Mechanism of intestinal TET3-mediated DNA oxidation and transcriptional regulation - PROJECT SUMMARY DNA methylation dynamics in mammals are an integral component of the epigenetic regulatory machinery. Methylated DNA functions as a repressive mark for gene expression and essential for cell fate determination during embryonic development. In adult tissues, DNA methylation at specific genomic loci is essential for the maintenance of cellular identity and function. Changes of DNA methylation profiles in intestinal epithelial cells (IECs) were observed upon colonization by commensal microbiota. However, how environmental cues including the microbiota, enteric pathogens and chemical stressors affect DNA methylation patterns in the intestinal epithelia, remains largely unclear. DNA methylation is catalyzed by DNA methyltransferases targeting cytosine at the 5th carbon position resulting in 5-methylcytosine (5mC). DNA methylation can be actively removed via successive DNA oxidations of 5mC into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC) catalyzed by the ten-eleven translocation (TET) family of DNA dioxygenases, TET1, TET2 and TET3. Both 5fC and 5caC can be excised by thymine DNA glycosylase (TDG) as part of the DNA base-excision repair mechanism resulting in unmodified cytosines. Both TET and TDG have been associated with high risk of intestinal bowel disease development. However, the role of TET-mediated DNA oxidations in intestinal epithelial physiology remains unexplored. We find that TET3 protects IECs against pro-inflammatory stressors including the enteric pathogen Salmonella typhimurium and the chemical-induced colitis model dextran sodium sulfate (DSS). We identified a TET3-dependent reprogramming of gene expression profiles in IECs in response to pro-inflammatory luminal stressors resulting in the upregulation of genes involved in Wnt and Notch signaling pathways. TET activity depends on the metabolite -ketoglutarate (KG) to oxidize 5mC into 5hmC, 5fC and 5caC. We hypothesize that in response to luminal signals such as KG, TET3-mediated DNA oxidations act as integral epigenetic and transcriptional regulators to maintain IEC homeostasis and protect from luminal stressors. Mechanistically, we propose a distribution of labor among TET-mediated DNA oxidations whereby 5fC and 5caC, whose levels are dynamically regulated by TETs and TDG, promote transcriptional pausing of stress- responsive genes involved in epithelial repair and innate defense programs, whose transcriptional pausing state may be released by TDG-mediated excision of 5fC/5caC followed by enrichment of 5hmC levels. To address this hypothesis, we generated mouse models with Tet3 specific deletion in IECs, Paneth cells or intestinal stem cells. We will implement diverse genome wide approaches to decipher gene networks whose expression is regulated by TET3-mediated DNA oxidations affecting transcriptional pausing in response to commensal microbiota and luminal stressors. This project is innovative because it will decipher the epigenetic and transcriptional mechanisms that are dependent on the KG-TET-TDG axis to maintain homeostasis and protect the intestinal epithelial lining against pro-inflammatory conditions.
