Microbial and dietary control of intestinal epithelial differentiation by HNF4A

项目摘要 关于肠上皮细胞（IEC）如何适应饮食和环境， 微生物群，以及这种适应背后的转录调控机制。我们长久以来- 长期目标是了解微生物群和饮食如何与肠上皮细胞沟通，以调节其 physiology.该提案的目的是利用功能基因组学、遗传学和生物化学 确定肠道适应微生物群和高脂肪的转录和细胞基础的方法 饮食.我们的初步研究表明，微生物群会改变肠上皮细胞对单一高脂肪膳食的反应，就像高脂肪膳食一样 无菌小鼠的膳食诱导了肠细胞特异性转录程序，而无菌小鼠的相同膳食诱导了肠细胞特异性转录程序。 常规小鼠抑制了这些程序，并刺激了肠干细胞特异性转录， 程序.这表明微生物群抑制肠干细胞分化为肠上皮细胞， 对高脂肪食物产生不同反应的细胞。然而，我们不知道长期高脂饮食的影响。 饮食单独或与微生物群组合对肠道适应的影响。我们和其他人已经表明， 核受体转录因子肝细胞核因子4 α（HNF 4A）对这两种物质都有反应， 微生物群和高脂肪饮食，并负责建立肠上皮细胞身份，将其定位为潜在的 整合这些外部刺激以调节IEC的分化。我们之前发现微生物群 抑制HNF 4A活性，但这种抑制的机制仍然未知。我们的初步数据 结果表明，微生物群增强了蛋白激酶A（PKA）活性和与HNF 4A的相互作用。我们还 观察到HNF 4A在PKA调节位点被磷酸化以破坏IEC中的DNA结合。我们将测试我们的 高脂饮食和微生物群相互作用抑制肠干细胞分化为 通过PKA抑制HNF 4A的表达。首先，我们将确定微生物群和高脂肪饮食 单细胞介导HNF 4A交互抑制肠干细胞向肠上皮细胞分化 喂食高脂肪或低脂肪饮食的Hnf 4afl/fl和Hnf 4aDIEC无菌小鼠中的RNA-seq和组织学。二是 确定微生物群是否通过PKA抑制HNF 4A，通过给予PKA的药理学抑制剂， 利用生物化学技术追踪HNF 4A磷酸化、DNA 结合和靶基因表达。预期成果将以几种方式垂直推进该领域。 首先，他们将扩大我们对微生物群和高脂肪饮食如何相互作用地调节丰度的认识。 以及HNF 4A在适应性IEC分化中的作用。第二，他们将识别 微生物群调节HNF 4A活性的分子机制，这可能导致新的工具来激活 HNF 4A活性这些结果将对我们的领域产生积极的影响，通过发现机制， 肠适应不同的刺激和识别药物以调节肠生理学来治疗疾病。

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.