Transcriptional control and enhancer recruitment in mouse and human intestinal secretory differentiation

项目总结 肠道分泌细胞(SEC：杯状细胞(Gob)、内分泌细胞(EE)、内分泌细胞(Paneth)和簇状细胞(Tuft)) 消化、代谢、屏障和其他生理功能。肠道干细胞(ISCs)补充了这两种细胞 吸收细胞和SEC上皮细胞，但SEC多样性和普查的起源在很大程度上是未知的。因为 分离和表征中间前体细胞状态一直很困难，目前大部分 对小鼠和人类SEC血统的了解取决于对小鼠的遗传学研究。浅谈形态和 功能基础，四种成熟的SEC细胞类型被认为是不同的，它们的起源可以追溯到共同的 Atoh1表达的隐窝祖细胞(SEC-Pro)，虽然有些小鼠的EE和TUFT-但不是Gob或泛细胞 在缺少SEC特异性转录因子(Tf)的情况下产生。细胞的命运通常由细胞决定- 驱动特定转录程序的特定顺式调控元件(CRE-主要是增强子) 在细胞限制性因子的指导下。了解原语SEC-Pro如何执行后续的CRE 和细胞命运决定，我们开发了两个并行和强大的实验系统：(1)捕获 体内标记的早期小鼠SEC衍生物用于深入表征基因和单个... 细胞分辨率，以及(2)2D人类ISC样细胞在体外沿着完整的SEC谱系分化。两者都有 模型产生了大量的多势和双势前兆，因此允许原创的机械洞察力 进入相应的转录和Cre状态。我们的三个具体目标综合了来自 破译人和鼠SEC细胞表观遗传学基础和调控转铁蛋白逻辑的两个平行模型 差异化。AIM 1使用前体和子代SEC细胞的mRNA和开放染色质图谱 用于描述特定中间状态的显著特征的微分连续体，先前映射 未知的TF驱动的转变，区分细胞自主和外部因素，从而得出一个统一的 哺乳动物SEC血统多样性的模型。考虑到零星发表的研究结果，我们的 初步数据表明，Gob和潘并不是真正不同的细胞类型，而是不同版本的 多功能SEC最终产物，其中特定的局部信号作用于相同的开放染色质补充，以 驱动器井口或平移功能。目的2应用一套体外和体内实验来检验特定的假说 表皮生长因子(EGF)，特别是典型的Wnt信号有利于泛态，而骨骼 形态发生蛋白(BMP)信号驱动Gob状态。我们会研究如何以其他方式招聘 特定信号通路的GOB或泛选择性Cre可能是这些流体细胞状态的基础。AIM 3用途 功能增益型和功能损失型策略，以解决先前关于TF驱动的挑战性问题 人体SEC细胞，特别是EE和TUFT细胞的规格。总体而言，这项建议的一个特别优点是 对共同决定中间和最终细胞的特定细胞CRE和TF的深入机械研究 在小鼠和人类SEC血统中的潜力。

PROJECT SUMMARY Intestinal secretory (Sec: goblet (Gob), enteroendocrine (EE), Paneth (Pan), and tuft) cells serve vital digestive, metabolic, barrier, and other physiologic functions. Intestinal stem cells (ISCs) replenish both absorptive and Sec epithelial cells, but the origins of Sec diversity and census are largely unknown. Because it has been difficult to isolate and characterize intermediate precursor cell states, much of the current understanding of the mouse and human Sec lineages rests on mouse genetic studies. On morphologic and functional grounds, the four mature Sec cell types are considered distinct and their origins trace to a common ATOH1-expressing crypt progenitor (Sec-Pro), although some mouse EE and tuft –but not Gob or Pan– cells arise in the absence of this Sec-specifying transcription factor (TF). Cell fates are generally dictated by cell- specific cis-regulatory elements (CREs – mainly enhancers) that drive particular transcriptional programs under the direction of cell-restricted TFs. To understand how primitive Sec-Pro execute subsequent CRE and cell fate decisions, we have developed two parallel and powerful experimental systems: (1) Capture of labeled early mouse Sec derivatives in vivo for deep characterization of gene and enhancer activity at single- cell resolution, and (2) Differentiation of 2D human ISC-like cells in vitro along the full Sec lineage. Both models yield large numbers of multi- and bi-potential precursors, hence allowing original mechanistic insights into the corresponding transcriptional and CRE states. Our three Specific Aims integrate findings from the two parallel models to decipher the epigenetic basis and regulatory TF logic of mouse and human Sec cell differentiation. Aim 1 uses mRNA and open-chromatin profiles of precursor and descendant Sec cells within a differentiation continuum to characterize the salient features of specific intermediate states, map previously unknown TF-driven transitions, distinguish cell-autonomous from -extrinsic factors, and thus derive a unifying model for mammalian Sec lineage diversity. Considered in the light of scattered published findings, our preliminary data suggest that Gob and Pan are not genuinely distinct cell types, but different versions of a versatile Sec end-product where specific local signals act on the same complement of open chromatin to drive Gob or Pan features. Aim 2 applies a suite of in vitro and in vivo experiments to test specific hypotheses that epidermal growth factor (EGF) and especially canonical Wnt signaling favor the Pan state, while bone morphogenetic protein (BMP) signaling drives the Gob state. We will examine how alternative recruitment of Gob- or Pan-selective CREs by specific signaling pathways might underlie these fluid cell states. Aim 3 uses gain- and loss-of-function strategies to address previously challenging questions about TF-driven specification of human Sec, especially EE and tuft, cells. Overall, a particular strength of this proposal is the deeply mechanistic investigation of cell-specific CREs and TFs that together dictate interim and final cell potential in the murine and human Sec lineage.