KANSL3 directs transcriptional programs essential for hepatic metabolism and differentiation [scRNA-seq]

Understanding the molecular mechanisms governing liver homeostasis and disease progression is crucial for developing new therapeutic strategies for chronic liver diseases and liver cancer, with emerging evidence underscoring the key role of epigenetic regulation. Here, we show that hepatic deletion of the epigenetic regulator KANSL3, a component of the NSL complex, causes early-onset chronic liver disease, distinct from metabolic dysfunction-associated steatohepatitis (MASH). Loss of KANSL3 leads to biliary hyperplasia and early hepatic fibrosis, indicating its vital role in liver structure and function. KANSL3 acts as a master regulator of hepatocyte gene expression through histone acetylation, impacting global liver transcriptional networks. scRNA-seq analysis uncovers that KANSL3 deletion disrupts hepatocyte differentiation in vivo and in hepatic organoids. Ultimately, chronic KANSL3 loss alters the liver microenvironment and promotes an immunosuppressive premalignant state linked to hepatocellular carcinoma (HCC). Overall design: Comparison of liver cells isolated from control mice or mice with liver-specific knockout of Kansl3 of two diferent ages. Neonatal experiment: Control (n=3) and Kansl3 LKO (n=3) neonatal (P7) livers were isolated. One piece of the liver was used for BEC isolation and the other for hepatocyte isolation. For BEC isolation single cell suspensions were prepared by 0.25mg/ml collagenase (Sigma C2674) digest at 37°C for 1 hr. For the BECs population Zombie NIR (Biolegend, 423105) negative (live cells), CD45/CD31/CD11b- and EpCAM+ cells were sorted. For the hepatic stellate cell (HSC) enriched population Zombie NIR (Biolegend, 423105) negative (live cells), CD45/CD31/CD11b/EpCAM- cells were sorted. For hepatocyte isolation, single cell suspensions were prepared by 0.8mg/ml collagenase (Sigma C2674) and 50 U/µl) DNase I (Applichem) in DMEM) digest at 37°C for 30 min. For the hepatocyte enriched population Zombie NIR negative, large cells were sorted. For the scRNA-seq experiment, BECs, hepatocytes and HSC populations were isolated from three AlbCre and three Kansl3 LKO mice. For each population, the samples from each genotype were pooled. The BECs, hepatocytes and HSC populations were mixed in an 1:1:1 ratio. This cell mix was directly subjected to 10x scRNA-seq library preparation. One 10x reaction was performed per genotype. A total of 16,000 cells per genotype were processed using the 10X Genomics protocol CG00052 RevB from the Chromium Single Cell 3' Library & Gel Bead Kit v2 (PN-120237). The libraries were quantified using a fragment analyzer and pooled for multiplexed sequencing on a NovaSeq 6000. Old liver experiment: 90-week-old control (n=2) and Kansl3 LKO (n=2) livers were isolated and digested in 0.8mg/ml collagenase (Sigma C2674) and 50 U/µl) DNase I (Applichem) in DMEM) at 37°C for 30 min. Cell suspension was cryopreserved in FCS supplemented with 1% DMSO and stored in liquid nitrogen. Frozen sample thawed and viable cells were enriched using the LeviCell 1.0 device (Levitas Bio), selecting the option “large cells”. All samples had a viability of approximately 70%. The cells were then prepared for 10x scRNA-seq library preparation. Two 10x reactions were performed per genotype, one for each animal. A total of 16,000 cells per genotype were processed according to the 10X Genomics protocol CG000204 RevD using the Chromium Single Cell 3' Library & Gel Bead Kit v3.1 (PN-1000121). Libraries were quantified with a fragment analyzer and pooled for multiplexed sequencing on a NovaSeq 6000.