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• W2896741666 abstract "•CAR activation stimulates a massive liver transcriptional response•Target genes control drug detoxification, general metabolism, and liver growth•CAR stimulates genes through coactivation and histone acetylation•CAR inhibits genes when it shares their enhancers with other nuclear receptors The constitutive androstane receptor (CAR/Nr1i3) regulates detoxification of drugs and other xenobiotics by the liver. Binding of these compounds, activating ligands, causes CAR to translocate to the nucleus and stimulate genes of detoxification. However, CAR activation also changes metabolism and induces rapid liver growth. To explain this gene regulation, we characterized the genome-wide early binding of CAR; its binding partner, RXRα; and the acetylation that they induced on H4K5. CAR-linked genes showed either stimulation or inhibition and regulated lipid, carbohydrate, and energy metabolism, as well as detoxification. Stimulation of expression increased, but inhibition did not decrease, H4K5Ac. Transcriptional inhibition occurred when CAR bound with HNF4α, PPARα, or FXR on the same enhancers. Functional competition among these bound nuclear receptors normally coordinates transcriptional resources as metabolism shifts. However, binding of drug-activated CAR to the same enhancers adds a new competitor that constitutively alters the normal balance of metabolic gene regulation. The constitutive androstane receptor (CAR/Nr1i3) regulates detoxification of drugs and other xenobiotics by the liver. Binding of these compounds, activating ligands, causes CAR to translocate to the nucleus and stimulate genes of detoxification. However, CAR activation also changes metabolism and induces rapid liver growth. To explain this gene regulation, we characterized the genome-wide early binding of CAR; its binding partner, RXRα; and the acetylation that they induced on H4K5. CAR-linked genes showed either stimulation or inhibition and regulated lipid, carbohydrate, and energy metabolism, as well as detoxification. Stimulation of expression increased, but inhibition did not decrease, H4K5Ac. Transcriptional inhibition occurred when CAR bound with HNF4α, PPARα, or FXR on the same enhancers. Functional competition among these bound nuclear receptors normally coordinates transcriptional resources as metabolism shifts. However, binding of drug-activated CAR to the same enhancers adds a new competitor that constitutively alters the normal balance of metabolic gene regulation. The constitutive androstane receptor (CAR/Nr1i3), an important nuclear receptor (NR) transcription factor in hepatocytes, regulates genes that detoxify drugs and xenobiotics. CAR controls an unusually strong transcriptional response that also regulates liver growth and cell proliferation, promotes cancer, and alters glucose metabolism and cholesterol homeostasis (Kobayashi et al., 2015Kobayashi K. Hashimoto M. Honkakoski P. Negishi M. Regulation of gene expression by CAR: an update.Arch. Toxicol. 2015; 89: 1045-1055Crossref PubMed Scopus (44) Google Scholar, Tian et al., 2011Tian J. Huang H. Hoffman B. Liebermann D.A. Ledda-Columbano G.M. Columbano A. Locker J. Gadd45β is an inducible coactivator of transcription that facilitates rapid liver growth in mice.J. Clin. Invest. 2011; 121: 4491-4502Crossref PubMed Scopus (47) Google Scholar). CAR-binding activators—drugs and xenobiotics—are agonist ligands that stimulate translocation to the nucleus. Following translocation, CAR heterodimerizes with RXR to bind a characteristic DNA site within transcriptional enhancers (Choi et al., 1997Choi H.S. Chung M. Tzameli I. Simha D. Lee Y.K. Seol W. Moore D.D. Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR.J. Biol. Chem. 1997; 272: 23565-23571Crossref PubMed Scopus (231) Google Scholar, Wei et al., 2000Wei P. Zhang J. Egan-Hafley M. Liang S. Moore D.D. The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism.Nature. 2000; 407: 920-923Crossref PubMed Scopus (561) Google Scholar). This direct repeat 4-binding site (DR4) consists of two 6-bp half sites that bind RXR and CAR, separated by 4 bp. The so-called phenobarbital response element (PBRE) was the first CAR site to be characterized and is −2.3 kb from the transcription start of Cyp2b10, a gene with exceptional induction by CAR activators (Kawamoto et al., 1999Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene.Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (470) Google Scholar). The halogenated hydrocarbon TCPOBOP (1,4-bis[2-(3,5)-dichoropyridyloxy] benzene), a specific ligand for mouse CAR, induces transcription of characteristic target genes from very low basal levels (Columbano et al., 2005Columbano A. Ledda-Columbano G.M. Pibiri M. Cossu C. Menegazzi M. Moore D.D. Huang W. Tian J. Locker J. Gadd45beta is induced through a CAR-dependent, TNF-independent pathway in murine liver hyperplasia.Hepatology. 2005; 42: 1118-1126Crossref PubMed Scopus (75) Google Scholar, Locker et al., 2003Locker J. Tian J. Carver R. Concas D. Cossu C. Ledda-Columbano G.M. Columbano A. A common set of immediate-early response genes in liver regeneration and hyperplasia.Hepatology. 2003; 38: 314-325Crossref PubMed Scopus (69) Google Scholar, Tzameli et al., 2000Tzameli I. Pissios P. Schuetz E.G. Moore D.D. The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear receptor CAR.Mol. Cell. Biol. 2000; 20: 2951-2958Crossref PubMed Scopus (341) Google Scholar). Moreover, the dramatic growth response to a single dose doubles the liver mass by 18 hr (Tian et al., 2011Tian J. Huang H. Hoffman B. Liebermann D.A. Ledda-Columbano G.M. Columbano A. Locker J. Gadd45β is an inducible coactivator of transcription that facilitates rapid liver growth in mice.J. Clin. Invest. 2011; 121: 4491-4502Crossref PubMed Scopus (47) Google Scholar). Several close relatives of CAR are important hepatocyte regulators with similar binding sites: homodimeric HNF4α and RXR heterodimeric partners PPARα, FXR, LXRα/β, PXR, and THRα/β. HNF4α is the central regulator of hepatocyte phenotype (Fang et al., 2012Fang B. Mane-Padros D. Bolotin E. Jiang T. Sladek F.M. Identification of a binding motif specific to HNF4 by comparative analysis of multiple nuclear receptors.Nucleic Acids Res. 2012; 40: 5343-5356Crossref PubMed Scopus (68) Google Scholar). CAR and the other RXR partners control metabolic processes within this phenotype. PPARα regulates genes of fatty acid oxidation and transport following activation by ligands such as unsaturated fatty acids, eicosanoids, and clofibrate drugs (Rakhshandehroo et al., 2007Rakhshandehroo M. Sanderson L.M. Matilainen M. Stienstra R. Carlberg C. de Groot P.J. Muller M. Kersten S. Comprehensive analysis of PPARalpha-dependent regulation of hepatic lipid metabolism by expression profiling.PPAR Res. 2007; 2007: 26839Crossref PubMed Scopus (157) Google Scholar). FXR, activated by bile acids, controls cholesterol catabolism by regulating genes of bile acid transporters, apolipoproteins, and carbohydrate and amino acid metabolism (Kim and Moore, 2017Kim K.H. Moore D.D. Regulation of liver energy balance by the nuclear receptors farnesoid X receptor and peroxisome proliferator activated receptor alpha.Dig. Dis. 2017; 35: 203-209Crossref PubMed Scopus (12) Google Scholar). LXR—activated by oxidized cholesterol derivatives—stimulates genes of fatty acid and triglyceride synthesis and cholesterol conversion to bile acids (Boergesen et al., 2012Boergesen M. Pedersen T.A. Gross B. van Heeringen S.J. Hagenbeek D. Bindesboll C. Caron S. Lalloyer F. Steffensen K.R. Nebb H.I. et al.Genome-wide profiling of liver X receptor, retinoid X receptor, and peroxisome proliferator-activated receptor alpha in mouse liver reveals extensive sharing of binding sites.Mol. Cell. Biol. 2012; 32: 852-867Crossref PubMed Scopus (156) Google Scholar). PXR, like CAR, is a drug and xenobiotic receptor. Ligands for CAR and PXR are generally distinct but their gene regulation overlaps (Cui and Klaassen, 2016Cui J.Y. Klaassen C.D. RNA-Seq reveals common and unique PXR- and CAR-target gene signatures in the mouse liver transcriptome.Biochim. Biophys. Acta. 2016; 1859: 1198-1217Crossref PubMed Scopus (40) Google Scholar). The thyroid hormone receptor (THR) regulates liver genes of lipid, fatty acid, and steroid metabolism (Grontved et al., 2015Grontved L. Waterfall J.J. Kim D.W. Baek S. Sung M.H. Zhao L. Park J.W. Nielsen R. Walker R.L. Zhu Y.J. et al.Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.Nat. Commun. 2015; 6: 7048Crossref PubMed Scopus (65) Google Scholar). All these NRs activate transcription via their ligand-binding domains (LBDs), which directly bind p160 coactivators. As part of transcriptional activation, the p160s acetylate histones 3 and 4 at several sites, particularly histone 3 lysine 9 (H3K9) and H4K5 (Li et al., 2003Li X. Wong J. Tsai S.Y. Tsai M.J. O'Malley B.W. Progesterone and glucocorticoid receptors recruit distinct coactivator complexes and promote distinct patterns of local chromatin modification.Mol. Cell. Biol. 2003; 23: 3763-3773Crossref PubMed Scopus (199) Google Scholar, Spencer et al., 1997Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. et al.Steroid receptor coactivator-1 is a histone acetyltransferase.Nature. 1997; 389: 194-198Crossref PubMed Scopus (1026) Google Scholar). In addition, the p160s bind another coactivator, CBP, that acetylates H3K27 (Pasini et al., 2010Pasini D. Malatesta M. Jung H.R. Walfridsson J. Willer A. Olsson L. Skotte J. Wutz A. Porse B. Jensen O.N. et al.Characterization of an antagonistic switch between histone H3 lysine 27 methylation and acetylation in the transcriptional regulation of Polycomb group target genes.Nucleic Acids Res. 2010; 38: 4958-4969Crossref PubMed Scopus (224) Google Scholar, Tie et al., 2009Tie F. Banerjee R. Stratton C.A. Prasad-Sinha J. Stepanik V. Zlobin A. Diaz M.O. Scacheri P.C. Harte P.J. CBP-mediated acetylation of histone H3 lysine 27 antagonizes Drosophila Polycomb silencing.Development. 2009; 136: 3131-3141Crossref PubMed Scopus (331) Google Scholar). Without activating ligands, most NRs can also repress transcription through recruitment of corepressors that deacetylate histones. However, activated NRs stimulate some genes, whereas they inhibit others. This alternate type of inhibition has an important effect on phenotype, but the mechanism is poorly understood. CAR dimerizes with RXR and binds to DNA like other NRs, but distinctive features simplify its effects and make CAR a valuable tool for examining transcriptional regulation in vivo. (1) Most NRs modulate activation and repression through their LBDs, but the CAR LBD has an the unusual conformation that prevents binding of corepressors. CAR is thus a pure transcriptional activator (Suino et al., 2004Suino K. Peng L. Reynolds R. Li Y. Cha J.Y. Repa J.J. Kliewer S.A. Xu H.E. The nuclear xenobiotic receptor CAR: structural determinants of constitutive activation and heterodimerization.Mol. Cell. 2004; 16: 893-905Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). (2) CAR resides in the cytoplasm unless ligand or signal transduction activates nuclear transport (Kawamoto et al., 1999Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene.Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (470) Google Scholar). CAR-initiated transcription depends on this translocation. Hence, basal activity is very low but activation of target genes is rapid. (3) CAR lacks an N-terminal activation domain. Such domains provide a second activation mechanism for other NRs. In this article, we present the chromatin immunoprecipitation sequencing (ChIP-seq) characterization of CAR, with parallel genome-wide characterization of RXR binding, chromatin H4K5 acetylation, and DNaseI hypersensitive sites (DHS). To correlate these dynamic modifications of chromatin with gene expression, we defined the hepatocyte transcriptome, its response to CAR activation, and the association of specific CAR binding sites to regulated genes. The analyses showed distinctive differences between gene stimulation and inhibition by CAR and suggested that inhibition was related to binding of a second NR. We therefore characterized HNF4α binding and its relationship to CAR, followed by analysis of RXR dimeric partners from published datasets. The following study of CAR thus reveals a competitive antagonism among activating NRs that controls the dynamic balance of liver metabolic regulation. CAR activation in the liver transcriptionally regulates genes of detoxification and metabolism while stimulating growth and cell proliferation. The transcriptional responses