Brinkman, J. A. | bioRxiv Science

bioRxiv2026

Chromatin State Distinguishes Injury-Responsive from State-Stabilizing Transcriptional Programs in Hybrid Hepatocytes

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-{kappa}B- 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.

developmental biology↗

bioRxiv2025

Menin-MLL1 complex cooperates with NF-Y to promote HCC survival

Identification of new therapeutic targets in hepatocellular carcinoma (HCC) remains critical. Chromatin regulating complexes are frequently mutated or aberrantly expressed in HCC, suggesting dysregulation of chromatin environments is a key feature driving liver cancer. To investigate whether the altered chromatin state in HCC cells could be targeted, we designed and utilized an epigenome-focused CRISPR library that targets genes involved in chromatin regulation. This focused approach allowed us to test multiple HCC cell lines in both 2D and 3D growth conditions, which revealed striking differences in the essentiality of genes involved in ubiquitination and multiple chromatin regulators vital for HCC cell survival in 2D but whose loss promoted growth in 3D. We found the core subunits of the menin-MLL1 complex among the strongest essential genes for HCC survival in all screens and thoroughly characterized the mechanism through which the menin-MLL1 complex promotes HCC cell growth. Inhibition of the menin-MLL1 interaction led to global changes in occupancy of the complex with concomitant decreases in H3K4me3 and expression of genes involved in PI3K/AKT/mTOR signaling pathway. Menin inhibition affected chromatin accessibility in HCC cells, revealing that increased chromatin accessibility at sites not bound by menin-MLL1 was associated with the recruitment of the pioneer transcription factor complex NF-Y. A CRISPR/Cas9 screen of chromatin regulators in the presence of menin inhibitor SNDX-5613 revealed a significantly increased cell death when combined with NFYB knockout. Together these data show that menin-MLL1 is necessary for HCC cell survival and cooperates with NF-Y to regulate oncogenic gene transcription.

cancer biology↗