Microbial and dietary control of intestinal epithelial differentiation by HNF4A

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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.