Molecular and Chromatin Accessibility Programs Underlying Epithelial Injury and Impaired Regeneration in Neonatal...

Neonatal necrotizing enterocolitis (NEC) is a severe gastrointestinal disorder with high mortality, characterized by epithelial cell injury and compromised epithelial repair. The mechanisms underlying defective epithelial regeneration remain poorly understood despite advances in single-cell omics. Addressing these challenges is essential for elucidating the pathogenesis of NEC and identifying therapeutic targets to restore epithelial regeneration and replace the damaged epithelial layer.

Using a well-established neonatal mouse model of NEC induced by formula feeding, hypoxia, and lipopolysaccharide, we applied an integrated multi-omics framework to map epithelial injury at transcriptomic, chromatin accessibility, and spatial levels. These included bulk RNA sequencing, single-nucleus RNA sequencing (snRNA-seq), single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), and multiplexed error-robust fluorescence in situ hybridization (MERFISH) for spatial transcriptomics. Complementary in vitro experiments and in vivo mouse models were utilized to evaluate NEC phenotypes, intestinal tissue morphology, and organoid formation.

Changes in cell type composition, transcriptional network remodeling, and chromatin accessibility were observed in the small intestine of neonatal mice with NEC. Chromatin accessibility significantly changed in epithelial cells, highlighting their pivotal roles in NEC. A marked reduction in intestinal stem cells (ISCs) and transit-amplifying cells, along with an increased proportion of enteroendocrine cells, indicates disrupted epithelial regeneration and functional differentiation. These changes correlated with disrupted WNT signaling and stem cell maintenance genes (eg, Lgr5, Smoc2, Axin2) and activation of inflammatory and hypoxia-related pathways (eg, Il6, Tnfα). The epigenetic regulator Ezh2 was identified as a critical factor in maintaining LGR5+ ISCs and epithelial homeostasis. Knockdown of Ezh2 reduced stemness and proliferation-related gene expression and exacerbated inflammation. Reactivation of WNT signaling restored Ezh2 and Lgr5 expression, improving intestinal regeneration.

This study provides a comprehensive multi-omics atlas of epithelial injury in experimental NEC and reveals Ezh2 as a key regulator of LGR5+ ISC identity and regeneration. By integrating chromatin, transcriptomic, and spatial information, our findings highlight previously unrecognized mechanisms of ISC failure in NEC and support therapeutic strategies targeting Ezh2 and WNT signaling to restore epithelial integrity.