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- W1997228575 abstract "The nuclear receptor constitutive androstane receptor (CAR) acts as a xenobiotic sensor and regulates the expression of enzymes, such as several cytochromes P450s and the UDP-glucuronosyltransferase (UGT) type 1A1. CAR binds as a heterodimer with the retinoid X receptor (RXR) to specific DNA sites, called response elements (REs). Clusters of CAR REs, referred to as phenobarbital response enhancer modules (PBREMs), have been identified in several CAR target genes. In this study we confirm that REs formed by direct repeats of two AGTTCA hexamers with 4 spacing nucleotides are optimal for the binding of CAR-RXR heterodimers. In addition, we found that the heterodimers also form complexes on everted repeat-type arrangements with 8 spacing nucleotides. We also observed that CAR is able to bind DNA as a monomer and to interact in this form with different coregulators even in the presence of RXR. Systematic variation of the nucleotides 5′-flanking to both AGTTCA hexamers showed that the dinucleotide sequence modulates the DNA complex formation of CAR monomers and CAR-RXR heterodimer by a factor of up to 20. The highest preference was found for the sequence AG and lowest for CC. The increased DNA affinity of CAR is mediated by the positively charged arginines 90 and 91 located in the carboxyl-terminal extension of the DNA-binding domain of the receptor. Furthermore, we show that one of the three CAR REs of the human UGT1A1 PBREM is exclusively bound by CAR monomers and this is regulated by ligands that bind to this nuclear receptor. This points to a physiological role for CAR monomers. Therefore, both CAR-RXR heterodimers and CAR monomers can contribute to the gene activating function of PBREMs in CAR target genes. The nuclear receptor constitutive androstane receptor (CAR) acts as a xenobiotic sensor and regulates the expression of enzymes, such as several cytochromes P450s and the UDP-glucuronosyltransferase (UGT) type 1A1. CAR binds as a heterodimer with the retinoid X receptor (RXR) to specific DNA sites, called response elements (REs). Clusters of CAR REs, referred to as phenobarbital response enhancer modules (PBREMs), have been identified in several CAR target genes. In this study we confirm that REs formed by direct repeats of two AGTTCA hexamers with 4 spacing nucleotides are optimal for the binding of CAR-RXR heterodimers. In addition, we found that the heterodimers also form complexes on everted repeat-type arrangements with 8 spacing nucleotides. We also observed that CAR is able to bind DNA as a monomer and to interact in this form with different coregulators even in the presence of RXR. Systematic variation of the nucleotides 5′-flanking to both AGTTCA hexamers showed that the dinucleotide sequence modulates the DNA complex formation of CAR monomers and CAR-RXR heterodimer by a factor of up to 20. The highest preference was found for the sequence AG and lowest for CC. The increased DNA affinity of CAR is mediated by the positively charged arginines 90 and 91 located in the carboxyl-terminal extension of the DNA-binding domain of the receptor. Furthermore, we show that one of the three CAR REs of the human UGT1A1 PBREM is exclusively bound by CAR monomers and this is regulated by ligands that bind to this nuclear receptor. This points to a physiological role for CAR monomers. Therefore, both CAR-RXR heterodimers and CAR monomers can contribute to the gene activating function of PBREMs in CAR target genes. Nuclear receptors (NRs) 1The abbreviations used are: NR, nuclear receptor; CAR, constitutive androstane receptor; DBD, DNA-binding domain; DR, direct repeat; ER, everted repeat; GST, glutathione