Early Epigenetic Drivers of Revival Stem Cell Emergence in Intestinal Injury - PROJECT SUMMARY/ABSTRACT Radiation-induced intestinal injury is a frequent and debilitating complication of abdomino-pelvic cancer therapy, often resulting in malabsorption, infection, and chronic gastrointestinal dysfunction. The intestinal epithelium regenerates through dedifferentiation events that give rise to revival stem cells (RevSCs), which replenish lost LGR5⁺ intestinal stem cells (ISCs) following injury. While the identity and regenerative potential of RevSCs are increasingly well understood, the earliest chromatin remodeling events and mechanisms that initiate their formation are not. Preliminary scRNA-seq data from our lab show that Bmi1-YFP⁺ epithelial cells, derived from Bmi1-CreERT2; Rosa26eYFP mice, form a distinct RevSC cluster as early as 24 hours post-irradiation, a timepoint significantly earlier than previous reports of RevSCs emerging from 48-96h. Thus, employing this mouse model provides a unique mechanistic window for studying the early emergence and regulation of RevSCs. We observed widespread chromatin accessibility changes at RevSC loci within 3 hours following injury from ATAC-seq data on intestinal epithelial cells (IECs), indicating promoter and enhancer remodeling as a key early event. Given the role of CBP/p300 histone acetyltransferases in maintaining enhancer activity, I hypothesize that injury-induced chromatin remodeling and a transient reduction in CBP/p300-dependent acetylation following injury, direct IECs toward a “pre-RevSC” state, which can be investigated through the lens of the Bmi1-lineage, enabling subsequent RevSC formation. I will test my hypothesis with two SPECIFIC AIMS. Aim 1 will define the transcriptional and chromatin dynamics that drive RevSC emergence by combining single-nucleus multi-ome (snRNA+snATAC-seq) and CUT&RUN profiling in both whole intestinal tissues, capturing broad, lineage- independent changes, and Bmi1-YFP⁺ IECs to investigate drivers of RevSC emergence. Aim 2 will determine how CBP/p300 inhibition alters enhancer accessibility and promotes RevSC formation and lineage plasticity using in vitro organoid models and in vivo pharmacologic and genetic perturbations. This work will generate a high-resolution atlas of early regenerative chromatin states and uncover enhancer-centric mechanisms that govern epithelial reprogramming. Importantly, it will elucidate how enhancer accessibility and histone modifications orchestrate cell fate transitions during intestinal regeneration, uncovering early epigenetic regulators of RevSC formation. These findings will identify molecular markers of regenerative potential and inform strategies to enhance mucosal repair in radiation enteropathy and inflammatory bowel disease. As part of a structured training plan, I will receive hands-on instruction in single-nucleus multi-omics, enhancer mapping, and organoid-based assays, complemented by formal coursework, clinical shadowing, and mentorship from physician-scientists. This integrated approach will provide the technical, analytical, and professional foundation necessary for a successful career as a physician-scientist in gastroenterology.
