Liver regeneration CRISPR screen identifies E3 ligase NEURL1B regulates hepatocyte mitosis by destabilizing microtubule organizing centers.

BACKGROUND AND AIMS: The proliferation of hepatocytes is essential for liver regeneration. Hepatocytes harbor supernumerary centrosomes, which can lead to multipolar spindles, chromosome misalignment and prolonged mitosis. How hepatocytes overcome these mitotic challenges to efficiently regenerate is unclear. METHODS: To understand how hepatocytes cope with mitotic challenges, we established a high-quality in vivo CRISPRi screen in regenerating mouse liver. By cross-referencing with gene essentiality data in cell lines, we identified genes required specifically by hepatocytes for proliferation. Knockout mice, RNA sequencing and proteomic approaches were used to understand the function of the candidate gene and the mechanism in regulating hepatocyte proliferation. RESULT: NEURL1B, a rarely studied E3 ligase, is specifically required for hepatocyte cell division in vivo. Inhibiting NEURL1B results in enlarged, polyploid hepatocytes, impaired liver repopulation, and resistance to liver cancer. This dependence on NEURL1B arises from a unique spindle pole organization mechanism used by hepatocytes. At the start of mitosis, hepatocytes exhibit multiple microtubule-organizing centers (MTOCs) organized by NUMA1. These MTOCs undergo extensive remodeling, including clustering and inactivation to form bipolar spindles. NEURL1B localizes to and destabilizes MTOCs, with NUMA1 identified as a substrate. By destabilizing MTOCs, NEURL1B facilitates inactivation of extra MTOCs and enables mitosis progression. Human single nucleus RNA-seq and cell culture data support a conserved role for NEURL1B in human hepatocytes. CONCLUSION: Our approach to studying mitosis in vivo reveals a unique mechanism used by hepatocytes to manage supernumerary centrosomes and identifies NEURL1B as a critical regulator of this pathway, paving the way to new treatments for liver regeneration and cancer.