Dynamics of chromatin marks and the role of EZH2/JMJD3/P300 during hepatoblast differentiation

Bi-potential hepatoblasts give rise to hepatocytes with the default fate choice, while undergo a regulatory fate transition to cholangiocytes. We aimed to understand how the “default” vs “regulatory” cell fate differentiations were modulated at the chromatin level. We performed chromatin immunoprecipitation sequencing (ChIP-seq) of hepatobiliary lineages to profile promoter and enhancer associated histone modifications H3K4me3, H3K27me3, H3K27ac and H3K4me1. We also performed in vivo gene manipulation and ex vivo intervention of related histone modifiers were performed to validate their roles in hepatoblast differentiation. We discovered that removal of H3K27me3 from bivalency on promoters was associated with activation of genes related to hepatoblast-cholangiocyte fate transition. In vivo gene manipulation and ex vivo intervention with H3K27me3-modifying enzymes demonstrated that Ezh2 and Jmjd3 specifically regulated hepatoblast-cholangiocyte cell fate transition. In addition, establishments of active and primed enhancers were associated with the developmental processes of both hepatocytes and cholangiocytes, and knockout of P300 inhibited these lineages development. Overall design: To investigate the dynamics of chromatin marks during hepatoblast differentiation, we dissected the Sox9-CreER;Rosa-tdTomato fetal livers in mouse E12.5 and E17.5 embryos. DLK+ and tdTomato+ cells were sorted by FACS for hepatic cells and cholangiocytes isolation. We performed Low-cell-number native ChIP-seq (N-ChIP) using antibodies against H3K4me3, H3K27me3, H3K4me1 and H3K27ac. We performed CUT&RUN ChIP-seq using antibodies against EZH2. We also performed bulk cell RNA-seq to E17.5 hepatocytes and cholangiocytes. To investigate the role of EZH2/JMJD3/P300 during hepatoblast differentiation, we dissected Alfp-Cre;Jmjd3-f/f, Alfp-Cre;Ezh2-f/f, Alfp-Cre;P300-f/f mouse livers and corresponding control groups at P0. DLK+ hepatocytes and EpCAM+ cholangiocytes were isolated by FACS. We also dissected mouse livers at E12.5 and cultured liver lobes for 2 days with treatment of DMSO (vehicle), 2 µM GSKJ4 or 1 µM GSK126. We performed single cell RNA-seq (scRNA-seq) to these cells using Smart-seq2 method.