PROJECT SUMMARY Active intestinal stem cells (aISCs) are tasked with maintaining the highly proliferative intestinal epithelium, a tissue with absorptive, secretory, and barrier functions that are critical to homeostasis and survival. Dysregulation of aISCs can lead to inflammation and cancer, while loss of aISCs due to cytotoxic, pathogenic, or radiation- induced injury can threaten epithelial integrity and pose a severe health risk. aISC injury is a common side effect of chemotherapeutic or radiation treatment of cancer. In response to aISC injury, the intestinal epithelium has the remarkable capacity to regenerate its stem cell pool through dedifferentiation of mature epithelial cells and their progenitors, which function as facultative ISCs (fISCs). However, little is understood about the genetic mechanisms that permit this high level of cellular plasticity. One way cells are able to simultaneously modulate the expression of many genes is through chromatin modification. Ten-eleven translocation methylcytosine dioxygenases (TET enzymes) are involved in DNA demethylation through the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), a process associated with gene activation. TET enzymes have well- known functions in maintaining stem cell-specific gene expression in mouse embryonic stem cells, and published data from our lab demonstrate that Tet1 is enriched in adult mouse ISCs and intestinal epithelial progenitors. My preliminary data show that the loss of Tet1 results in aberrant lineage specification, suggesting a central role for Tet1 in changing cell identity. The central hypothesis of this proposal is that Tet1 facilitates ISC identity transitions through activation of cell type specific gene expression. I will test this hypothesis with the following specific aims: Aim 1 will establish the genetic requirement for Tet1 during fISC dedifferentiation following aISC injury using both targeted aISC ablation and cytotoxic injury models. Post-injury regeneration will be assessed by histology and organoid growth assays. Aim 2A will characterize Tet1’s gene regulatory mechanisms during differentiation through genomic, epigenomic, and transcriptomic analysis of inducible Tet1 knockout (Tet1iKO) mice. Aim 2B will use the same approaches to describe Tet1’s gene regulatory mechanisms during dedifferentiation in Tet1iKO mice that have suffered ISC injury. This project will determine how Tet1 facilitates changes in cell identity and provide me with training towards my goal of obtaining a research-focused faculty position studying chromatin regulatory mechanisms of maintaining stem cell function and establishing cell identity. This work will advance the understanding of regenerative mechanisms and provide insight into intestinal epithelial responses to damage and injury.