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

肠道上皮的DNA损伤与许多病理条件有关，从化疗/放射性肠病到暴露于细菌遗传毒素和慢性炎症/生物失调背景下的活性氧物种。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，上皮的动态平衡周转是由隐窝底部的一群循环的肠道干细胞(ISCs)维持的。由于这些ISCs对DNA损伤引起的细胞死亡高度敏感，上皮再生是由抗DNA损伤的“储备ISC”群体驱动的。在之前的资助阶段，我们证明了MSI家族RNA结合蛋白的激活是储备ISCs进入细胞周期的必要条件和充分条件，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存在争议，最近的发现表明，一系列致力于谱系的上皮细胞(Paneth细胞、运输放大的肠细胞前体细胞和分泌/肠内分泌谱系细胞(EECs)一旦暴露在利基环境中，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，该模型含有 内源EEC特异性Chga基因座(ChgaCreER2aTomato)控制下的CreERT2-2a-td番茄盒。我们的初步数据表明，该等位基因忠实地捕获了整个EEC谱系中的细胞，从不成熟的祖细胞到成熟的EEC。此外，对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ChgaCreER2a番茄群体，这表明该群体是这一过程中唯一需要的群体。在这里，我们测试了这样的假设，即DNA损伤后EEC谱系细胞需要再生，并且这一过程受特定的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学的“不归路点”，在此之后它们的可塑性就会丧失。为了解决这些假设，我们将新的转基因小鼠模型与单细胞基因组和功能分析相结合，包括可诱导的MSI2-HyperTRIBE等位基因，它使我们能够在体内识别罕见的EEC谱系细胞中的直接MSI2结合靶点，以及组蛋白H2B-GFP脉冲追逐分析，它使我们能够评估EEC谱系细胞的潜在干细胞潜力如何随着年龄的变化而变化。最终，这个方案中的实验使用最先进的单细胞基因组和功能方法来洞察从罕见但令人难以置信的强大细胞群中再生上皮的分子基础。这项工作的发现将向 制定有针对性的策略，预防性地预防肠道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.