Transcription factor HNF4A activity is suppressed by microbiota and inhibits IFN beta production

Abstract

Intestinal epithelial cells (IECs) separate the intestinal lumen from lamina propria resident immune cells, positioning IECs as mediators of microbial signals to the immune system. Understanding these microbe-IEC-immune interactions may represent potential therapeutic targets for immunological diseases. We previously discovered that microbiota regulate the activity of the nuclear receptor transcription factor Hepatocyte nuclear factor 4 alpha (HNF4A). Microbiota suppress HNF4A-activated genes in zebrafish and lead to genome-wide reduction of HNF4A DNA occupancy in mouse IECs. HNF4A activates lipid metabolism and epithelial differentiation gene programs, while also suppressing inflammatory and immune response gene programs in the intestine. We therefore hypothesized that microbial suppression of HNF4A contributes to activation of immunological pathways. We conducted an unbiased coimmunoprecipitation-mass-spectrometry analysis comparing HNF4A interacting partners in IECs from germ-free and conventionalized (germ-free mice colonized with SPF feces for 2 weeks) mice. HNF4A interacted with significantly more proteins in germ-free IECs, including several proteins involved in promotion of transcription and chromatin regulation, consistent with HNF4A being more active in germ-free mice. Our HNF4A and H3k27ac ChIP-seq data from IECs derived from germ-free and conventionalized mice identified that HNF4A binds ubiquitously to IEC enhancers, but also revealed a small number of enhancers that were activated by microbiota with no HNF4A binding. Motif enrichment analysis showed that these enhancers were enriched for binding sites for Interferon regulatory factors (IRFs). This raises the interesting possibility that there is a selective disadvantage to colocalizing IRF and HNF4A occupancy at a given enhancer, and suggests that these transcriptional regulatory pathways may otherwise communicate. To test this, we used a luciferase reporter assay in HEK293T cells to probe the impact of HNF4A on the IFNβ promoter, which is controlled by IRF3/7. HNF4A potently suppressed IFNβ promoter activity following viral stimulation. HNF4A DNA-binding activity is necessary for this suppression; however, there is no evidence HNF4A binds the IFNβ promoter, suggesting HNF4A likely induces a repressor of the IFNβ promoter. Together these results indicate that HNF4A is both a target of microbiota suppression and a potent suppressor of IFNβ production. Though IRF bound enhancers appear to be insulated against HNF4A binding in IECs, our results show that these two transcriptional pathways communicate, potentially representing a novel mechanism through which microbiota influence innate immunity. NIH P01-DK094779 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.