ABSTRACT Intestinal stem cells (ISCs) reside in intestinal crypts and undergo continual proliferation, self-renewal, and differentiation in order to maintain function of the intestinal epithelium, which is replaced approximately once a week throughout adult life. Precise regulation of cell fate decisions between self-renewal and differentiation is critical for ISC homeostasis and damage response, but the role of chromatin regulation in balancing these decisions remains poorly characterized. Our currently funded K01 focuses on dissecting the genomic regulatory network of Tet1, which catalyzes DNA demethylation and is specifically enriched in ISC populations. Preliminary data from these studies demonstrate that loss of Tet1 results in remarkably rapid crypt hyperplasia and increased ISC function, which occurs within 5 days of Tet1 ablation. The central hypothesis of the proposed study is that Tet1 negatively regulates ISC activity by promoting differentiation. To test this hypothesis and expand on our K01, we will: (1) characterize the phenotypic role of Tet1 in ISC proliferation and differentiation in vivo, at 5 days and 4 weeks post-ablation (Aim 1) and (2) map the Tet1-dependent genomic landscape of 5-hydroxymethylcytosine (5hmC), the primary product of TET1 catalytic activity, in ISCs, progenitors, and post-mitotic cells (Aim 2). Collectively, these Aims will expand on and integrate with our ongoing K01 studies to provide sufficient preliminary data for future, R01-funded investigation of Tet1-mediated mechanisms involved in ISC homeostasis and regeneration.