ONECUT2 regulates RANKL-dependent Enterocyte and Microfold cell differentiation in the small intestine; a multi-omics study [bulk RNA-seq]

Microfold (M) cells reside in the intestinal epithelium of Peyer’s patches. Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. By using single-cell RNA sequencing, we identified an M cell precursor population. We linked precursor-specific gene expression to transcription factor motif content in cis-regulatory elements with our newly developed computational tool SCEPIA, uncovering a high expression of and motif activity for the transcription factor ONECUT2. Subsequent in vitro and in vivo perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling to support Enterocyte differentiation, thereby restricting M cell lineage specification. This study sheds new light on the mechanism regulating mucosal immunity and cell fate balance, and provides a powerful blueprint for investigation of cell fate switches in the intestinal epithelium. Bulk RNA-sequencing analyses of mouse small intestinal organoids treated with RANKL for 6 days, alone or in combination with CSRM617, or control organoids grown in conventional organoid culture medium. Replicates correspond to three independent differentiation experiments.