Brinkman, J. A.; Hantleys, F.; Raab, J. R.; Gracz, A. D.

Liver injury induces rapid transcriptional responses in hepatocytes, yet the chromatin features that distinguish injured hepatocytes from healthy hepatocytes remain poorly understood. Using an integrated functional genomics approach combining bulk RNA-seq, ATAC-seq, and CUT&Tag profiling of H3K27ac and H3K27me3, we define the transcriptional and chromatin landscape of Sox9-expressing hepatocytes, which exhibit gene expression consistent with both hepatocyte and biliary identity. Under homeostatic conditions, Sox9+ hybrid hepatocytes (HybHeps) are rare and confined to the periportal space, while chronic injury induces an expansion of Sox9+ metaplastic hepatocytes (MetHeps). We identify three classes of differentially expressed genes associated with injury-responsive, state-associated, or shared regulatory programs and demonstrate that these classes are governed by distinct chromatin mechanisms. Injury-responsive transcription is driven primarily by dynamic chromatin accessibility remodeling at NF-kB- and AP-1- enriched regulatory elements, while state-associated and shared programs are reinforced through selective H3K27ac and H3K27me3 modification with comparatively stable accessibility. Relative to conventional hepatocytes, HybHeps encode a permissive chromatin landscape at injury-responsive loci under homeostatic conditions, consistent with epigenetic priming that facilitates rapid inflammatory activation. Projection of mouse-derived gene programs onto a human liver single-cell atlas encompassing both healthy and diseased hepatocytes confirms that SOX9-expressing hepatocytes preferentially engage injury-associated inflammatory modules while attenuating hepatocyte metabolic identity programs. Together, these findings define a chromatin-based regulatory dichotomy between inflammatory responsiveness and hybrid hepatocyte cell state stability, providing mechanistic insight into how differentiated epithelial cells integrate inflammatory signals while preserving cell state.