could reflect direct regulation by CAR or regulation by CAR-induced factors. The responses could also be consequences of general processes like cell proliferation or of indirect mechanisms like depletion of coregulators. To discriminate direct regulation, we carried out ChIP-seq analysis of CAR binding in liver 3 hr after treatment with TCPOBOP (Figures 1 and S1). The responses are sexually dimorphic, so analysis was simplified by an exclusive focus on young mature female mice without dietary manipulation (Ledda-Columbano et al., 2003Ledda-Columbano G.M. Pibiri M. Concas D. Molotzu F. Simbula G. Cossu C. Columbano A. Sex difference in the proliferative response of mouse hepatocytes to treatment with the CAR ligand, TCPOBOP.Carcinogenesis. 2003; 24: 1059-1065Crossref PubMed Scopus (42) Google Scholar, Waxman et al., 1985Waxman D.J. Dannan G.A. Guengerich F.P. Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes.Biochemistry. 1985; 24: 4409-4417Crossref PubMed Scopus (560) Google Scholar). CAR antibodies have been problematic, but our prior studies characterized a CAR-binding antibody suitable for conventional ChIP (Tian et al., 2011Tian J. Huang H. Hoffman B. Liebermann D.A. Ledda-Columbano G.M. Columbano A. Locker J. Gadd45β is an inducible coactivator of transcription that facilitates rapid liver growth in mice.J. Clin. Invest. 2011; 121: 4491-4502Crossref PubMed Scopus (47) Google Scholar), and we established highly specific ChIP-seq conditions with this antibody (see Transparent Methods). The specificity is exemplified by the demonstration of 3 CAR-binding enhancers in the region upstream of Cyp2b10 (Figures 1A and 1B), including the PBRE within CAR peak 3 (Kawamoto et al., 1999Kawamoto T. Sueyoshi T. Zelko I. Moore R. Washburn K. Negishi M. Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene.Mol. Cell. Biol. 1999; 19: 6318-6322Crossref PubMed Scopus (470) Google Scholar). The binding coincides with strong transcriptional stimulation (Figure 1C). CAR detection was complemented by analysis of RXRα, promoter marker H3K4Me3, DNaseI hypersensitivity (DHS), enhancer marker H4K5Ac, and RNA sequencing (RNA-seq). The enhancer marker H4K5Ac was chosen because it directly reflects transcriptional activation by NR (Li et al., 2003Li X. Wong J. Tsai S.Y. Tsai M.J. O'Malley B.W. Progesterone and glucocorticoid receptors recruit distinct coactivator complexes and promote distinct patterns of local chromatin modification.Mol. Cell. Biol. 2003; 23: 3763-3773Crossref PubMed Scopus (199) Google Scholar, Spencer et al., 1997Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. et al.Steroid receptor coactivator-1 is a histone acetyltransferase.Nature. 1997; 389: 194-198Crossref PubMed Scopus (1026) Google Scholar) and we validated its application by comparing with more common markers of enhancer activation, H3K9Ac and H3K27Ac (Figure S2). For all 3, CAR bound to “valleys” within a field of acetylation. H4K5Ac provided the strongest enhancer detection, the highest discrimination between promoters and enhancers, and greatest changes following TCPOBOP treatment. The relative sensitivity of H4K5Ac detection is exemplified by the changes surrounding CAR enhancer peak 1 (Figure S2B). Because it marked the largest number of enhancer regions, H4K5Ac also provided valuable discrimination of specific and non-specific binding peaks. Many studies have used DHS to mark enhancers and their activation. However, most enhancer regions of CAR-regulated genes showed only moderate levels of DHS, which was much more prominent at promoters of unrelated genes (Figure S2). Compilation of sequence tags around CAR showed an almost identical distribution of RXR binding in the center of DHS regions. CAR, RXR, and DHS were surrounded by regions of H4K5Ac, which, however, was centrally depleted (Figure 1D). Because of their sensitivity and complementary relationships, we integrated analysis of NR and H4K5Ac. DHS was analyzed separately, and an additional chromatin modification, H3K4Me3, was used only as a marker of active promoters. TCPOBOP treatment strongly stimulated CAR binding of the PBRE (peak 3) and two novel enhancers (peaks 1 and 2) near the Cyp2b10 promoter (Figures 1A and 1B). RXR