Biliary epithelial cells (BECs) form bile ducts in the liver and are facultative liver stem cells that establish a ductular reaction (DR) to support liver regeneration following injury. Liver damage induces periportal LGR5+ putative liver stem cells that can form BEClike organoids, suggesting that RSPO-LGR4/5-mediated WNT/b-catenin activity is important for a DR. We addressed the roles of this and other signaling pathways in a DR by performing a focused CRISPRbased loss-of-function screen in BEC-like organoids, followed by in vivo validation and single-cell RNA sequencing. We found that BECs lack and do not require LGR4/5-mediated WNT/b-catenin signaling during a DR, whereas YAP and mTORC1 signaling are required for this process. Upregulation of AXIN2 and LGR5 is required in hepatocytes to enable their regenerative capacity in response to injury. Together, these data highlight heterogeneity within the BEC pool, delineate signaling pathways involved in a DR, and clarify the identity and roles of injury-induced periportal LGR5+ cells.

Highlights d Hepatocytes throughout the liver contribute to liver homeostasis and repair d AXIN2 + pericentral hepatocytes do not comprise bona fide liver stem cells d Hepatocytes upregulate AXIN2 and LGR5 during regeneration d Ablation of AXIN2 + hepatocytes only transiently disrupts the pericentral zone

Dimethylation of histone H3 lysine 9 (H3K9me2) is an important epigenetic mark associated with transcription repression. Here, we identified PHF8, a JmjC-domain-containing protein, as a histone demethylase specific for this repressing mark. Recombinant full-length wild type protein could remove methylation from H3K9me2, but mutation of a conserved histidine to alanine H247A abolished the demethylase activity. Overexpressed exogenous PHF8 was colocalized with B23 staining. Endogenous PHF8 was also colocalized with B23 and fibrillarin, two well-established nucleolus proteins, suggesting that PHF8 is localized in the nucleolus and may regulate rRNA transcription. Indeed, PHF8 bound to the promoter region of the rDNA gene. Knockdown of PHF8 reduced the expression of rRNA, and overexpression of the gene resulted in upregulation of rRNA transcript. Concomitantly, H3K9me2 level was elevated in the promoter region of the rDNA gene in PHF8 knockdown cells and reduced significantly when the wild type but not the catalytically inactive H247A mutant PHF8 was overexpressed. Thus, our study identified a histone demethylase for H3K9me2 that regulates rRNA transcription.