Redox Heterogeneity in Mouse Embryonic Stem Cells Individualizes Cell Fate Decisions I
How cellular diversity is created is a key question in biology. Embryonic stem cells (ESCs) are pluripotent cells that can develop into any cell type in the body. Yet, the regulatory mechanisms that govern cell fate decisions remain largely unknown. Moreover, although genetically identical, individual ESCs are strikingly heterogeneous regarding their differentiation capacity but the molecular basis for this phenotypic heterogeneity is unclear. We now demonstrate that pluripotent mouse ESCs (mESCs) display large natural variations in mitochondrial reactive oxygen species (ROS) levels that individualize their nuclear redox state and redox-sensitive H3K4me3 landscape. By using intracellular redox probes, we sorted pluripotent and differentiating mESCs based on their nuclear redox environments into subpopulations and analysed their differentiation capacity. We show that more reduced mESCs are biased towards the mesendodermal fate and form the primitive streak upon differentiation. This mesendodermal specification is due to increased Wnt signaling pathway activity, mediated via a ROS-specific regulatory mechanism of p53-dependent expression of the Wnt ligand Wnt3, and increased H3K4me3 levels. We demonstrate that formation of a primitive streak-like structure by differentiating mESCs is initiated by increased mitochondrial ROS and p53 stabilization, followed by antioxidant Nrf2 response pathway activation that leads to a reduced nuclear redox environment, allowing for the accumulation of ROS-sensitive H3K4me3 marks and activation of Wnt3 and other mesendoderm-related gene expression. In summary, our study shows that natural variations in endogenous ROS levels individualize the H3K4me3 landscape of mESCs, and through gene expression changes govern their cell fate decision at the onset of gastrulation. Overall design: To investigate the potential downstream effects of a naturally more oxidized nuclear redox state, we generated a more oxidizing environment in an entire population of pluripotent E14Tg2a mESCs by exposing them to a non-lethal dose of 500 mM H2O2 for 30 min. To explore whether and how transient exposure of mESC to peroxide affects gene expression patterns, we conducted RNA seq-analysis from three independent H2O2-time-course experiments, analyzing samples before (0 h) as well as defined time points after the treatment (0.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 24 h). 0.5 h time point was collected immediately after the 30 min treatment with hydrogen peroxide. File names: numbers 1, 2 and 3 following the respective time point indicate biological replicates. Cells 24 h after the treatment with hydrogen peroxide were compared with untreated cells at the 24 h time point. Comparative gene expression profiling analysis of RNAseq data for pluripotent E14Tg2a cells at various time points after peroxide treatment vs. untreated cells before the treatment (0 h) or 24 h after starting the experiment.