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.

Paneth细胞（PC），该细胞位于活性加密柱柱（CBC）肠道干细胞之间 （ISC），在肠道中提供几个重要的稳态功能，包括i）调节肠 微生物组通过分泌抗菌胡椒体和ii）作为利基细胞提供生长因子和 信号与邻近的CBC细胞。最近的证据表明，秘密细胞（投产为PC）可以 恢复到CBC状态，以在受伤后重新填充肠。 CBC细胞高度依赖于规范 Wnt信号传导以驱动增殖和自我更新。 PC同样存在于高温环境中，并显示 强大的核β-catenin指示了典型的Wnt激活 - 但它们不会增殖。这提高了 问题：潜在的规范Wnt激活如何在相邻的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指定的转录级联（ATOH1-GFI1-SPDEF） 包括PC的肠道秘书细胞。我们最近的出版物表明SPDEF（Sam尖头领域 通常在PC中而不是CBC表示的ETS因子）可以抑制β-catenin染色质结合和 选定靶基因的转录激活。这些结果支持我们的总体假设，即SPDEF 移动β-catenin的染色质靶标，以调节干细胞与Paneth细胞命运。我们将测试这个 三个目标的假设。目标1：定义CBCS与CBCS中β-catenin和SPDEF靶标的景观。 Paneth细胞。我们将使用RNA和染色质免疫沉淀 - （芯片）SEQ来定义功能 LGR5+ CBC干细胞和PC中的β-catenin和SPDEF的靶标。这个目标将为我们提供第一个汇编 肠中SPDEF和β-catenin的细胞特异性靶标，并测试SPDEF是否需要 AIM 2：确定干细胞小众中β-catenin的染色质靶标。 SPDEF改变β-catenin靶标的转录机制。我们将探测物理 SPDEF和β-catenin蛋白复合物之间的相互作用，以及转录的变化 机械调节CBC与PC基因。这个目标将严格检验我们关于如何 SPDEF改变了β-catenin的靶标，对于此过程的任何合理靶向治疗至关重要 干涉。 AIM 3：测试SPDEF的作用：β-catenin的相互作用在受损的再生中 地下室。在这里，我们将使用单细胞RNA-Seq（SCRNA-SEQ）和芯片来表征损伤引起的反向， 并确定β-catenin染色质结合如何变化作为反向过程的一部分。接下来，我们会的 定义对SPDEF下调的要求和充分性，以使秘书细胞反向 受伤后干细胞。最后，我们将使用一种新颖的体外损伤逆转 - 再生方法 类器官测试SPDEF在调节β-catenin定向CBC恢复中的作用。这个目标将提供 单细胞分辨率来定义地下室内细胞可塑性的机理以及SPDEF和 再生中的β-catenin/wnt信号传导。