S-transferase; PBREM, phenobarbital response enhancer module; RE, response element; RXR, retinoid X receptor; TIF2, transcription intermediary factor 2; NCoR, nuclear corepressor; T3R, thyroid hormone receptor; UGT, UDP-glucuronosyltransferase; VDR, vitamin D receptor; DR, direct repeat; DOTAP, N-[1-(2,3-dioleoyloxy)propyl)]-N,N,N-trimethylammonium methylsulfate.1The abbreviations used are: NR, nuclear receptor; CAR, constitutive androstane receptor; DBD, DNA-binding domain; DR, direct repeat; ER, everted repeat; GST, glutathione S-transferase; PBREM, phenobarbital response enhancer module; RE, response element; RXR, retinoid X receptor; TIF2, transcription intermediary factor 2; NCoR, nuclear corepressor; T3R, thyroid hormone receptor; UGT, UDP-glucuronosyltransferase; VDR, vitamin D receptor; DR, direct repeat; DOTAP, N-[1-(2,3-dioleoyloxy)propyl)]-N,N,N-trimethylammonium methylsulfate. are a large family of transcription factors (48 human members) that have critical roles in nearly all aspects of vertebrate development and adult physiology by transducing the effects of small, lipophilic compounds into transcriptional response (1Chawla A. Repa J.J. Evans R.M. Mangelsdorf D.J. Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1679) Google Scholar). The existence of a highly conserved DNA-binding domain (DBD) and a carboxyl-terminal, structurally conserved ligand-binding domain define the family (2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schütz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6043) Google Scholar). The ligand-binding domains of most NRs consist of 12 α-helices that form a characteristic, 3-layer sandwich. The most carboxyl-terminal helix, helix 12, contains the activation function 2 domain, which serves as a molecular switch by interacting in the agonistic conformation of the ligand-binding domain with coactivator proteins that activate target gene transcription via further protein-protein interactions (3Rachez C. Freedman L.P. Gene (Amst.). 2000; 246: 9-21Crossref PubMed Scopus (282) Google Scholar). NRs can also contribute to gene silencing via the interaction with corepressor proteins that in turn contact histone deacetylases (4Polly P. Herdick M. Moehren U. Baniahmad A. Heinzel T. Carlberg C. FASEB J. 2000; 14: 1455-1463Crossref PubMed Google Scholar). The receptors for estrogen, progesterone, testosterone, cortisol, aldosterone, 1α,25-dihydroxyvitamin D3, thyroid hormone, and all-trans-retinoic acid are classical endocrine NRs (5Carlberg C. Biofactors. 1999; 10: 91-97Crossref PubMed Scopus (45) Google Scholar). However, most of the superfamily members were cloned before their specific ligands were known and were described as orphan NRs (6Blumberg B. Evans R.M. Genes Dev. 1998; 12: 3149-3155Crossref PubMed Scopus (284) Google Scholar). Subsequently, some of these compounds, which act as ligands for these orphan NRs, have been identified (1Chawla A. Repa J.J. Evans R.M. Mangelsdorf D.J. Science. 2001; 294: 1866-1870Crossref PubMed Scopus (1679) Google Scholar). One of these adopted orphan NRs is the constitutive androstane receptor (CAR; NR1I3 (7Committee Nuclear-Receptor Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar)), which has recently been implicated in mediating the effects of xenobiotics on the expression of enzymes, such as cytochrome P450s (8Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (645) Google Scholar) and UDP-glucuronosyltransferase (UGT) (9Sugatani J. Kojima H. Ueda A. Kakizaki S. Yoshinari K. Gong Q.H. Owens I.S. Negishi M. Sueyoshi T. Hepatology. 2001; 33: 1232-1238Crossref PubMed Scopus (330) Google Scholar). In contrast to classical endocrine NRs that show a very selective ligand binding with Kd values in the order of 1 nm or lower (10Herdick M. Bury Y. Quack M. Uskokovic M. Polly P. Carlberg C. Mol. Pharmacol. 