2 levels were generally lower and displayed large fluctuations. We are currently investigating the correlation between these changes and cell fate outcomes. In summary, our single cell live imaging approach allows deciphering the quantitative relationship between SOX2 dynamics and cell fate outcomes.
 Deluz et al; Genes Dev. 2016 Nov 15;30(22):2538-2550
 Thomson et al; Cell. 2011 Jun 10;145(6):875-89
Swiss National Science Foundation (grant no. PP00P3_144828)
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