Genetic regulation of active and reserve intestinal stem cell states

活性和储备肠道干细胞状态的基因调控

• 批准号: 10026771
• 负责人: SCOTT T MAGNESS
• 金额: $ 7.4万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-19 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10026771
• 关键词: Ablation; Alleles; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Cell Cycle Regulation; Cell Differentiation process; Cell Proliferation; Cell Survival; Cells; Cessation of life; DNA Repair; Data; Dose; Epithelial; Epithelial Cells; Epithelium; Equilibrium; Fluorescence; G1 Arrest; G1 Phase; Generations; Genes; Genetic; Genotoxic Stress; Health; Human; Image; Immunoblotting; Intestines; Label; Length; Link; Measures; Mediating; Mitosis; Mus; Pathway interactions; Pharmacology; Phase; Phase Transition; Phenocopy; Phenotype; Physiological; Process; Proliferating; Property; Radiation; Radio; Regenerative response; Regulation; Resistance; S Phase Arrest; Secretory Cell; Series; Signal Transduction; System; Testing; Tissues; Ubiquitination; Work; base; cell type; epithelium regeneration; experimental study; in vivo; intestinal crypt; intestinal epithelium; irradiation; monolayer; notch protein; outcome prediction; overexpression; radiation resistance; response; self-renewal; stem cells; transcription factor

ABSTRACT: Complete physiologic renewal of intestinal epithelium occurs about every week and is driven by actively proliferating ISCs (aISCs) located in the epithelial-crypt base. A rare subset cells in the crypt, still not completely defined but that are collectively known as `reserve' ISCs (rISCs), are `quiescent' or slowly dividing, and can convert into aISCs when certain health conditions or radio- or chemotherapeutic exposures damage and deplete the native aISC pool. The rISC to aISC conversion process is traditionally studied in mice where irradiation (IR) is used to deplete aISCs and induce rISCs. Here, rISCs are defined by their resistance to IR-induced death, and then by their `plastic' ability to generate actively dividing ISC progeny, which replenish the aISC pool and drive subsequent epithelial regeneration. The mechanisms conferring rISC radio-resistance and plasticity are unknown. In prior work we demonstrated that the transcription factor Sox9 is required for the generation and function of rISCs in mice. Lineage tracing with a Sox9CreERT2 driver showed that after IR, all regenerating epithelium is derived from cells that expressing Sox9, and epithelium-specific genetic ablation of Sox9 profoundly impeded epithelial regeneration and cell survival post-IR. These findings indicate that Sox9-dependent mechanisms govern rISC function. Growing evidence in the stem cell and radiation fields suggest that slowing the cell-cycle rate in dividing cells can enhance radioresistance after IR-exposure, and can modulate cell fate commitment versus self-renewal decisions. We have found that elevated Sox9 levels are associated with slowly dividing cells in the crypt, and that Sox9-overexpression in rapidly dividing aISCs can slow or halt their proliferation. We hypothesize that Sox9-expression levels modulate cell-cycle progression to determine and diversify rISC (Sox9HI) and aISC (Sox9LO) functions in the intestinal crypt. If supported by the results of our experiments, this study will uncover the underlying pathways governing rISC radioresistance and plasticity, and could provide a unifying mechanism describing whether and how rISC properties exist among a broad range of cell types in the crypt. Aim 1: Assess the effects of increasing Sox9 levels on cell-cycle progression in ISCs. Aim 2: Determine if Sox9-mediated G1-elongation confers radio-resistance. Aim 3: Determine if Sox9- mediated G1-elongation confers a `secretory precursor' rISC phenotype.

摘要：肠上皮的完整生理更新大约每周都会发生 由位于上皮键基碱的积极增殖的ISC（AISC）驱动。一个罕见的子集细胞 地下室，尚未完全定义，但被共同称为“储备” ISC（RISC）是 “静止”或缓慢分裂，并且可以在某些健康状况或无线电或无线电状态或 化学疗法暴露会损坏并耗尽本地AISC池。 RISC到AISC转换 传统上，在使用辐照（IR）来耗尽AISC并诱导RISC的小鼠中进行了过程。 在这里，riscs是通过对IR诱导的死亡的抵抗来定义的，然后是其“塑料”能力 产生主动分裂的ISC后代，该后代补充AISC池并随后驱动上皮 再生。提供RISC放射性和可塑性的机制尚不清楚。在先验 我们的工作证明了转录因子SOX9是需要的 小鼠中的risc。用Sox9Creert2驱动器进行谱系跟踪表明，IR后，所有再生上皮 源自表达SOX9的细胞，以及Sox9的上皮特异性遗传消融 IR后IR阻碍上皮再生和细胞存活。这些发现表明Sox9依赖性 机制控制RISC功能。干细胞和辐射场中越来越多的证据表明 在分裂细胞中降低细胞周期速率可以增强IR暴露后的放射线，并且可以 调节细胞命运承诺与自我更新决策。我们发现Sox9水平升高 与地下室中缓慢分裂的单元相关，而Sox9-Over表达在快速分裂中 AISC可以减慢或停止它们的扩散。我们假设Sox9-表达水平调节 细胞周期的进展，以确定和多样化RISC（SOX9HI）和AISC（SOX9LO）功能 肠道隐窝。如果在我们的实验结果的支持下，这项研究将发现 统治RISC辐射抗性和可塑性的基础途径，可以提供统一 描述RISC属性是否以及如何在广泛的细胞类型中存在的机制 地下室。目标1：评估SOX9水平增加对ISC细胞周期进展的影响。目标2： 确定SOX9介导的G1延误是否赋予了放射性。目标3：确定Sox9-是否 介导的G1延段赋予了“分泌前体” RISC表型。