binding and acetylation in control liver indicated that these enhancers were active before CAR binding. Treatment also stimulated transcription of an upstream gene (Cyp2b10us, with 2 spliced isoforms), 5.2 kb from Cyp2b10 (cloned by RACE; GenBank: MF399062, MF399063). Cyp2b10us is a long non-coding RNA without significant open reading frames and lacks homology with the Cyp2b10 transcription unit. Of note, the induction of Cyp2b10us demonstrates that CAR-binding enhancers link to regulation of multiple genes. Two closely related P450 genes, Cyp2b13 and Cyp2b9, are 164 and 276 kb, respectively, from Cyp2b10. Each gene had 2 CAR peaks near the promoter, one with and one without RXR. CAR strongly induced Cyp2b10 and Cyp2b10us, whereas Cyp2b13 and Cyp2b9 were both downregulated but with different time courses (Figure 1C). Induced CAR binding therefore associates with both increased and decreased transcription from different target genes. Gadd45b, another strongly induced candidate gene (Tian et al., 2011Tian J. Huang H. Hoffman B. Liebermann D.A. Ledda-Columbano G.M. Columbano A. Locker J. Gadd45β is an inducible coactivator of transcription that facilitates rapid liver growth in mice.J. Clin. Invest. 2011; 121: 4491-4502Crossref PubMed Scopus (47) Google Scholar), associated with more distant CAR-binding enhancers within adjacent Gng7, 29–60 kb from the Gadd45b promoter (Figure S1A). In addition to strong CAR-binding peaks, TCPOBOP also induced weak but significant CAR binding to other RXR sites within Gng7, a change noted throughout the genome. Upregulation of another candidate target, Myc, associated with induced CAR peaks at +343 and +361 kb (Figure S1B), whereas the intervening gene Pvt1 showed downregulation. Direct CAR regulation of Myc is possible, since even more distant enhancers control this gene (Uslu et al., 2014Uslu V.V. Petretich M. Ruf S. Langenfeld K. Fonseca N.A. Marioni J.C. Spitz F. Long-range enhancers regulating Myc expression are required for normal facial morphogenesis.Nat. Genet. 2014; 46: 753-758Crossref PubMed Scopus (80) Google Scholar) and other regulated genes are far more distant from the induced CAR peaks. Because of the Myc relationship, we used 400 kb as the cutoff for linking CAR peaks to regulated genes. Candidate analysis detected induced CAR binding near numerous regulated genes and validated the ChIP-seq analysis. We therefore compiled a genome-wide CAR peak set (Feng et al., 2012Feng J. Liu T. Qin B. Zhang Y. Liu X.S. Identifying ChIP-seq enrichment using MACS.Nat. Protoc. 2012; 7: 1728-1740Crossref PubMed Scopus (629) Google Scholar). Although significant peaks associated with virtually all known CAR targets, they were overshadowed by thousands of much stronger peaks that did not link to regulated genes. These latter peaks were restricted to a small fraction of the genome where similar peaks were detected with control antibodies. We therefore defined 624 genomic segments that could not be analyzed by ChIP-seq (see Transparent Methods and Table S1). These segments—totaling 17.4 mb, 0.7% of the mm9 genome including 62 genes (Table S2)—were filtered before analysis of the remaining 2,553 mb. Analysis of the remaining genome with MACS2 software (Feng et al., 2012Feng J. Liu T. Qin B. Zhang Y. Liu X.S. Identifying ChIP-seq enrichment using MACS.Nat. Protoc. 2012; 7: 1728-1740Crossref PubMed Scopus (629) Google Scholar) generated a set of 37,000 CAR peaks with p value < 0.001 (Figures 2A and S3A). Such large peak sets are common, but the number of peaks presents a significant barrier to correlation with specific gene regulation, so secondary cutoffs were derived from binding strength. Elimination of the bottom 2% of the range of peak strength reduced the peak number to 5,400, whereas elimination of 3% left only 3,500 peaks. RXR (9,970 peaks) and H4K5Ac (54,727 peaks) sets (Figures 2B, 2C, S3B, and S3C) were similarly restricted to the top 97% of the range of binding strength. The ratio of top 97% to top 98% peaks was 0.65 for CAR and 0.73 for RXR. The CAR ratio is a little smaller but does not extrapolate to a big difference between expected and observed binding sites. True CAR peaks have another defining property, binding induced by