Interplay Between GH-regulated, Sex-biased Liver Transcriptome and Hepatic Zonation Revealed by Single-Nucleus RNA Sequencing.

The zonation of liver metabolic processes is well-characterized; however, little is known about the cell type-specificity and zonation of sexually dimorphic gene expression or its growth hormone (GH)-dependent transcriptional regulators. We address these issues using single-nucleus RNA-sequencing of 32 000 nuclei representing 9 major liver cell types. Nuclei were extracted from livers from adult male and female mice; from males infused with GH continuously, mimicking the female plasma GH pattern; and from mice exposed to TCPOBOP, a xenobiotic agonist ligand of the nuclear receptor CAR that perturbs sex-biased gene expression. Analysis of these rich transcriptomic datasets revealed the following: 1) expression of sex-biased genes and their GH-dependent transcriptional regulators is primarily restricted to hepatocytes and is not a feature of liver nonparenchymal cells; 2) many sex-biased transcripts show sex-dependent zonation within the liver lobule; 3) gene expression is substantially feminized both in periportal and pericentral hepatocytes when male mice are infused with GH continuously; 4) sequencing nuclei increases the sensitivity for detecting thousands of nuclear-enriched long-noncoding RNAs (lncRNAs) and enables determination of their liver cell type-specificity, sex-bias and hepatocyte zonation profiles; 5) the periportal to pericentral hepatocyte cell ratio is significantly higher in male than female liver; and 6) TCPOBOP exposure disrupts both sex-specific gene expression and hepatocyte zonation within the liver lobule. These findings highlight the complex interconnections between hepatic sexual dimorphism and zonation at the single-cell level and reveal how endogenous hormones and foreign chemical exposure can alter these interactions across the liver lobule with large effects both on protein-coding genes and lncRNAs.