Sex-Differential Responses of Tumor Promotion-Associated Genes and Dysregulation of Novel Long Noncoding RNAs in Constitutive Androstane Receptor-Activated Mouse Liver

Abstract

Xenobiotic agonists of constitutive androstane receptor (CAR) induce many hepatic drug metabolizing enzymes, but following prolonged exposure, promote hepatocellular carcinoma, most notably in male mouse liver. Here, we used nuclear RNA-seq to characterize global changes in the mouse liver transcriptome following exposure to the CAR-specific agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), including changes in novel long noncoding RNAs that may contribute to xenobiotic-induced pathophysiology. Protein-coding genes dysregulated by 3 h TCPOBOP exposure were strongly enriched in KEGG pathways of xenobiotic and drug metabolism, with stronger and more extensive gene responses observed in female than male liver. After 27 h TCPOBOP exposure, the number of responsive genes increased >8-fold in males, where the top enriched pathways and their upstream regulators expanded to include factors implicated in cell cycle dysregulation and hepatocellular carcinoma progression (cyclin-D1, oncogenes E2f, Yap, Rb, Myc, and proto-oncogenes β-catenin, FoxM1, FoxO1, all predicted to be activated by TCPOBOP in male but not female liver; and tumor suppressors p21 and p53, both predicted to be inhibited). Upstream regulators uniquely associated with 3 h TCPOBOP-exposed females include TNF/NFkB pathway members, which negatively regulate CAR-dependent proliferative responses and may contribute to the relative resistance of female liver to TCPOBOP-induced tumor promotion. These responses may be modified by the many long noncoding liver RNAs we show are dysregulated by TCPOBOP or pregnane-X-receptor agonist exposure, including lncRNAs proximal to CAR target genes Cyp2b10, Por, and Alas1. These data provide a comprehensive view of the CAR-regulated transcriptome and give insight into the mechanism of sex-biased susceptibility to CAR-dependent mouse liver tumorigenesis.

Impact of 3-dimensional genome organization, guided by cohesin and CTCF looping, on sex-biased chromatin interactions and gene expression in mouse liver

“…Sex differences in mouse and human liver are linked to the sex-specific pattern of growth hormone secretion by the pituitary gland [69][70][71]. While we have demonstrated the presence of proximal and distal sex-biased looping in mouse liver, it is not clear how dynamic these loops are in response to factors that dysregulate sexbiased gene expression following alterations in hormone patterns [72] or xenobiotic exposure [60]. In an extreme example of dynamic looping, complete loss of cohesin followed by its reintroduction (achieved by auxin-induced targeted degradation and withdrawal) establishes that virtually all loops are lost after 40 min then regained within an hour of auxin withdrawal [22].…”

“…Despite identifying ~1,000 sex-biased binding sites for CTCF or cohesin, the majority are distal from enhancers or genes with known sex bias. Although these distal sites lack any obvious link to sex-biased gene expression in wild type liver, they could have a priming effect and contribute to sex-specific responses to hepatic stressors, such as high fat diet [59] or xenobiotic exposure [60]. Just as short-term feeding of a high fat diet can leave a lasting "epigenetic memory" in the form of epigenetic modifications [61], sex-biased CTCF/cohesin binding sites may prime each sex for differential looping patterns in response to subsequent chemical exposure or dietary stressors.…”

“…Cumulative frequency curves show the percent of each group on the y-axis within the distance to the nearest TSS indicated on the x-axis (in kilobases). TSS for protein coding (RefSeq) and liver lncRNA genes were considered [60]. P values for comparisons between male-and female-biased peaks of the same class are shown in the upper left of each plot (KS t-test).…”

Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to metabolic stress

“…Comparison of the PPARA-responsive lncRNAs to lncRNAs responsive to agonist ligands of two other nuclear receptors in mouse liver, namely CAR and PXR (Lodato et al, 2017), identified 8 lncRNAs that are induced by all three nuclear receptors and 11 lncRNAs repressed by all three receptors. Forty five other lncRNAs were induced, or were repressed, in common by PPARA and CAR, and 30 other lncRNAs were either induced or repressed in common by PPARA and PXR (Table S1C).…”

“…Down-regulation of lncRNA lncOb reduces leptin, leading to a leptin responsive form of obesity (Dallner et al, 2019). Other studies found that lncRNAs are regulated during adipocyte differentiation (Yuan et al, 2019a;Yuan et al, 2019b), are expressed in liver in a sex-dependent manner when regulated by growth hormone (Melia et al, 2016;Melia and Waxman, 2019), and can be strongly induced by xenobiotic exposure (Lodato et al, 2017). A compartment-specific transcriptional profiling approach revealed that lncRNA PAXIP1-AS1 regulates pulmonary arterial hypertension by modulating smooth muscle cell function (Jandl et al, 2019).…”