Heterogeneous Sox2 transcriptional dynamics mediate pluripotency maintenance in mESCs in response to LIF signaling perturbations

The LIF signaling pathway and its regulation of internal factors like Sox2 is crucial for maintaining self-renewal and pluripotency in mESCs. However, the direct impact of LIF signaling on Sox2 transcriptional dynamics at the single-cell level remains elusive. Here, we employ PP7/PCP-mediated live imaging to analyze the transcriptional dynamics of Sox2 under perturbation of the LIF signaling pathway at single-cell resolution. Removal of the LIF ligand or addition of a JAK inhibitor heterogeneously affects the cell population, reducing the number of Sox2-active cells, rather than completely abolishing Sox2 expression. Moreover, Sox2-active cells under LIF perturbation exhibit significant reductions in mRNA production per cell. This reduction is characterized by decreased size and frequency of transcriptional bursting, resulting in shorter duration of Sox2 activity. Notably, cells with reduced or absent Sox2 expression demonstrate a significant loss in pluripotency, indicating that a reduction in Sox2 transcription (rather than a complete loss) is sufficient to trigger the transition from embryonic to an early differentiated state. In LIF-perturbed cells with Sox2 expression reduced to about 50% of non-perturbed levels, we observe a binary behavior, with cells either retaining or losing pluripotency-associated traits. Lastly, we find Sox2 expression is transcriptionally inherited across cell cycles, with Sox2-active mother cells more likely to reactivate Sox2 after mitosis compared to Sox2-inactive cells. This robust transcriptional memory is observed independent of LIF signaling perturbation. Our findings provide new insights into the transcriptional regulation of Sox2, advancing our understanding of the quantitative thresholds of gene expression required for pluripotency maintenance and highlighting the power of single-cell approaches to unravel dynamic regulatory mechanisms.