2000; 57: 1206-1217PubMed Google Scholar), CAR binds a variety of structurally diverse compounds that display a relatively low affinity (Ki in the order of 1 μm) (11Moore L.B. Parks D.J. Jones S.A. Bledsoe R.K. Consler T.G. Stimmel J.B. Goodwin B. Liddle C. Blanchard S.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar). Interestingly, CAR differs from most other NRs by having a strong constitutive activity in the absence of ligand. This can be reduced by the binding of the inverse agonist 5α-androstan-3α-ol or potentiated by the agonists 5β-pregnane-3,20-dione (11Moore L.B. Parks D.J. Jones S.A. Bledsoe R.K. Consler T.G. Stimmel J.B. Goodwin B. Liddle C. Blanchard S.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar) and 6-(4-chlorophenyl)imidazo[2,1-b](1,3)thiazole-5-carbaldehyde o-3,4-dichlorobenzyl)-oxime (CITCO) (12Maglich J.M. Parks D.J. Moore L.B. Collins J.L. Goodwin B. Billin A.N. Stoltz C.A. Kliewer S.A. Lambert M.H. Willson T.M. Moore J.T. J. Biol. Chem. 2003; 278: 17277-17283Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Nuclear receptor responsive genes are defined through the presence of binding sites for particular NRs, referred to as response elements (REs), in their promoter regions (13Carlberg C. Eur. J. Biochem. 1995; 231: 517-527Crossref PubMed Scopus (170) Google Scholar, 14Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar). CAR has been shown to form heterodimers with the retinoid X receptor (RXR) on REs that are formed by a direct repeat (DR) of hexameric binding sites (15Sueyoshi T. Kawamoto T. Zelko I. Honkakoski P. Negishi M. J. Biol. Chem. 1999; 274: 6043-6046Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). Early reports indicated that CAR preferred to bind DR5-type REs (16Baes M. Gulick T. Choi H.-S. Martinoli M.G. Simha D. Moore D.D. Mol. Cell. Biol. 1994; 14: 1544-1552Crossref PubMed Scopus (409) Google Scholar, 17Choi H.S. Chung M. Tzameli I. Simha D. Lee Y.K. Seol W. Moore D.D. J. Biol. Chem. 1997; 272: 23565-23571Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). However, recently it has been shown that CAR-RXR heterodimers bind optimally to DR4-type REs (8Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (645) Google Scholar, 18Xie W. Barwick J.L. Simon C.M. Pierce A.M. Safe S. Blumberg B. Guzelian P.S. Evans R.M. Genes Dev. 2000; 14: 3014-3023Crossref PubMed Scopus (460) Google Scholar). The closest relatives of CAR, the vitamin D receptor (VDR) and the pregnane X receptor, are also known to bind REs that are formed by two hexameric sites in an everted repeat (ER) arrangement (19Blumberg B. Sabbagh W. Juguilon H. Bolado J. van Meter C.M. Ong E.S. Evans R.M. Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (812) Google Scholar, 20Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Invest. 1998; 102: 1016-1023Crossref PubMed Scopus (1365) Google Scholar, 21Schräder M. Nayeri S. Kahlen J.P. Müller K.M. Carlberg C. Mol. Cell. Biol. 1995; 15: 1154-1161Crossref PubMed Scopus (120) Google Scholar). A DNA complex formation of CAR for ER binding has not yet been described. The crystal structure of a DNA-bound thyroid hormone receptor (T3R)-RXR heterodimer (22Rastinejad F. Perlmann T. Evans R.M. Sigler P.B. Nature. 1995; 375: 203-211Crossref PubMed Scopus (467) Google Scholar) demonstrated a head-to-tail arrangement of the receptors with the T3R DBD binding to the downstream motif and the RXR DBD binding to the upstream motif. Moreover, this study showed that ∼65% of the DNA contacts of the T3R-RXR heterodimer are meditated by T3R and explained why this receptor is able to act as a monomer as well (23Quack M. Carlberg C. Biochem. J. 2001; 360: 387-393Crossref PubMed Google Scholar, 24Schräder M. Becker-Andre M. Carlberg C. J. Biol. Chem. 1994; 269: 6444-6449Abstract Full Text PDF PubMed Google Scholar). It is inferred that CAR-RXR heterodimers show the same type of DNA binding and polarity as T3R-RXR heterodimers (22Rastinejad F. Perlmann T. Evans R.M. Sigler P.B. Nature. 