Project Description Inflammation, cancer, and other common disorders of the digestive tract reflect dysfunction in the dynamics of intestinal stem (ISC) and other crypt cells. Improved treatments require a better understanding of how Lgr5+ ISC, progenitor, and differentiated cell states may be promoted or disfavored. Recent findings substantially revise the traditional view that movement from ISC intestinal bipotential progenitor (IBP) transit-amplifying (TA) cells is strictly linear and unidirectional. Instead, crypts appear to be highly dynamic units, where cells interconvert with surprising ease and various dedifferentiating progenitors are the main source of crypt regeneration after ISC injury. One gene particularly implicated in ISC functions encodes the Wnt-dependent transcription factor (TF) ASCL2. In the last funding period, we examined chromatin states critically in diverse resting crypt populations and in cells captured in the act of dedifferentiating into ISC; those findings made notable mechanistic contributions toward the emerging view of crypt dynamics in relation to chromatin states. To build on the advances, we engineered a new mouse Ascl2 allele to flag its expression with a fluorescent label, identify binding sites by epitope-tagged chromatin immunoprecipitation (ChIP), and delete the gene at will. Contrary to a published report, we find that Ascl2 is dispensable for resting ISC function. It is, however, essential (a) for crypts to replenish Lgr5+ ISC when the native pool is damaged, and (b) for cells with constitutive Wnt activity (Apc-/-) to form sizable tumors. Moreover, both Ascl2-/- and Ascl2+/- ISC differentiate prematurely into IBP, suggesting that ASCL2 levels dictate cell exit from the ISC compartment. These animal models and preliminary data provide the background and tools to ask fundamental mechanistic questions about the determinants of ISC vs. IBP vs. TA identity. Aim 1 examines wild-type and Ascl2-/- ISC and isolated regenerating crypt cells using state-of-the-art epigenome methods to identify crucial, ASCL2-dependent steps that favor the ISC state in native Lgr5+ and dedifferentiating cells. We will also ask which crypt populations can dedifferentiate and, in organoid cultures, which extrinsic signals promote rapid ASCL2-dependent crypt regeneration. Aim 2 tackles the problem that dedifferentiation of tumor cells limits the potential of ablating tumor-initiating (TIC, `cancer stem') cells as a treatment for cancer. We will test the hypotheses that ASCL2 drives distinct transcriptional programs in tumor and normal ISC, and that it is required to maintain tumors by dedifferentiation when TIC are killed. These studies could have important future clinical applications. Aim 3 tests the hypothesis that Lgr5+ cells in the crypt base are intrinsically poised at the junction between ISC and IBP states, and that ASCL2 levels determine ISC vs IBP identity. This original and cohesive investigation of labile crypt cell s...