TCPOBOP treatment. A cutoff ratio of 1.5 (treated/control) excluded nonstimulated peaks, and further reduced the peak set to ∼3,000. As a final discriminator, overlap with H4K5Ac-positive regions selected ∼2,500 peaks within regions of active transcriptional regulation (Figure 2D). CAR peaks fell into 2 classes, RXR:CAR and CAR-only peaks. De novo analysis with MEME-ChIP software (Bailey et al., 2015Bailey T.L. Johnson J. Grant C.E. Noble W.S. The MEME suite.Nucleic Acids Res. 2015; 43: W39-W49Crossref PubMed Scopus (617) Google Scholar) showed that the RXR:CAR and CAR-only sites were different (Figure 2E). The complete RXR:CAR peak set generated an NR-like CAR half-site motif, but a smaller set—263 peaks that represented the top 90% of the binding strength range—generated a complete CAR-DR4 motif in which the second repeat was nearly identical to the CAR half-site motif. Moreover, an RXR half-site motif, derived from RXR-only peaks, was similar to the first repeat of the DR4 motif. Scanning with the FIMO program (Bailey et al., 2015Bailey T.L. Johnson J. Grant C.E. Noble W.S. The MEME suite.Nucleic Acids Res. 2015; 43: W39-W49Crossref PubMed Scopus (617) Google Scholar) then detected matches (p < 0.001) to the CAR DR4 motif in 1,936 of the RXR:CAR peaks, averaging 1.7 motifs per peak. MEME-ChIP analysis of the CAR-only peak set did not derive an NR half-site but instead generated a CEBP motif (Figure 2E). The distribution of CEBP motifs showed central localization in both RXR:CAR and CAR-only sets with a much more significant correlation to the latter (Figure 2F). In contrast, DR4 motifs showed central localization only in RXR:CAR peaks. MEME analysis also derived FOXA motifs from RXR:CAR peaks (E = 1.9e-10), but positional analysis did not show the central localization that would indicate a close association with CAR (not illustrated). We then compared CAR, RXR, and H4K5Ac in 3 peak sets: RXR:CAR, CAR-only, and RXR-only peaks (Figure 2G). Stimulation of CAR binding was clear in both RXR:CAR and CAR-only sets. Binding of RXR to RXR:CAR sites increased only 5%, confirming that RXR previously bound as a heterodimer to different partners, or as a homodimer, before treatment. Moreover, the moderate increase of H4K5Ac indicated that induced CAR bound predominantly to previously active enhancers. Competition for limiting RXR is often invoked as a mechanism for transcriptional downregulation, but the binding of RXR to non-CAR sites was unchanged, ruling out this mechanism. However, the net 5% reduction of H4K5Ac on RXR-only sites was moderate but highly significant. This finding suggested an alternate inhibitory mechanism in which new CAR binding competed for H4K5-acetylating cofactors. The next studies related the 2,100 RXR:CAR and 400 CAR-only peaks to TCPOBOP-regulated genes. From the 24,000 unique genes in the mm9 Refseq annotation, RNA-seq libraries characterized a 12,000-gene hepatocyte transcriptome and sequential changes through the beginning of S phase at 24 hr (Figure 3A, Table S3). We began by correlating CAR binding with gene expression at 3 hr and found this time interval adequate to study most upregulation, but not downregulation. The variety of sequential regulatory patterns made the problem complex because no single time point was adequate to compare global gene regulation with CAR binding. Separate analysis of multiple time points, however, would have required a prohibitive number of replicate RNA-seq libraries and divided the peaks into small correlations. We therefore used the entire time series to determine the average expression over 24 hr, a normalized hourly value calculated from expression at 0, 3, 6, 12, 18, and 24 hr after TCPOBOP treatment, and used these inclusive 24-hr average values for most correlations. The analysis exploited the quantitative features of RNA-seq to integrate global changes in the transcriptome with individual genes and pathways. Total transcriptional changes were summed from the individual transcript changes in Table S3 (Figure 3B). A comparison of stimulation/inhibition ratios demonstrated that net 24-hr average transcriptional changes of RNA mass (reads/gene) were balanced when cut off at a 1.2-fold ratio, suggesting