1995; 375: 203-211Crossref PubMed Scopus (467) Google Scholar, 25Gronemeyer H. Moras D. Nature. 1995; 375: 190-191Crossref PubMed Scopus (66) Google Scholar). However, the investigation of natural CAR responding genes indicated that a single CAR RE seems to be insufficient for mediating the regulatory role of the receptor and that more likely at least two CAR REs in close proximity to each other are necessary. These multiple CAR RE clusters are commonly called phenobarbital response enhancer modules (PBREMs). The CYP2B6 gene contains two DR4-type REs with an additional binding site for the transcription factor NF-1 (8Honkakoski P. Zelko I. Sueyoshi T. Negishi M. Mol. Cell. Biol. 1998; 18: 5652-5658Crossref PubMed Scopus (645) Google Scholar), whereas the PBREM of the UGT1A1 gene is formed by three CAR REs (9Sugatani J. Kojima H. Ueda A. Kakizaki S. Yoshinari K. Gong Q.H. Owens I.S. Negishi M. Sueyoshi T. Hepatology. 2001; 33: 1232-1238Crossref PubMed Scopus (330) Google Scholar). The authors demonstrated that the three REs of the UGT1A1 PBREM are necessary for full ligand responsiveness, but how they act together is still unknown. Although T3R preferentially acts as a heterodimer, the T3R monomer bound to DNA has been shown to be a fully competent transcription factor (23Quack M. Carlberg C. Biochem. J. 2001; 360: 387-393Crossref PubMed Google Scholar, 24Schräder M. Becker-Andre M. Carlberg C. J. Biol. Chem. 1994; 269: 6444-6449Abstract Full Text PDF PubMed Google Scholar). Monomer binding in other members of the NR superfamily (e.g. NGFI-B (26Wilson T.E. Fahrner T.J. Johnston M. Milbrandt J. Science. 1991; 252: 1296-1300Crossref PubMed Scopus (479) Google Scholar) and ROR (27Carlberg C. Hooft van Huijsduijnen R. Staple J.K. DeLamarter J.F. Becker-Andre M. Mol. Endocrinol. 1994; 8: 757-770Crossref PubMed Scopus (191) Google Scholar)) appears to be mediated by the Grip box, which is a carboxyl-terminal extension of the DBD (28Zhao Q. Khorasanizadeh S. Miyoshi Y. Lazar M.A. Rastinejad F. Mol. Cell. 1998; 1: 849-861Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). This box provides receptor-specific interfaces for an interaction with the nucleotides that flank the 5′ end of the hexameric binding motif. It is known that the DNA binding affinity of T3R monomers is strongly modulated by the two nucleotides that directly flank the hexameric binding motif (24Schräder M. Becker-Andre M. Carlberg C. J. Biol. Chem. 1994; 269: 6444-6449Abstract Full Text PDF PubMed Google Scholar). Subsequently, it has been shown that these two 5′-flanking nucleotides also modulate the DNA complex formation of T3R-RXR and VDR-RXR heterodimers (29Quack M. Frank C. Carlberg C. J. Cell. Biochem. 2002; 86: 601-612Crossref PubMed Scopus (33) Google Scholar). However, neither the influence of the 5′-flanking nucleotides for CAR-RXR heterodimer binding nor the possibility of CAR monomer binding has been addressed. In this study, the functional profile of CAR-RXR heterodimers in relation to CAR monomers was investigated. We found that CAR preferentially binds as a heterodimer with RXR to DR4- and ER8-type REs. Surprisingly, we also observed CAR-DNA monomer complexes even in the presence of RXR. CAR monomers and heterodimers showed to interact with coactivator and corepressor proteins. Moreover, we observed that both CAR monomers and CAR-RXR heterodimers have preferences for the dinucleotide that flank the 5′ of the hexameric DNA binding sites. These preferences were mediated by amino acids Arg-90 and Arg-91 of the carboxyl-terminal extension of the DBD. Finally, we indicate a possible physiological role for CAR monomers contributing to the functionality of human UGT1A1 gene promoter PBREM. Protein Expression Vectors—Full-length cDNAs for human CAR (16Baes M. Gulick T. Choi H.