USP4‐Dependent CHAF1B Stabilization Regulates Distinct SETDB1 Ubiquitin States Linked to AKT T308 Signaling and Lipogenic Remodeling in HCC

Durable responses to current therapies remain limited in hepatocellular carcinoma (HCC), highlighting the need to identify regulators of malignant progression. By integrating multi-omics analyses, spatial transcriptomics, clinical specimens, and multiple models, we identified chromatin assembly factor 1B (CHAF1B) as a functional regulator of HCC phenotypes. Gain- and loss-of-function of CHAF1B altered proliferative, migratory, clonogenic, and tumorigenic phenotypes. LC-MS/MS, DIA proteomics, and cell-based assays revealed CHAF1B-associated lipogenic remodeling characterized by SREBP1C nuclear localization, lipogenic gene/protein induction, and lipid-droplet accumulation. Mechanistically, the WD40 repeat-containing region of CHAF1B contributed to its association with UHRF1 and SETDB1, supporting UHRF1-associated K63-linked ubiquitination and CRM1/exportin-1-dependent cytoplasmic redistribution of SETDB1. Conversely, CHAF1B depletion enhanced SETDB1 association with VHL and favored a predominantly K11-associated degradative ubiquitin state linked to proteasomal SETDB1 loss. SETDB1 redistribution and catalytic activity were associated with AKT T308-linked signaling. A focused CRISPR-based screen of deubiquitinases identified USP4 as an upstream regulator of CHAF1B protein homeostasis. USP4 depletion or Akebia saponin D (ASD) increased K48-linked ubiquitination of CHAF1B, reduced CHAF1B protein abundance, attenuated AKT T308-linked signaling, and suppressed malignant and lipogenic phenotypes. These findings reveal distinct ubiquitin-dependent states governing SETDB1 stability and identify USP4-dependent CHAF1B stabilization as an upstream regulatory node in HCC.