Sex differences in pituitary growth hormone (GH) secretion (pulsatile in males vs near continuous/persistent in females) impart sex-dependent expression to hundreds of genes in adult mouse liver. Signal transducer and activator of transcription (STAT) 5, a GH-activated transcription factor that is essential for liver sexual dimorphism, is dynamically activated in direct response to each male plasma GH pulse. However, the impact of GH-induced STAT5 pulses on liver chromatin accessibility and downstream transcriptional events is unknown. In this study, we investigated the impact of a single pulse of GH given to hypophysectomized mice on local liver chromatin accessibility (DNase hypersensitive site analysis), transcription rates (heterogeneous nuclear RNA analysis), and gene expression (quantitative polymerase chain reaction and RNA sequencing) determined 30, 90, or 240 minutes later. The STAT5-dependent but sex-independent early GH response genes Igf1 and Cish showed rapid, GH pulse-induced increases in chromatin accessibility and gene transcription, reversing the effects of hypophysectomy. Rapid increases in liver chromatin accessibility and transcriptional activity were also induced in hypophysectomized male mice for some (Ces2b, Ugt2b38) but not for other liver STAT5-dependent male-biased genes (Cyp7b1). Moreover, in pituitary-intact male mice, Igf1, Cish, Ces2b, and Ugt2b38 all showed remarkable cycles of chromatin opening and closing, as well as associated cycles of induced gene transcription, which closely followed each endogenous pulse of liver STAT5 activity. Thus, the endogenous rhythms of male plasma GH pulsation dynamically open and then close liver chromatin at discrete, localized regulatory sites in temporal association with transcriptional activation of Igf1, Cish, and a subset of STAT5-dependent male-biased genes.

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.

NK cell activity is regulated by the integration of positive and negative signals. One important source of these signals for human NK cells is the KIR family which includes both members that transduce positive and those that generate negative signals. KIR3DL1 inhibits NK cell activity upon engagement by its ligand HLA-Bw4. The highly homologous KIR3DS1 is an activating receptor, which has implicated in the outcome of a variety of pathological situations. However, unlike KIR3DL1, direct binding of KIR3DS1+ cells to HLA has not been demonstrated. We analyzed four key amino acid differences between KIR3DL1*01502 and KIR3DS1*013 to determine their role in KIR binding to HLA. Single substitutions of these residues dramatically reduced binding by KIR3DL1. In the reciprocal experiment, we found that the rare KIR3DS1 allotype KIR3DS1*014 binds HLA-Bw4 even though it differs from KIR3DS1*013 at only one of these positions (138). This reactivity was unexpectedly dependent on residues at other variable positions, as HLA-Bw4 binding was lost in receptors with KIR3DL1-like residues at both positions 199 and 138. These data provide the first evidence for the direct binding of a KIR3DS1+ cells to HLA-Bw4, and highlights the key role for position 138 in determining ligand specificity of KIR3DS1. They also reveal that KIR3DS1 reactivity and specificity is dictated by complex interactions between the residues in this region, suggesting a unique functional evolution of KIR3DS1 within the activating KIR family.

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.

No abstract

Many xenobiotics are metabolized in mammalian liver by pathways regulated by constitutive androstane receptor (CAR). Here, we identify early gene targets of mouse liver CAR and investigate their associated, CAR‐induced changes in local chromatin accessibility and transcription factor (TF) recruitment. Genes induced or repressed by the CAR agonist TCPOBOP were identified by RNA‐Seq after 3 or 27 hr. Chromatin sites that dynamically open or close following CAR activation (changes in DNase I hypersensitive sites, ΔDHS) were identified by DNase‐Seq. Genes dysregulated in 3 hr TCPOBOP‐treated mouse liver were enriched in KEGG pathways for retinol and drug metabolism (DAVID analysis; p<E‐15). IPA identified activated CAR and its agonists as the top predicted upstream regulators of these gene responses. After 27 hr TCPOBOP treatment, many more genes were identified and the predicted upstream regulators expanded to include cyclin D and vitamin D, indicating significant secondary transcriptional regulation. DNase‐Seq identified ~3,600 ΔDHS that were opened or closed by 3 hr TCPOBOP treatment. These ΔDHS were not randomly distributed: mapping to their putative gene targets (i.e., nearest gene within 10 kb) revealed enrichment (12.4‐fold, p<E‐45) of the TCPOBOP‐induced DHS for genes inducible by CAR. Thus, chromatin opening is associated with activation of nearby CAR target genes. Genomic sequences in the 3 hr TCPOBOP‐inducible DHS regions were enriched for TF binding motifs for CAR and for CCAAT‐enhancer binding protein (CEBP), suggesting that CEBPs cooperate with CAR to open chromatin and up regulate target gene expression. Studies designed to test this hypothesis are in progress and will be reported. Grant support: NIH ES024421.

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.