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

肠上皮的DNA损伤与多种病理情况有关，包括化疗/放射性肠病、慢性炎症/生态失调背景下暴露于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内的微生物群至关重要。在没有损伤的情况下，上皮的稳态更新是由隐窝基部的循环肠干细胞（ISCs）维持的。由于这些ISC对DNA损伤诱导的细胞死亡高度敏感，因此上皮细胞再生是由抗DNA损伤的“储备ISC”群体驱动的。在之前的资助期内，我们证明了RNA结合蛋白Msi家族的激活对于储备ISCs的细胞周期进入是必要和充分的，因此对于DNA损伤的再生反应至关重要。然而，这一群体的确切身份一直是争论的主题，最近的研究结果表明，许多谱系性上皮细胞(Paneth细胞、转运扩增肠细胞祖细胞和分泌/肠内分泌谱系细胞（EECs）一旦暴露于生态位环境中，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，我们产生了一种新的小鼠模型，其中含有a

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.