Assembly of a dynamic spatio-temporal map of gene expression regulation during intestinal stem cell differentiation to enterocytes

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Loss of transcriptional control and concomitant aberrant gene expression are hallmarks of cancer and developmental disorders. Precise gene expression regulation throughout development ensures cell fate specification and differentiation towards the formation of healthy tissues. Transcription regulation is achieved by lineage-determining transcription factors (TFs) binding to cis-regulatory DNA elements of their target genes, thereby inducing cell type-specific gene expression programs. In the multicellular context of developing embryos or renewing adult tissues, gene expression is influenced by the cellular position and signaling environment. To understand the regulatory mechanisms ensuring healthy tissue formation, it is therefore essential to resolve cell type-specific developmental gene regulation spatially and temporally in such a multicellular system.In the mammalian intestinal epithelium, a spatial differentiation hierarchy guides directional movement of differentiating intestinal stem cells (ISCs). Recapitulating this behavior in vitro, mouse small intestinal organoids provide a well-defined 3D tissue model to study spatiotemporal gene expression regulation during ISC differentiation. Building upon recent developments in sequencing-based Micro-C and lattice light-sheet microscopy, this proposal combines cutting-edge genomics with live-cell imaging from the tissue to the single-molecule level to probe space and time dimensions of gene expression regulation during differentiation in intestinal organoids. Focusing on the differentiation pathway from ISCs to enterocytes, which are responsible for nutrient uptake, this project addresses the following aims:1) Characterization of cell type-specific chromatin organization in the intestinal epithelium to reveal changes in chromatin structure during differentiation.2) Long-term visualization of TFs driving cell-fate decisions and correlation with target gene expression to reveal their dynamics relative to cell type and position within intestinal organoids.3) Dynamic biophysical and quantitative TF characterization relative to cell type and position within intestinal organoids to conclude a data-driven model for cell fate-determining gene regulatory mechanisms.This work will provide the first spatiotemporal map of lineage-determining TFs inducing cell type-specific gene expression programs to drive ISC differentiation to enterocytes. It will further reveal how cell fate specification is modulated by chromatin reorganization, TF abundance and DNA-binding behavior within the multicellular environment of intestinal organoids. Furthermore, this work will set the technological groundwork for future studies to understand the mechanisms causing disease during tissue formation and renewal.