Tet1 safeguards lineage allocation in intestinal stem cells.

Intestinal stem cells (ISCs) balance self-renewal and differentiation to maintain the intestinal epithelial barrier, which is replaced weekly throughout adult life. Genetic control of ISC differentiation is well-defined relative to transcription factor (TF) activity, but less is known regarding the role of chromatin regulation in ISC biology. Prior work from our lab and others has shown that Tet1, a chromatin modifying enzyme involved in DNA demethylation, is specifically enriched in ISCs and early secretory progenitors. While constitutive loss of Tet1 is associated with defects in early postnatal ISC development, its role in adult ISC biology remains unknown. Here, we show that Tet1 safeguards ISC fate decisions by reducing sensitivity to extrinsic signaling. Inducible, intestine-specific Tet1 knockout mice (Tet1iKO) exhibit environmentally sensitive phenotypes, including absorptive differentiation bias and premature expression of mature absorptive transcripts in ISCs. These phenotypes are largely "silenced" in animals housed in a high-level barrier facility, where Tet1iKO epithelium closely resembles controls. Despite the lack of baseline phenotype in these conditions, Tet1iKO mice retain increased sensitivity to pro-differentiation signaling. In vivo, succinate administration induces increased tuft and goblet cell hyperplasia in the absence of Tet1, while Tet1iKO organoids cultured with IL-4 or DAPT exhibit increased tuft and enteroendocrine cell specification, respectively. While ATAC-seq of Tet1iKO ISCs reveals minimal changes in chromatin accessibility, footprinting analysis suggests increased binding of lineage-specific TFs and CTCF even in the absence of cellular phenotypes. Together, our data demonstrate that Tet1 serves as a "buffer" against ISC differentiation and suggest that it does so in a lineage agnostic manner that is not dependent on changes in chromatin accessibility.