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

转录控制缺失和伴随的基因表达异常是癌症和发育障碍的标志。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规范和向健康组织形成的分化。转录调控是通过决定谱系的转录因子（TFs）结合其靶基因的顺式调控DNA元件来实现的，从而诱导细胞类型特异性基因表达程序。在胚胎发育或成人组织更新的多细胞环境中，基因表达受到细胞位置和信号环境的影响。因此，为了了解确保健康组织形成的调控机制，有必要在这样一个多细胞系统中解决细胞类型特异性发育基因在空间和时间上的调控。在哺乳动物肠上皮中，空间分化层级指导肠干细胞（ISCs）的定向运动。在体外，小鼠小肠类器官为研究ISC分化过程中的时空基因表达调控提供了一个定义良好的三维组织模型。基于基于测序的Micro-C和晶格光片显微镜的最新发展，本研究将前沿基因组学与从组织到单分子水平的活细胞成像相结合，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。本项目主要研究肠上皮细胞向负责营养摄取的肠上皮细胞的分化途径，旨在：1)表征肠上皮细胞类型特异性染色质组织，揭示分化过程中染色质结构的变化。2)长期可视化tf驱动细胞命运决定及其与靶基因表达的相关性，揭示其在肠道类器官内与细胞类型和位置相关的动态。3)肠道类器官中与细胞类型和位置相关的动态生物物理和定量TF表征，建立细胞命运决定基因调控机制的数据驱动模型。这项工作将提供第一个谱系决定tf诱导细胞类型特异性基因表达程序的时空图谱，以驱动ISC向肠细胞分化。这将进一步揭示在肠道类器官的多细胞环境中，染色质重组、TF丰度和dna结合行为如何调节细胞命运规范。此外，这项工作将为未来的研究奠定技术基础，以了解在组织形成和更新过程中引起疾病的机制。

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.