Epigenetic mechanisms of intestinal stem cell fate conversion in response to severe radiation injury

ABSTRACT Gastrointestinal acute radiation syndrome (GI-ARS) results in high mortality after radiological disasters and there is no FDA-approved countermeasure to mitigate its effects. The pathophysiology of GI-ARS derives from severe damage of intestinal crypt epithelial cells, with associated villus shortening, epithelial desquamation, and loss of barrier function. Sepsis and death can occur if endogenous regeneration of the intestinal epithelial barrier is not successful. Strategies to augment this post-radiation intestinal regenerative capacity may yield strategies to mitigate the toxicity of GI-ARS at time points after the first 24 hours post-radiation exposure. Under normal conditions, multipotent intestinal stem cells marked with the protein Lgr5 (Lgr5+ ISCs) support the multilineage repopulation of the high-turnover intestinal epithelium from their position at the base of the small intestinal crypt. These ISCs are ablated after high-dose radiation exposure. However, we and others have shown that a “revival stem cell” population (revSC), marked by the protein Clusterin (Clu), arises in the intestinal epithelium after severe radiation injury. However, only a subpopulation of these Clu+ revSCs can regenerate the radiation-ablated ISC population to support the restoration of the intestinal epithelial layer. Such stem cell-associated cell state changes typically rely upon rapid epigenetic and transcriptional reprogramming, but the mechanisms underlying the transition of revSCs to new Lgr5+ ISCs that are required for repopulating the irradiated small intestines remain poorly understood. This proposal endeavors to comprehensively examine the epigenetically-regulated and transcriptionally-dependent transitions, and the molecular factors upon which these transitions occur during these cell state changes. We will utilize well-defined genetically engineered lineage tracing and reporter mouse models that permit specific isolation of unirradiated Lgr5+ ISCs, radiation- induced Clu+ rev-SCs, and post-radiation rev-SC-derived Lgr5+ ISCs. Aim 1 proposes to perform an integrated analysis of RNA-seq and ATACseq data derived from flow-sorted populations of mouse intestinal cells harvested 3 and 4 days after in vivo abdominal radiation (14 Gy, ~LD50), testing the hypothesis that each cell state critical to small intestine radiation response exhibits discrete epigenetic regulatory and transcriptional landscapes. Aim 2 proposes to employ CRISPR screening using a library containing sgRNAs against epigenetic and transcriptional regulators, with a FACS-based CRISPR screen on irradiated mouse organoids bearing Clu-CreER; LSL-tdTomato reporter to identify regulators of revSC induction 3 days after irradiation and a dropout screen to identify those regulators essential for post-revSC repopulation of small intestinal epithelial cells. The results of this project will identify crucial epigenetic regulators of post-radiation intestinal recovery. The ultimate objective is to unveil new mitigation strategies for GI-ARS after radiological disasters by promoting the regeneration of new Lgr5+ ISCs post-irradiation to restore homeostasis of the damaged small intestines.