a constant level of transcriptional elongation. In contrast, transcript number (reads/gene/kb) showed a 5% net increase of transcriptional initiation that presumably reflected the synthetic requirements of massive liver growth. With this moderate 1.2-fold cutoff, 97% of CAR peaks linked to regulated genes. In contrast, a 2-fold cutoff restricted analysis to the most prominent gene regulations associated with CAR, but linked only 37% of the binding peaks. A 1.2-fold change in gene expression may seem to be a small effect, but can actually reflect a very large increase in transcriptional initiation if the gene is strongly expressed (see Table S3). Moreover, changes of this magnitude for serum proteins, the high-abundance products of the liver transcriptome, have important biological effects. The vast majority of CAR peaks were in distant regulatory elements within 400 kb of regulated genes (Figures 3C). The median peak-promoter distances for up- and downregulated genes were similar, 34 and 40 kb, respectively. However, for 2-fold regulation, median peak locations were significantly closer to upregulated (18 kb) and farther from downregulated genes (50 kb). To assess the biological significance of CAR peak-associated gene expression, functional enrichment analysis (Heinz et al., 2010Heinz S. Benner C. Spann N. Bertolino E. Lin Y.C. Laslo P. Cheng J.X. Murre C. Singh H. Glass C.K. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities.Mol. Cell. 2010; 38: 576-589Abstract Full Text Full Text PDF PubMed Scopus (4773) Google Scholar) compared gene number and statistical significance to ∼5,000 profiles (Figure 3D). We also quantified the changes in transcript number for each response. The expected correlations of upregulated genes to xenobiotic and drug metabolism were clear and significant. Surprisingly, the correlations to lipid metabolism, the insulin-responsive IRS signal transduction pathway, and mitochondrial genes had higher statistical significance and accounted for larger changes in net transcription. In addition, downregulation dominated the nonxenobiotic responses. CAR induction therefore significantly reprioritized metabolic gene expression in addition to stimulating xenobiotic metabolism. Correlation by distance associated CAR-binding elements with 6,400 regulated genes. To examine mechanisms, however, we simplified the relationships to the two regulated genes closest to each CAR-binding peak, a set of ∼2,500 genes (Figure 4A, Table S4), which accounted for ∼60% of the total transcriptional change. Significantly, both closer and farther genes in the pairs contributed substantial regulation (Figure 4B). In early studies with the full RXR:CAR peak set, correlation of chromatin changes with gene expression gave biphasic curves, an upward region of positive correlation for increased expression and a flat region of noncorrelation for decreased expression. To resolve these discordant effects, we defined 4 peak subsets with unambiguous relationships to up- or downregulation (Figure 4C). The subsets consisted of peaks associated only with a single regulated gene within 400 kb (single up, n = 94; single down, n = 179) or with 2 genes within 400 kb that had parallel regulation (dual up, n = 282; dual down, n = 572). The remaining peaks had an ambiguous relationship to gene expression, since they associated with oppositely regulated genes (dual opposite, defined by relative distance of the regulated genes as up down, n = 257; or down up, n = 214). The inclusive subsets together comprised 80% of all induced RXR:CAR peaks. The main reason for exclusion was when one of the linked genes only showed a transient expression change at 3 hr. Analysis of H4K5Ac in subsets revealed a clear difference between up- and downregulation (Figure 4C). The former had increased acetylation, the expected effect of an activating N" @default.
• W2896741666 created "2018-10-26" @default.
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• W2896741666 date "2018-11-01" @default.
• W2896741666 modified "2023-09-26" @default.
• W2896741666 title "Binding of Drug-Activated CAR/Nr1i3 Alters Metabolic Regulation in the Liver" @default.
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