TET-mediated DNA oxidations in mucosal innate defense

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PROJECT SUMMARY DNA methylation pattern in the genome of intestinal epithelial cells (IECs) can be altered by gut microbiota. How the functions of various IEC types are affected by such epigenetic changes under homeostatic and stress conditions remain unclear. DNA methylation is a repressive epigenetic mark that can be actively reversed by the Ten-Eleven Translocation family of enzymes TET1, TET2 and TET3. TETs are DNA dioxygenases that successively oxidize methylated DNA - 5-methylcytosine (5mC) - into 5-hydroxy-methylcytosine (5hmC), 5- formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised by DNA based excision repair factors leading to unmodified cytosines. TET enzymes were recently implicated as new risk factors in inflammatory bowel disease (IBD) patients, but the role of TET-mediated DNA oxidation in homeostasis and in response to environmental stressors are unknown. Preliminary data show that human IECs display an elevation of 5hmC DNA oxidation upon infection by invasive pathogen, and mouse IECs lacking Tet3 had reduction of 5hmC abundance in ileal IECs. scRNA-Seq suggests that mouse Tet3 is the most abundant TET enzyme in IECs, especially in Paneth cells. Tet3DIEC mice had reduced mature Paneth cells, increased susceptibilities to inflammation caused by enteric pathogen or barrier-disrupting chemical. The project tests a central hypothesis that TET3-mediated DNA oxidations play dual roles in guiding IEC differentiation and promoting anti-microbial response via 5hmC-induced permissive chromatin while restraining differentiation via 5fC- and 5caC-induced transcriptional pausing. Aim 1 will use genome wide approaches to identify intestinal stem cell (ISC) and Paneth cell specific TET3 and DNA oxidation gene regulatory networks under homeostasis and in responding to distinct cellular stressors such as pathogen and chemical. Aim 2 will characterize how ISC and Paneth cell specific TET3-mediated DNA oxidation regulate mucosal inflammatory response. This MPI project, utilizing complementary expertise in epigenetics and intestinal biology to address how DNA oxidations regulate the epigenome in response to stressors to help resolve inflammation. The idea that TET-mediated DNA oxidations may be an integral mucosal innate immune component to cope with oxidative stresses during infection and inflammation is novel. Elucidating TET functions in specific IEC types may exert major impact on human gastrointestinal mucosal immunology and diseases. If TET enzymes are indeed uncovered by this research as key mediator and regulator of inflammatory responses, as predicted by literature and our preliminary data, the outcome can be of high relevance to translational medicine.