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

转录控制的丧失和伴随的基因表达异常是癌症和发育障碍的特征。在整个发育过程中，精确的基因表达调控确保了细胞命运的指定和分化，以形成健康的组织。转录调控是通过谱系决定的转录因子(TF)与其靶基因的顺式调控DNA元件结合，从而诱导特定细胞类型的基因表达程序来实现的。在发育胚胎或更新成人组织的多细胞环境中，基因表达受到细胞位置和信号环境的影响。因此，为了理解确保健康组织形成的调控机制，在这样一个多细胞系统中解决特定细胞类型的发育基因调控是至关重要的。在哺乳动物的肠道上皮中，空间分化层次指导分化的肠道干细胞(ISCs)的定向运动。在体外总结这一行为，小鼠小肠类器官提供了一个明确的3D组织模型来研究ISC分化过程中的时空基因表达调控。在基于测序的Micro-C和晶格光片显微镜的最新发展的基础上，这一建议将尖端基因组学与从组织到单分子水平的活细胞成像相结合，以探索肠道类器官分化过程中基因表达调控的空间和时间维度。重点放在从ISCs到肠细胞的分化途径上，这是负责营养吸收的途径，该项目的目标如下：1)表征肠上皮细胞特定类型的染色质组织，以揭示细胞分化过程中染色质结构的变化；2)长期可视化转录因子驱动细胞命运的决定及其与靶基因表达的相关性，以揭示其与细胞类型和在肠道器官中的位置相关的动态变化；3)与细胞类型和在肠道器官中的位置相关的动态生物物理和定量表征，以得出决定细胞命运的基因调控机制的数据驱动模型。这项工作将提供第一张由谱系决定的转录因子诱导细胞类型特异性基因表达程序的时空图，以推动ISC向肠细胞分化。它将进一步揭示在肠道类器官的多细胞环境中，染色质重组、转铁蛋白丰度和DNA结合行为是如何调节细胞命运的。此外，这项工作将为未来了解组织形成和更新过程中的致病机制的研究奠定技术基础。

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.