Background: Regeneration of the intestinal epithelium following injury depends on the precise spatiotemporal communication between intestinal epithelial and stromal cells. Our previous data show that normally quiescent reserve intestinal stem cells marked by B-cell-specific Moloney murine leukemia virus integration site (BMI1⁺) facilitate intestinal epithelial regeneration upon radiation injury by activating Musashi-1. This process is initiated by the activation of Sonic Hedgehog (SHH) signaling in the injured intestinal epithelium. Here, we present results demonstrating that transcriptomic and epigenetic changes in the epithelial and stromal cells are pertinent to successful intestinal epithelial regeneration. Aim: To determine how changes in and interaction between stromal and epithelial cells facilitate the remodeling of the intestinal epithelium upon injury. Methods: To assess the transcriptomic and epigenetic changes in the intestinal epithelium upon injury, Bmi1Cre^ER;Rosa26^eYFP and Pdgfra-Cre;mTmG mice were irradiated with 12 Gy of total body γ irradiation (TBI) or sham-irradiated. Proximal small intestines were collected over the four days post-TBI and analyzed with scRNA-seq, ATAC-seq, RT-PCR, and Western blotting. We used cyclopamine and the PORCUPINE inhibitor LGK974 to abolish the activity of SHH and WNT pathways, respectively. Results: Our results demonstrate that radiation injury increases SHH signaling in the intestinal epithelial cells, which activates stromal cell proliferation and expression of WNT factors, concluding in the regeneration of the intestinal epithelium. Analyzing YFP⁺ (BMI1⁺) cells via scRNA-seq shows that 24hr post-TBI, epithelial cells are a heterogeneous population, with a proportion of them possessing characteristics of fetal-like cells. ATAC-seq of enriched intestinal epithelial cells (CD45^- CD326⁺) combined with in silico analysis of H3Kme1 and H3K27ac ChIP-seq identified differentially open chromatin peaks between sham and irradiated mice at 3, 24, and 96hr post-irradiation, demonstrating distinct pathways activation at analyzed time points. We identified many genes in the TCF4/WNT signaling pathway with transcriptionally active promoters. Furthermore, analysis of stromal cells from irradiated Pdgfra-Cre;mTmG mice demonstrates activation of the WNT signaling expression program within 24 hours of irradiation – coinciding with observed changes in epithelial cells. Consequently, SHH and WNT signaling inhibitors block the activation of the respective pathways and inhibit intestinal epithelium's regenerative capacity. Conclusion: Radiation injury modifies transcriptomic and epigenetic programs of epithelial and stromal cells, and coordination between these cell populations is paramount for intestinal epithelial regeneration.

Robert Bronstein

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