Mechanisms of intestinal stem cell differentiation and plasticity.

潘氏细胞（PC），位于活跃的隐窝基底柱状（CBC）肠干细胞之间 肠上皮细胞（ISC）在肠道中起几种重要的稳态功能，包括i）调节肠上皮细胞的功能， ii）作为小生境细胞提供生长因子，和 向相邻的CBC细胞发送信号。最近的证据表明，分泌细胞（注定成为PC）可以 恢复到CBC状态，以在受伤后重新填充肠道。CBC细胞高度依赖于典型的 Wnt信号传导促进增殖和自我更新。PC同样存在于高Wnt环境中， 一种强有力的细胞核β-连环蛋白，表明典型的Wnt激活--但它们不增殖。这就提出了一个 问题：有效的经典Wnt激活如何在相邻上皮细胞中产生如此不同的结果？ 我们先前发表的结果确定了一个转录级联（Atoh 1-Gfi 1-SPDEF）， 肠分泌细胞包括PC。我们最近的出版物表明，SPDEF（SAM指向域 ETS因子），通常在PC中表达，但在CBC中不表达，可以抑制β-连环蛋白染色质结合， 选择靶基因的转录激活。这些结果支持了我们的总体假设，即SPDEF 转移β-连环蛋白的染色质靶点以调节干细胞与潘氏细胞的命运。我们将测试这个 三个目标的假设。目的1：定义CBCs与CBCs中β-连环蛋白和SPDEF靶点的分布。 潘氏细胞。我们将使用RNA-和染色质免疫沉淀-（ChIP-）序列来定义功能性 Lgr 5 + CBC干细胞和PC中β-连环蛋白和SPDEF的靶点。这一目标将使我们的第一个纲要， SPDEF和β-连环蛋白在肠道中的细胞特异性靶点，并测试SPDEF是否是必需的， 足以转移干细胞龛中β-连环蛋白的染色质靶点。目标2：确定 SPDEF改变β-catenin靶点转录的机制。我们将探测 SPDEF和β-catenin蛋白复合物之间的相互作用，以及转录水平的变化， 机制调控CBC与PC基因。这一目标将严格检验我们的机械假说， SPDEF改变了β-连环蛋白的靶点，并且对于任何合理的靶向该过程的治疗是必不可少的。 干预目的3：测试SPDEF：β-连环蛋白相互作用在受损的神经再生中的作用。 地穴在这里，我们将使用单细胞RNA-seq（scRNA-seq）和ChIP来表征损伤诱导的逆转， 并确定β-连环蛋白染色质结合作为逆转过程的一部分如何变化。接下来我们就 定义下调SPDEF以实现分泌细胞逆转为 损伤后的干细胞最后，我们将使用一种新的体外损伤-逆转-再生方法， 类器官以测试SPDEF在调节β-连环蛋白指导的CBC恢复中的作用。这一目标将提供 单细胞分辨率，以确定隐窝内细胞可塑性的机制和SPDEF的作用， 再生中的β-连环蛋白/WNT信号传导。

Paneth cells (PCs), which reside intercalated among the active crypt base columnar (CBC) intestinal stem cells (ISCs), serve several important homeostatic functions in the gut, including i) regulation of the intestinal microbiome by secretion of antimicrobial peptides and ii) serving as niche cells to provide growth factors and signals to adjacent CBC cells. Recent evidence has suggested that secretory cells (fated to become PCs) can revert to a CBC state to repopulate the intestine following injury. CBC cells are highly dependent on canonical Wnt signaling to drive proliferation and self-renewal. PCs likewise exist in a high-Wnt environment, and show robust nuclear β-catenin indicative of canonical Wnt activation--but they do not proliferate. This raises the question: how does potent canonical Wnt activation produce such different results in adjacent epithelial cells? Our previously published results identified a transcriptional cascade (Atoh1-Gfi1-SPDEF) that specifies intestinal secretory cells including PCs. Our recent publications indicate that SPDEF (SAM Pointed Domain ETS Factor), which is normally expressed in PCs but not CBCs, can repress β-catenin chromatin binding and transcriptional activation of select target genes. These results support our overarching hypothesis that SPDEF shifts the chromatin targets of β-catenin to regulate stem cell vs. Paneth cell fate. We will test this hypothesis in three aims. Aim 1: Define the landscape of β-catenin and SPDEF targets in CBCs vs. Paneth cells. We will use RNA- and Chromatin Immunoprecipitation- (ChIP-) seq to define functional targets of β-catenin and SPDEF in Lgr5+ CBC stem cells and PCs. This aim will give us the first compendium of cell-specific targets of SPDEF and β-catenin in the intestine, and test whether SPDEF is necessary and sufficient to shift the chromatin targets of β-catenin in the stem cell niche. Aim 2: Determine the mechanism by which SPDEF changes transcription of β-catenin targets. We will probe the physical interaction between SPDEF and β-catenin protein complexes, as well as the changes to transcriptional machinery regulating CBC vs. PC genes. This aim will rigorously test our mechanistic hypothesis about how SPDEF alters β-catenin targets, and is essential for any rational targeting of this process for therapeutic intervention. Aim 3: Test the role for SPDEF:β-catenin interaction in regeneration of damaged crypts. Here, we will use single-cell RNA-seq (scRNA-seq) and ChIP to characterize injury-induced reversion, and to determine how β-catenin chromatin binding changes as part of the reversion process. Next, we will define the requirement and sufficiency for downregulation of SPDEF to achieve reversion of secretory cells into stem cells following injury. Finally, we will use a novel in vitro injury-reversion-regeneration methodology in organoids to test the role for SPDEF in regulating β-catenin directed CBC recovery. This aim will provide single-cell resolution to define the mechanism of cellular plasticity within the crypt and the role of SPDEF and β-catenin/WNT signaling in regeneration.