Mechanisms of intestinal stem cell differentiation and plasticity.

肠道干细胞分化和可塑性的机制。

• 批准号: 9788430
• 负责人: NOAH Freeman SHROYER
• 金额: $ 56.09万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788430
• 关键词: Affinity; Binding; Binding Sites; Cell Line; Cells; ChIP-on-chip; ChIP-seq; Chromatin; Columnar Cell; Complex; Down-Regulation; ETS Domain; Environment; Epithelial Cells; GFI1 gene; Genes; Genetic Transcription; Growth Factor; Human; In Vitro; Infection; Injury; Intestines; LGR5 gene; Lead; Measures; Mediating; Methodology; Multiprotein Complexes; Mus; Mutation; Natural regeneration; Normal Cell; Nuclear; Organoids; Paneth Cells; Process; Publications; Publishing; RNA; Radiation; Recovery; Regulation; Resolution; Role; SAM Domain; Secretory Cell; Signal Transduction; Specific qualifier value; Stem cells; TCF Transcription Factor; Tertiary Protein Structure; Testing; Therapeutic Intervention; Transcriptional Activation; WNT Signaling Pathway; antimicrobial peptide; base; beta catenin; chemotherapy; chromatin immunoprecipitation; gut microbiome; mutant; novel; overexpression; prevent; progenitor; protein complex; self-renewal; side effect; single-cell RNA sequencing; stem cell differentiation; stem cell niche

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.

潘氏细胞 (PC)，位于活跃的隐窝基底柱状 (CBC) 肠干细胞之间 (ISC)，在肠道中发挥多种重要的稳态功能，包括 i) 肠道调节 ii) 作为生态位细胞提供生长因子和 最近的证据表明，分泌细胞（注定会成为 PC）可以向邻近的 CBC 细胞发出信号。 损伤后恢复到 CBC 状态以重新填充肠道的 CBC 细胞高度依赖于规范。 驱动 PC 增殖和自我更新的 Wnt 信号同样存在于高 Wnt 环境中，并且表明。 强大的核β-连环蛋白表明典型的Wnt激活——但它们不增殖。 问题：有效的经典 Wnt 激活如何在邻近的上皮细胞中产生如此不同的结果？ 我们之前发表的结果确定了一个转录级联 (Atoh1-Gfi1-SPDEF)，它指定 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并 这些结果支持我们的总体假设：SPDEF。 改变 β-连环蛋白的染色质靶标来调节干细胞与潘氏细胞的命运，我们将对此进行测试。 三个目标的假设 目标 1：定义 CBC 与 SPDEF 目标的情况。 我们将使用 RNA 和染色质免疫沉淀 (ChIP-) seq 来定义功能。 Lgr5+ CBC 干细胞和 PC 中 β-catenin 和 SPDEF 的靶点这一目标将为我们提供第一个概要。 SPDEF 和 β-catenin 在肠道中的细胞特异性靶标，并测试 SPDEF 是否必要以及 足以改变干细胞生态位中 β-连环蛋白的染色质靶标。 目标 2：确定 SPDEF 改变 β-catenin 靶标转录的机制我们将探讨其物理机制。 SPDEF 和 β-catenin 蛋白复合物之间的相互作用，以及转录的变化 CBC 与 PC 基因的调节机制 这一目标将严格检验我们关于如何调节 CBC 基因的机制假设。 SPDEF 改变 β-连环蛋白靶标，对于合理靶向该过程以进行治疗至关重要 目标 3：测试 SPDEF：β-连环蛋白相互作用在受损再生中的作用。 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在逆转过程中如何变化。 定义下调 SPDEF 以实现分泌细胞逆转为的要求和充分性 最后，我们将使用一种新颖的体外损伤恢复再生方法。 类器官来测试 SPDEF 在调节 β-连环蛋白指导的 CBC 恢复中的作用。 单细胞分辨率来定义隐窝内细胞可塑性的机制以及 SPDEF 和 再生中的 β-连环蛋白/WNT 信号传导。