Transcription factors (TFs) mediate gene expression changes during differentiation and development. However, how TF biophysical properties and abundance dynamically regulate specific cell state transitions remains poorly understood. Using automated live-cell single-molecule tracking (SMT) in intestinal organoid models, we revealed an expression-level-independent decrease in the fraction of immobile sex-determining region Y box 9 (SOX9) molecules during differentiation from ∼48% to ∼38%, largely dependent on DNA binding. Strikingly, long-term SOX9 overexpression caused organoids to transition from budding to spheroid morphology accompanied by increased proliferation and a loss in gene expression signatures for intestinal identity and function. In this fetal-like reprogrammed state, a larger fraction of partially self-interacting SOX9 molecules (∼61%) binds to DNA. Our results suggest context-dependent SOX9 single-molecule dynamics during adult intestinal differentiation and fetal-like reversion in consequence to long-term SOX9 overexpression. Our work underpins the power of our automated live-cell SMT framework to generate testable hypotheses toward unraveling molecular mechanisms underlying tissue-level phenotypes.
a2213-6711