Single nucleus RNA-seq gene expression profiling for protein coding and lncRNA genes using livers of male and female mice, with GH infusion, and following TCPOBOP exposure

Single nucleus RNA-seq gene expression profiling was carried out for protein coding and lncRNA genes using nuclei extracted from livers from adult male and female mice; from male mice infused with GH continuously for one week, mimicking the female plasma GH pattern; and from male and female mice exposed to TCPOBOP, a xenobiotic agonist ligand of the nuclear receptor CAR, which perturbs sex-biased gene expression in the liver. Our analysis of these rich single nucleus, mouse liver transcriptomic datasets, comprised of 32,000 liver nuclei representing 9 major liver cell types, revealed: 1) the expression of sex-biased genes and their key GH-dependent transcriptional regulators is primarily restricted to hepatocytes and is largely not a feature of liver non-parenchymal cells; 2) many sex-biased transcripts show sex-dependent zonation within the liver lobule; 3) gene expression is substantially feminized in both 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 on both protein-coding genes and lncRNAs. Overall design: Single nuclei suspensions were generated from frozen liver tissue from adult male and female ICR (CD-1) mice that were treated with 3 mg/kg TCPOBOP dissolved in corn oil, or corn oil vehicle, for 27 hr, or adult male mice that were treated with continuous growth hormone given as an infusion (20 ng rat GH per hr per gram body weight) for 7 days. Single nuclei suspensions were processed through the standard 10x Genomics library preparation protocol to generate single nucleus RNA-seq sequencing libraries, used to investigate the sex bias in gene expression, cell type specificity and liver zonation expression profiles of expressed protein coding genes and lncRNAs under five different biological conditions. Frozen liver tissue (livers from n=4 mice per group, frozen tissue pooled per condition) was homogenized, washed and filtered to generate single nuclei suspensions and subsequently 10x Genomics sequencing libraries for the following six groups: male and female mice injected with vehicle (control); male and female mice given TCPOBOP at 3 mg/kg body weight and then euthanized 27 h later; and male mice treated with growth hormone (GH) given as a continuous infusion using a subcutaneous Alzet osmotic minipump for 7 days, and its untreated male liver control.