High-resolution, in vivo genome binding interactions of the mouse and human constitutive androstane receptors in rodent liver reveal species differences and novel cancer-related target genes

The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor orchestrating xenobiotic drug metabolism and endobiotic energy homeostasis. Emerging roles for species differences in CAR's regulation of hepatocyte proliferation and as an effector of hepatocarcinogenesis remain poorly understood, although several lines of evidence support the concept that mouse CAR and human CAR differentially program these processes. In this investigation, we conducted whole genome mapping of CAR-DNA interactions using high resolution ChIP-exo protocols combined with an adenovirus delivery strategy allowing transient expression of the respective receptors in livers of transgenic mice. The results obtained were informative in several respects, revealing for example CAR binding to novel target genes, including Gdf15 and Foxo3, previously characterized as regulators of carcinogenic process, and identification of species differences in the genomic interactions of mouse and human receptors that program altered expression profiles for the proto-oncogenes, Myc and Bmf. The ChIP-exo analyses also allowed for characterization of high-resolution mCAR and hCAR binding motifs across the genome. Together, the results obtained help clarify CAR's role as a regulator of biological signaling processes involved in cell proliferation and tumorigenesis, and provide new insights into species variation in liver tumor promotion. Overall design: Adenovirus YFP-hCAR and YFP-mCAR constructs were delivered to CAR (-/-) knockout mice, total liver was harvested for ChIP-exo to investigate binding regions of hCAR and mCAR in C57/Bl6 mice. Following delivery, mice were treated by species specific CAR direct activators (1,4-bis[2-(3,5-dichloropyridyloxy)] benzene; TCPOBOP, for mouse; 6-(4-chlorophenyl: imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; CITCO, for human), or the indirect activator, phenobarbital. 3 biological replicates for each treatment. Total 12 samples. Adenovirus YFP-empty constructs (n=2) were used as controls for ChIP-exo analysis.