The basis for and function of enteroendocrine lineage plasticity in the intestinal DNA damage response

肠内分泌谱系可塑性在肠道DNA损伤反应中的基础和作用

• 批准号: 10399552
• 负责人: CHRISTOPHER Joachim LENGNER
• 金额: $ 43.24万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10399552
• 关键词: Address; Age; Alleles; Automobile Driving; Binding; Biological Assay; Cell Cycle; Cell Death; Cell Lineage; Cells; Chemotherapy and/or radiation; Chromatin; Chronic; Coupled; Coupling; DNA Damage; DNA Double Strand Break; Data; Development; Disease; Dose; Doxycycline; Endocrine; Enterocytes; Enteroendocrine Cell; Environment; Epigenetic Process; Epithelial; Epithelial Cells; Exhibits; Exposure to; Family; Funding; Future; Genome; Genomics; Histone H2B; Histones; In Vitro; Inflammation; Injury; Intestines; Label; Molecular; Mus; Mutagens; Natural regeneration; Organoids; Paneth Cells; Pathologic; Pathway interactions; Pharmaceutical Preparations; Physiologic pulse; Play; Population; Process; Property; Publishing; RNA; RNA Binding; RNA Editing; RNA-Binding Proteins; Reactive Oxygen Species; Regenerative capacity; Regenerative response; Reporter; Resistance; Resolution; Role; Secretory Cell; System; Testing; Therapeutic; Time; Transcript; Work; base; cell type; crosslinking and immunoprecipitation sequencing; dysbiosis; epithelium regeneration; experimental study; in vivo; insight; intestinal epithelium; intestinal injury; microbiota; mouse model; novel; progenitor; prophylactic; recombinase; regenerative; response; single cell analysis; stem cell niche; stem cell population; stem cells; therapeutic development; tool; transcriptome; transcriptome sequencing

DNA damage to the intestinal epithelium is associated with a number of pathological conditions, ranging from chemotherapy/radiation enteropathy to exposure to bacterial genotoxins and reactive oxygen species in the context of chronic inflammation/dysbiosis. Rapid and efficient epithelial regeneration is therefore critical for restoring barrier function and sequestering microbiota in the lumen. In the absence of injury, homeostatic turnover of the epithelium is maintained by a population of cycling intestinal stem cells (ISCs) at the crypt base. As these ISCs are highly sensitive to DNA damage-induced cell death, epithelial regeneration is driven by a DNA damage-resistant `reserve ISC' population. In the prior funding period, we demonstrated that activation of the Msi family of RNA binding proteins is both necessary and sufficient for cell cycle entry of reserve ISCs, and thus crucial for the regenerative response to DNA damage. However, the precise identity of this population has been a subject of contention, with recent findings suggesting that a host of lineage-committed epithelial cells (Paneth cells, transit-amplifying enterocyte progenitors, and secretory/enteroendocrine lineage cells (EECs) are capable of reverting to the ISC state once exposed to the niche environment. In our ongoing studies to characterize the reserve ISC, we generated a new mouse model harboring a CreERT2-2a-tdTomato cassette under control of the endogenous EEC-specific Chga locus (ChgaCreER2aTomato). Our preliminary data demonstrates that this allele faithfully captures cells across the EEC lineage, from immature progenitor to mature EEC. Further, lineage tracing from these cells verifies that a significant proportion of regeneration after DNA damaging injury is derived from the ChgaCreER2aTomato population, suggesting that this population is uniquely required for this process. Here, we test the hypothesis that EEC- lineage cells are required for regeneration after DNA damage and that this process is controlled by specific Msi-RNA interactions. Further, we hypothesize that cells of the EEC lineage reach an epigenetic `point of no return' after which their plasticity is lost. To address these hypotheses, we combine novel genetically modified mouse models with single cell genomic and functional assays, including an inducible Msi2-HyperTRIBE allele which enables the identification of direct Msi2 binding targets in rare EEC lineage cells in vivo, as well as histone H2B-GFP pulse-chase assays that enable us to assess how the latent stem cell potential of EEC lineage cells changes as a function of their age. Ultimately, the experiments in this proposal employ state-of- the art single cell genomic and functional approaches to gain insight into the molecular basis for epithelial regeneration from a rare but incredibly powerful cell population. Findings from this work will inform the development of targeted strategies to prophylactically guard against intestinal injury or to enhance the regenerative response post-injury.

肠上皮 DNA 损伤与许多病理状况有关，从化疗/放射性肠病到慢性炎症/生态失调背景下接触细菌基因毒素和活性氧。因此，快速有效的上皮再生对于恢复屏障功能和隔离管腔中的微生物群至关重要。在没有损伤的情况下，上皮细胞的稳态更新是由隐窝基底的一群循环肠干细胞（ISC）维持的。由于这些 ISC 对 DNA 损伤诱导的细胞死亡高度敏感，因此上皮再生是由抗 DNA 损伤的“储备 ISC”群体驱动的。在之前的资助期间，我们证明了 RNA 结合蛋白 Msi 家族的激活对于储备 ISC 进入细胞周期是必要且充分的，因此对于 DNA 损伤的再生反应至关重要。然而，该群体的精确身份一直是争论的主题，最近的研究结果表明，许多谱系定向上皮细胞（潘氏细胞、转运放大肠细胞祖细胞和分泌/肠内分泌谱系细胞（EEC））一旦暴露于生态位环境，就能够恢复到 ISC 状态。在我们正在进行的表征储备 ISC 的研究中，我们生成了一个新的小鼠模型 窝藏一个 CreERT2-2a-tdTomato 盒受内源 EEC 特异性 Chga 基因座 (ChgaCreER2aTomato) 控制。我们的初步数据表明，该等位基因忠实地捕获了整个 EEC 谱系的细胞，从不成熟的祖细胞到成熟的 EEC。此外，这些细胞的谱系追踪证实，DNA 损伤后的再生很大一部分来自 ChgaCreER2aTomato 群体，表明该群体是该过程所必需的。在这里，我们测试了以下假设：DNA 损伤后 EEC 谱系细胞需要再生，并且该过程由特定的 Msi-RNA 相互作用控制。此外，我们假设 EEC 谱系的细胞达到表观遗传的“不归点”，之后它们的可塑性就会丧失。为了解决这些假设，我们将新型转基因小鼠模型与单细胞基因组和功能测定相结合，包括可诱导的 Msi2-HyperTRIBE 等位基因，该等位基因能够在体内鉴定罕见 EEC 谱系细胞中的直接 Msi2 结合靶标，以及组蛋白 H2B-GFP 脉冲追踪测定，使我们能够评估 EEC 谱系细胞的潜在干细胞潜力如何随其细胞的变化而变化。 年龄。最终，该提案中的实验采用了最先进的单细胞基因组和功能方法，以深入了解罕见但强大的细胞群上皮再生的分子基础。这项工作的调查结果将告知 制定有针对性的策略来预防性预防肠道损伤或增强损伤后的再生反应。