Microbial and dietary control of intestinal epithelial differentiation by HNF4A

项目总结 关于肠上皮细胞(IECs)如何适应饮食和营养的认识存在很大差距 微生物区系，以及这种适应背后的转录调控机制。我们的长- 学期目标是了解微生物区系和饮食如何与肠道上皮沟通，以调节其 生理学。这项提议的目标是利用功能基因组、遗传和生化 确定肠道对微生物区系和高脂肪适应的转录和细胞基础的方法 节食。我们的初步研究表明，微生物区系改变了IECS对单一高脂肪食物的反应 无菌小鼠的膳食诱导肠细胞特异性转录程序，而相同的膳食在 常规化小鼠抑制这些程序并刺激肠道干细胞特异性转录 程序。这表明，微生物区系抑制了肠道干细胞向肠细胞的分化，产生了 对高脂肪食物有不同反应的细胞。然而，我们还不知道长期高脂肪饮食的影响。 饮食单独或与微生物区系联合使用对肠道适应的影响。我们和其他人已经证明 核受体转录因子肝细胞核因子4α(HNF4a)对两者都有反应 微生物区系和高脂肪饮食，并负责建立肠细胞身份，将其定位为潜在的 这些外部刺激的整合以调节IECs的分化。我们之前发现微生物区系 抑制HNF4a活性，但这种抑制的机制尚不清楚。我们的初步数据 结果表明，微生物区系提高了蛋白激酶A(PKA)的活性并增强了与HNF4a的相互作用。此外，我们 观察到的HNF4a在PKA调节的位点被磷酸化，从而破坏IECS中的DNA结合。我们将测试我们的 中心假设高脂饮食和微生物区系相互作用抑制肠道干细胞分化为 通过PKA抑制HNF4a对肠上皮细胞的作用。首先，我们将确定微生物区系和高脂肪饮食是否 单细胞通过HNF4a交互作用抑制肠干细胞向肠细胞分化 Hnf4afl/fl和Hnf4aDIEC灵知生菌小鼠喂饲高脂或低脂饮食的RNA序列和组织学。第二，我们将 确定微生物区系是否通过给予PKA的药理抑制剂来抑制HNF4a 利用生化技术追踪HNF4a磷酸化、DNA的变化 结合，和靶基因表达。预期结果将在几个方面垂直推进这一领域。 首先，它们将扩展我们对微生物区系和高脂肪饮食如何交互调节丰度的知识 和IEC类型的转录，以及HNF4a在适应性IEC分化中的作用。其次，他们将确定 微生物区系调节HNF4a活性的分子机制，这可能导致新的激活工具 HNF4a活性。这些结果将对我们的领域产生积极的影响，因为它发现了 肠道适应不同的刺激和识别药物来调节肠道生理以治疗疾病。

PROJECT SUMMARY There is a significant gap in knowledge of how intestinal epithelial cells (IECs) adapt to both diet and microbiota simultaneously, and the transcriptional regulatory mechanisms underlying this adaptation. Our long- term goal is to understand how microbiota and diet communicate with the intestinal epithelium to regulate its physiology. The objective of this proposal is to leverage functional genomic, genetic, and biochemical approaches to identify the transcriptional and cellular bases of intestinal adaptation to microbiota and high-fat diet. Our preliminary studies showed microbiota alter the response of IECs to a single high-fat meal, as high-fat meal in germ-free mice induced enterocyte-specific transcriptional programs, while the same meal in conventionalized mice suppressed those programs and stimulated intestinal stem cell-specific transcriptional programs. This suggests that microbiota suppress intestinal stem cell differentiation into enterocytes, yielding cells that mount differential responses to high-fat meal. Yet we do not know the effects of long-term high-fat diet alone or in combination with microbiota on intestinal adaptation. We and others have shown that the nuclear receptor transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) is responsive to both microbiota and high-fat diet, and is responsible for establishing enterocyte identity, positioning it as a potential integrator of these external stimuli to regulate differentiation of IECs. We previously discovered that microbiota suppressed HNF4A activity, but the mechanism of this suppression remains unknown. Our preliminary data showed that microbiota enhanced Protein Kinase A (PKA) activity and interaction with HNF4A. Further, we observed HNF4A is phosphorylated at a PKA regulated site to disrupt DNA binding in IECs. We will test our central hypothesis that high-fat diet and microbiota interactively suppress intestinal stem cell differentiation into enterocytes by inhibiting HNF4A through PKA. First, we will determine if microbiota and high-fat diet interactively suppress intestinal stem cell differentiation into enterocytes through HNF4A by using single-cell RNA-seq and histology in Hnf4afl/fl and Hnf4aDIEC gnotobiotic mice fed a high-fat or low-fat diet. Second, we will determine if microbiota suppress HNF4A through PKA by administering a pharmacological inhibitor of PKA to gnotobiotic mice and utilizing biochemical techniques to track alterations in HNF4A phosphorylation, DNA binding, and target gene expression. The expected outcomes will vertically advance the field in several ways. First, they will expand our knowledge of how microbiota and high-fat diet interactively regulate the abundance and transcription of IEC types, and the role of HNF4A in adaptive IEC differentiation. Second, they will identify molecular mechanisms by which microbiota regulate HNF4A activity, which can lead to new tools to activate HNF4A activity. These results would have a positive impact on our field by discovering mechanisms by which the intestine adapts to diverse stimuli and identifying drugs to modulate intestinal physiology to treat disease.