-S. Martinoli M.G. Simha D. Moore D.D. Mol. Cell. Biol. 1994; 14: 1544-1552Crossref PubMed Scopus (409) Google Scholar), human RXRα (30Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1256) Google Scholar), and chicken T 3 Rα (31Sap J. Munoz A. Damm K. Goldberg Y. Ghysdael J. Leutz A. Beug H. Vennström B. Nature. 1986; 324: 635-640Crossref PubMed Scopus (1018) Google Scholar) were subcloned into the T7/SV40 promoter-driven pSG5 expression vector (Stratagene, La Jolla, CA). The full-length cDNA for mouse CAR (17Choi H.S. Chung M. Tzameli I. Simha D. Lee Y.K. Seol W. Moore D.D. J. Biol. Chem. 1997; 272: 23565-23571Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) was subcloned into the T7/cytomegalovirus promoter-driven pCMX expression vector. The point mutants of RXR and human CAR were generated using the QuickChange point mutagenesis kit (Stratagene) and confirmed by sequencing. The helix 12 deletion mutants of RXR and human CAR were created by introducing a stop codon at amino acid positions 444 and 342, respectively, in the proteins. The same constructs were used for both T7 RNA polymerase-driven in vitro transcription/translation of the respective cDNAs and for viral promoter-driven overexpression of the respective proteins in mammalian cells. Glutathione S-transferase (GST) Fusion Protein Construct—The NR interaction domain of human TIF2, spanning amino acids 646 to 926 (32Voegel J.J. Heine M.J.S. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (948) Google Scholar), and of mouse NCoR, spanning amino acids 1679 to 2453 (33Hörlein A.J. Näär A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Söderström M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1700) Google Scholar), were subcloned into the GST fusion vector pGEX (Amersham Biosciences). Reporter Gene Constructs—Single copies of DR4(T/T) and its 30 5′ and 3′ variations, the three REs from the human UGT1A1 gene (9Sugatani J. Kojima H. Ueda A. Kakizaki S. Yoshinari K. Gong Q.H. Owens I.S. Negishi M. Sueyoshi T. Hepatology. 2001; 33: 1232-1238Crossref PubMed Scopus (330) Google Scholar), and the monomer binding site AGAGTTCA (monomer(T)AG) were each fused with the thymidine kinase (tk) promoter driving the firefly luciferase reporter gene. All 35 constructs were verified by sequencing. The core sequences of the REs are indicated in Figs. 2 and 6 and Table I.Fig. 6Contribution of CAR heterodimers and monomers to the activity of the UGT1A1 promoter PBREM. The sequence of the PBREM of the UGT1A1 gene with the three CAR binding sites RE1, RE2, and RE3 is indicated above. A, gel shift experiments were performed with in vitro translated human CAR (hCAR) and human RXR (hRXR) protein and 32P-labeled RE1, RE2, and RE3. Protein-DNA complexes were resolved from free probe through 8% non-denaturing polyacrylamide gels. Representative gels are shown. The percentage of protein-complexed DNA (in relation to maximal binding of CAR-RXR heterodimers to RE1) was quantified using a Fuji FLA-3000 reader. NS indicates nonspecific complexes. Reporter gene assays were performed with extracts from MCF-7 cells that were transiently transfected with a luciferase reporter construct containing the indicated dimeric and monomeric REs and an expression vector for human CAR. Cells were treated for 16 h with solvent, 10 μm 5β-pregnane-3,20-dione (B), 10 μm CITCO or 10 μm 5α-androstan-3α-ol (C). Data were normalized to the basal activity of DR4(T/T) without receptor overexpression. Columns represent means of at least three experiments and bars indicate standard deviations. Two-tail, paired Student's t test was performed and p values were calculated with reference to the respective solvent controls (*, p < 0.05; **, p < 0.01).