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. Overall design: 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.微褶皱（Microfold, M）细胞定位于派尔集合淋巴结（Peyer's patches）的肠上皮中。其具备从肠腔摄取并转运抗原至下方淋巴组织的独特能力，是调控肠道相关免疫应答的关键环节。本研究采用多组学策略，探究小鼠小肠类器官中驱动M细胞分化的分子机制。我们构建了体外M细胞分化过程中染色质可及性变化与转录因子动态调控的全面图谱，由此鉴定出大量细胞类型特异性调控元件及其关联的转录因子。借助单细胞RNA测序（single-cell RNA sequencing），我们鉴定出一类M细胞前体细胞群。我们依托自研的计算工具SCEPIA，将前体细胞特异性基因表达与顺式调控元件中的转录因子基序进行关联分析，发现转录因子ONECUT2呈现高表达状态且具备基序活性。后续体外与体内扰动实验表明，ONECUT2在RANK/RANKL信号通路下游发挥作用，以促进肠上皮细胞分化，进而限制M细胞谱系特化。本研究为调控黏膜免疫与细胞命运平衡的机制提供了新的见解，同时为探究肠上皮细胞的命运转换提供了极具价值的研究范式。整体实验设计：对经RANKL处理6天的小鼠小肠类器官（单独处理或联合CSRM617处理），以及采用常规类器官培养基培养的对照类器官进行批量RNA测序（bulk RNA-sequencing）分析。生物学重复对应3次独立的分化实验。