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

肠上皮细胞的DNA损伤与许多病理状况相关，从化疗/放射性肠病到慢性炎症/生态失调背景下暴露于细菌遗传毒素和活性氧。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，上皮细胞的稳态周转由隐窝基部的循环肠干细胞（ISCs）群体维持。由于这些ISC对DNA损伤诱导的细胞死亡高度敏感，因此上皮再生由DNA损伤抗性的“储备ISC”群体驱动。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ISCs进入细胞周期是必要的和足够的，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一个争论的主题，最近的研究结果表明，一个主机的谱系定向上皮细胞（潘氏细胞，transit-amplifying enterocyte progenitors，和分泌/肠内分泌谱系细胞（EECs）是能够恢复到ISC状态一旦暴露于生态位环境。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， CreERT 2 -2a-tdTomato盒在内源性EEC特异性Chga基因座（ChgaCreER 2aTomato）的控制下。我们的初步数据表明，该等位基因忠实地捕获整个EEC谱系的细胞，从未成熟的祖细胞到成熟的EEC。此外，来自这些细胞的谱系追踪证实了DNA损伤损伤后的显著比例的再生源自ChgaCreER 2aTomato群体，表明该群体是该过程唯一需要的。在这里，我们测试的假设，EEC-谱系细胞需要DNA损伤后的再生，这一过程是由特定的Msi-RNA相互作用控制。此外，我们假设EEC谱系的细胞达到了表观遗传的“不归路点”，之后它们的可塑性丧失。为了解决这些假设，我们将联合收割机新型遗传修饰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Msi 2-HyperTRIBE等位基因，其能够在体内鉴定罕见EEC谱系细胞中的直接Msi 2结合靶标，以及组蛋白H2 B-GFP脉冲追踪测定，其使我们能够评估EEC谱系细胞的潜在干细胞潜能如何随其年龄而变化。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，以深入了解从罕见但非常强大的细胞群中进行上皮再生的分子基础。这项工作的结果将为 开发靶向策略以预防肠损伤或增强损伤后的再生反应。

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.