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.

Changes in the CD-1 mouse uterine transcriptome during proestrus and estrus were investigated to help elucidate mechanisms of uterine tissue remodeling during the estrus cycle and their regulation by estrogen and progesterone in preparation of the uterus for pregnancy. Mice were staged beginning at 6 weeks of age, and uterine horns were harvested after monitoring two estrus cycles. Microarray analysis of whole uterine horn RNA identified 2428 genes differentially expressed in estrus compared to proestrus, indicating there is extensive remodeling of mouse uterus during the estrus cycle, affecting ~10% of all protein-encoding genes. Many (~50%) of these genes showed the same differential expression in independent analyses of isolated uterine lumenal epithelial cells. Changes in gene expression associated with structural alterations of the uterus included remodeling of the extracellular matrix, changes in cell keratins and adhesion molecules, activation of mitosis and changes in major histocompatibility complex class II (MHCII) presentation, complement and coagulation cascades, and cytochrome P450 expression. Signaling pathways regulated during the estrus cycle, involving ligand-gated channels, Wnt and hedgehog signaling, and transcription factors with poorly understood roles in reproductive tissues, included several genes and gene networks that have been implicated in pathological states. Many of the molecular pathways and biological functions represented by the genes differentially expressed from proestrus to estrus are also altered during the human menstrual cycle, although not necessarily at the corresponding phases of the cycle. These findings establish a baseline for further studies in the mouse model to dissect mechanisms involved in uterine tissue response to endocrine disruptors and the development of reproductive tract diseases.

Activation of the nuclear receptor and transcription factor CAR (Nr1i3) by its specific agonist ligand TCPOBOP (1, 4-bis[2-(3, 5-dichloropyridyloxy)]benzene) dysregulates hundreds of genes in mouse liver and is linked to male-biased hepatocarcinogenesis. To elucidate the genomic organization of CAR-induced gene responses, we investigated the distribution of TCPOBOP-responsive RefSeq coding and long noncoding RNA (lncRNA) genes across the megabase-scale topologically associating domains (TADs) that segment the genome, and which provide a structural framework that functionally constrains enhancer-promoter interactions. We show that a subset of TCPOBOP-responsive genes cluster within TADs, and that TCPOBOP-induced genes and TCPOBOP-repressed genes are often found in different TADs. Further, using DNase-seq and DNase hypersensitivity site (DHS) analysis, we identified several thousand genomic regions (ΔDHS) where short-term exposure to TCPOBOP induces localized changes (increases or decreases) in mouse liver chromatin accessibility, many of which cluster in TADs together with TCPOBOP-responsive genes. Sites of chromatin opening were highly enriched nearby genes induced by TCPOBOP and chromatin closing was highly enriched nearby genes repressed by TCPOBOP, consistent with TCPOBOP-responsive ΔDHS serving as enhancers and promoters that positively regulate CAR-responsive genes. Gene expression changes lagged behind chromatin opening or closing for a subset of TCPOBOP-responsive ΔDHS. ΔDHS that were specifically responsive to TCPOBOP in male liver were significantly enriched for genomic regions with a basal male bias in chromatin accessibility; however, the male-biased response of hepatocellular carcinoma-related genes to TCPOBOP was not associated with a correspondingly male-biased ΔDHS response. These studies elucidate the genome-wide organization of CAR-responsive genes and of the thousands of associated genomic sites where TCPOBOP exposure induces both rapid and persistent changes in chromatin accessibility.

Constitutive androstane receptor (CAR) (Nr1i3), a liver nuclear receptor and xenobiotic sensor, induces drug, steroid, and lipid metabolism and dysregulates genes linked to hepatocellular carcinogenesis, but its impact on the liver epigenome is poorly understood. TCPOBOP (1, 4-bis-[2-(3, 5-dichloropyridyloxy)]benzene), a halogenated xenochemical and highly specific CAR agonist ligand, induces localized chromatin opening or closing at several thousand mouse liver genomic regions, discovered as differential DNase-hypersensitive sites (ΔDHS). Active enhancer and promoter histone marks induced by TCPOBOP were enriched at opening DHS and TCPOBOP-inducible genes. Enrichment of CAR binding and CAR motifs was seen at opening DHS and their inducible drug/lipid metabolism gene targets, and at many constitutively open DHS located nearby. TCPOBOP-responsive cell cycle and DNA replication genes codependent on MET/EGFR signaling for induction were also enriched for CAR binding. A subset of opening DHS and many closing DHS mapping to TCPOBOP-responsive target genes did not bind CAR, indicating an indirect mechanism for their changes in chromatin accessibility. TCPOBOP-responsive DHS were also enriched for induced binding of RXRA, CEBPA, and CEBPB, and for motifs for liver-enriched factors that may contribute to liver-specific transcriptional responses to TCPOBOP exposure. These studies elucidate the enhancer landscape of TCPOBOP-exposed liver and the widespread epigenetic changes that are induced by both direct and indirect mechanisms linked to CAR activation. The global maps of thousands of environmental chemical-induced epigenetic changes described here constitute a rich resource for further research on xenochemical effects on liver chromatin states and the epigenome.

CAR and PXR are important xenobiotic sensors that rapidly induce and repress genes regulating diverse liver metabolic processes, including drug, steroid and lipid metabolism. The effects of specific activators of CAR and PXR on mouse liver chromatin accessibility were determined using DNase‐Seq to identify global changes in DNase hypersensitive sites (DHS) 3 hr after treatment of mice with TCPOBOP (CAR activation) or PCN (PXR activation). Differential DHS analysis (ΔDHS) identified 928 TCPOBOP‐induced DHS and 161 TCPOBOP‐repressed DHS, as well as 1,062 PCN‐induced DHS and 390 PCN‐repressed DHS, with some overlap between the two sets of ΔDHS. Thus, CAR and PXR rapidly induce large numbers of localized changes in chromatin accessibility. Several ΔDHS were confirmed by qPCR, including ΔDHS upstream of the CAR target gene Cyp2b10. The ΔDHS were not randomly distributed: mapping ΔDHS to their presumed targets (i.e., the nearest gene within 100 kb) revealed strong (11.4‐fold) enrichment (p<E‐19) of the TCPOBOP‐induced DHS in the set of TCPOBOP‐induced genes, identified in the same livers. The gene targets of TCPOBOP‐induced DHS also showed strong enrichment (5.9‐fold, p<0.005) in the set of TCPOBOP‐repressed genes. Thus, chromatin opening is associated with rapid activation as well as rapid repression of CAR target genes. We also found strong enrichment (11.7‐fold, p<E‐29) of target genes of PCN‐induced DHS in the PCN‐inducible gene set, whereas genes repressed by PCN were significantly enriched (6.4‐fold, p<E‐04) in PCN‐repressible DHS gene targets. Thus, CAR and PXR may repress genes by fundamentally different mechanisms: DHS opening for CAR vs. DHS closing for PXR.