Distinct SOX9 single-molecule dynamics characterize adult differentiation and fetal-like reprogrammed states in intestinal organoids

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 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 towards unraveling molecular mechanisms underlying tissue-level phenotypes. Contact: [e-mail] Overall design: Please check the following: Poly-A RNA-seq profiling of mouse small intestinal organoids stably expressing Halo-NLS (control organoids, 3 biological replicates) or Halo- or mEGFP-tagged Sox9 (organoids and "spheroids", 3 biological replicates each)