PROJECT SUMMARY The liver exhibits remarkable capacity for regeneration, but chronic injury or severe acute damage can overwhelm compensatory responses and result in liver failure. The need for transplantable livers regularly exceeds the donor pool, necessitating the development of new regenerative medicine-based therapies and a deeper understanding of the liver’s endogenous repair mechanisms. This proposal seeks a mechanistic understanding of gene regulatory networks underlying ductular reaction (DR), a damage response associated with a broad range of liver injury and disease. DR is defined by the proliferative expansion of biliary epithelial cells (BECs) and can involve context-dependent lineage conversion between mature hepatocytes and BECs that contributes to tissue regeneration. The genetic regulation of DR remains poorly understood, including how BECs balance proliferation and phenotypic plasticity. Sox9 is a transcription factor required for stem/progenitor cell function in a number of epithelial tissues and has been shown to establish cellular identity through genome-wide effects on the chromatin landscape. In the liver, Sox9 is required for timing of BEC specification in development and is broadly expressed in adult BECs. Our lab recently used a Sox9EGFP transgene to study BEC heterogeneity and showed that Sox9 is expressed at distinct levels in subpopulations of BECs and peribiliary hybrid hepatocytes (HybHeps) during homeostasis and cholestasis. New findings from our lab also demonstrate abnormalities in BECs of adult Sox9 knockout mice. The central hypothesis of this proposal is that Sox9 functions as a master regulator of DR, by inhibiting proliferation and promoting BEC identity. The following specific aims will test this hypothesis: Aim 1A will determine the role of Sox9 in damage induced BEC proliferation, through the use of the Sox9EGFP allele and (1) combined BEC/hepatocyte or (2) BEC-specific Sox9 knockout mouse models. Aim 1B will determine the role of Sox9 in bi-directional BEC-to-hepatocyte plasticity, through complementary in vivo lineage tracing and in vitro organoid assays. Aim 2A will map the chromatin regulatory landscape of DR, by integrating transcriptomics and chromatin assays in BEC subpopulations during liver injury. Aim 2B will determine the genomic regulatory impact of Sox9 in DR by applying single cell multi-omics to BEC- specific Sox9 knockout mouse models. The data generated in this project will provide fundamental mechanistic insight into genetic regulation of DR and identify regulatory nodes for therapeutic targeting to enhance regeneration in end stage liver disease.