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

## TL;DR

The study shows how the movement and binding of SOX9 molecules change during intestinal cell development and when cells are reprogrammed to a fetal-like state.

## Contribution

The novel use of automated live-cell single-molecule tracking reveals SOX9 dynamics linked to differentiation and reprogramming in intestinal organoids.

## Key findings

- SOX9 immobile fraction decreases during differentiation, independent of expression levels.
- Long-term SOX9 overexpression causes fetal-like reversion with increased proliferation and loss of intestinal identity.
- Automated single-molecule tracking in 2D monolayers reveals transcription factor dynamics underlying organoid phenotypes.

## Abstract

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.

•Heterogenous diffusion behavior of SOX9 across intestinal differentiation states•Concentration-independent decrease in chromatin-bound SOX9 upon differentiation•SOX9 overexpression leads to fetal-like reversion and more immobile SOX9 molecules•Automated SMT in 2D monolayers reveals TF dynamics underlying organoid phenotypes

Heterogenous diffusion behavior of SOX9 across intestinal differentiation states

Concentration-independent decrease in chromatin-bound SOX9 upon differentiation

SOX9 overexpression leads to fetal-like reversion and more immobile SOX9 molecules

Automated SMT in 2D monolayers reveals TF dynamics underlying organoid phenotypes

Walther and colleagues employed an automated live-cell single-molecule tracking pipeline to study the diffusive behavior of the transcription factor SOX9 during adult differentiation and fetal-like reprogrammed states in intestinal organoid models. The authors linked distinct fractions of chromatin-bound SOX9 molecules to specific cellular states in enteroid monolayers, thereby paving the way to unravel molecular mechanisms underlying differentiation and organoid phenotypes.