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IEfficiency of DNA binding and transactivation of CAR monomers and CAR-RXR heterodimers is modulated by flanking sequences to the downstream and upstream motifNNAGTTCAGAAGTTCATGNNAGTTCANNAGTTCATGAGAGTTCA→→ →→ →ABCDEFGAssay receptor ligandGel shift CAR solventGel shift CAR-RXR solventReporter gene CAR solventReporter gene CAR pregnanedioneGel shift CAR-RXR solventReporter gene CAR solventReporter gene CAR pregnanedioneMeanS.D.MeanS.D.MeanS.D.MeanS.D.MeanS.D.MeanS.D.MeanS.D.AA453ap < 0.05.816ap < 0.05.472bp < 0.01.11410ap < 0.05.1055110823720AC82bp < 0.01.201cp < 0.001.233cp < 0.001.516bp < 0.01.84592620514AG10011100110072261618725ap < 0.05.1508ap < 0.05.31925ap < 0.05.AT222bp < 0.01.171cp < 0.001.100cp < 0.001.243cp < 0.001.15111bp < 0.01.14715ap < 0.05.29534ap < 0.05.CA164bp < 0.01.433bp < 0.01.314cp < 0.001.636ap < 0.05.10114102822821CC63bp < 0.01.130bp < 0.01.242cp < 0.001.546bp < 0.01.615ap < 0.05.655ap < 0.05.12912ap < 0.05.CG193bp < 0.01.241bp < 0.01.517bp < 0.01.11411ap < 0.05.692ap < 0.05.805ap < 0.05.17915CT71bp < 0.01.131cp < 0.001.122cp < 0.001.292cp < 0.001.668ap < 0.05.668ap < 0.05.14718ap < 0.05.GA363ap < 0.05.646ap < 0.05.463bp < 0.01.1078ap < 0.05.1004100722616GC71bp < 0.01.1112ap < 0.05.921121616733ap < 0.05.927ap < 0.05.20526ap < 0.05.GG288ap < 0.05.473cp < 0.001.455bp < 0.01.1019ap < 0.05.701061813610GT91bp < 0.01.281cp < 0.001.241cp < 0.001.546bp < 0.01.9012931019916TA262ap < 0.05.413bp < 0.01.323bp < 0.01.677bp < 0.01.672ap < 0.05.763ap < 0.05.1727TC61bp < 0.01.101cp < 0.001.192cp < 0.001.424cp < 0.001.839735ap < 0.05.18319TG223bp < 0.01.344bp < 0.01.95819818894113624120TT82bp < 0.01.161bp < 0.01.72cp < 0.001.163cp < 0.001.752ap < 0.05.79619410a p < 0.05.b p < 0.01.c p < 0.001. Open table in a new tab In vitro translated human proteins were generated by coupled in vitro transcription/translation using rabbit reticulocyte lysate as recommended by the supplier (Promega, Madison, WI). Protein batches were quantified by test translations in the presence of [35S]methionine. The specific concentration of the receptor proteins was adjusted to ∼4 ng/μl (10 ng corresponds to approximately to 0.2 pmol) after taking the individual number of methionine residues per protein into account. Bacterial overexpression of GST, GST-TIF2-(646–926), and GST-NCoR-(1679–2453) was obtained from the Escherichia coli BL21-(DE3)pLysS strain (Stratagene) containing the respective expression plasmids. GST and GST-TIF2-(646–926) fusion protein expression were stimulated with 0.25 mm isopropyl-β-d-thiogalactopyranoside for 3 h at 37 °C and GST-NCoR-(1679–2453) expression was induced with 1.25 mm isopropyl-β-d-thiogalactopyranoside for 5 h at 25 °C. Proteins were purified and immobilized by glutathione-Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's protocol and were eluted in the presence of excess glutathione. Gel shift assays were performed with standard (∼10 ng) or the indicated amounts of the appropriate in vitro translated proteins. The proteins were incubated for 15 min in a total volume of 20 μl of binding buffer (10 mm Hepes, pH 7.9, 150 mm KCl, 1 mm dithiothreitol, 0.2 μg/μl poly(dI-dC), and 5% glycerol). For supershift experiments 3 μg of bacterially expressed GST, GST-TIF2-(646–926), and GST-NCoR-(1679–2453) proteins were added to the reaction mixture. Constant amounts (1 ng) of 32P-labeled double-stranded oligonucleotides (50,000 cpm) corresponding to one copy of a monomeric or dimeric RE (for core sequences see Figs. 1, 2, and 6) were then added and incubation was continued for 20 min at room temperature. Protein-DNA complexes were resolved by electrophoresis through 8% non-denaturing polyacrylamide gels in 0.5× TBE (45 mm Tris, 45 mm boric acid, 1 mm EDTA, pH 8.3) and quantified on a Fuji FLA-3000 reader (Tokyo, Japan) using Image Gauge software (Fuji). GST pull-down assay were performed with 50 μl of a 50% Sepharose bead slurry GST-TIF2-(646–926) or GST-NCoR-(1679–2453) (preblocked with 1 μg/μl bovine serum albumin) and 20 ng in vitro translated 35S-labeled NRs. Proteins were incubated in immunoprecipitation buffer (20 mm Hepes, pH 7.9, 200 mm KCl, 1 mm EDTA, 4 mm MgCl2, 1 mm dithiothretiol, 0.1% Nonidet P-40, and 10% glycerol) for 20 min at 30 °C. In vitro translated proteins that were not bound to GST fusion proteins were washed away with immunoprecipitation buffer. GST fusion protein-bound 35S-labeled NRs were resolved by electrophoresis through 10% SDS-polyacrylamide gels and quantified on a Fuji FLA-3000 reader using Image Gauge software. MCF-7 human breast cancer cells were seeded into 6-well plates (105 cells/ml) and grown overnight in phenol red-free Dulbecco's modified Eagle's medium supplemented with 5% charcoal-stripped fetal bovine serum. Plasmid DNA containing liposomes were formed by incubating 1 μg of a reporter plasmid and 1 μg of expression vectors for human CAR with 10 μg of DOTAP (Roth, Karlsruhe, Germany) for 15 min at room temperature in a total volume of 100 μl. After dilution with 900 μl of phenol red-free Dulbecco's modified Eagle's medium, the liposomes were added to the cells. Phenol red-free Dulbecco's modified Eagle's medium supplemented with 500 μl of 15% charcoal-stripped fetal bovine serum was added 4 h after transfection. At this time, either solvent, 10 μm 5β-pregnane-3,20-dione (Steraloids, Newport, RI), 10 μm CITCO (Biomol, Plymouth Meeting, PA), or 10 μm 5α-androstan-3α-ol (Steraloids) were also added. The cells were lysed 16 h after onset of stimulation using the reporter gene lysis buffer (Roche Diagnostics) and the constant light signal luciferase reporter gene assay was performed as recommended by the supplier (Canberra-Packard, Groningen, The Netherlands). The high amino acid sequence identity (54.5%) between the DBDs of human T3Rβ and human CAR allowed to use the crystal structure of hT3Rβ-hRXRα (Protein Data Bank number 2nll, Ref. 22Rastinejad F. Perlmann T. Evans R.M. Sigler P.B. Nature. 1995; 375: 203-211Crossref PubMed Scopus (467) Google Scholar) as a template for a human CAR-human RXR model. A molecular surface model was produced by the MSMS package (34Sanner M.F. Olson A.J. Spehner J.C. Biopolymers. 1996; 38: 305-320Crossref PubMed Google Scholar), which is included in the CHIMERA program package (35Huang C.C. Couch G.S. Pettersen E.F. Ferrin T.E. Pac. Symp. Biocomput. 1996; 1: 724Google Scholar). The visual model building was done with Xtalview (36McRee D.E. J. Struct. Biol. 1999; 125: 155-165Crossref Scopus (2020) Google Scholar). All the non-conserved amino acid side chains were built up following as close as possible the information of the hT3Rβ template. To determine the multiple DNA complex formation abilities of CAR, gel shift experiments were performed using equal molar quantities (0.2 pmol or ∼10 ng) of in vitro translated human CAR (hCAR) and human RXRα (hRXR) protein on DR1–DR6- and ER1–ER10-type REs (Fig. 1). The DR4-type element, which is a perfect repeat of two AGTTCA motifs (DR4(T/T)) and was originally derived from the enhancer of the rat pit-1 gene (37Rhodes S.J. Chen R. DiMattia G.E. Scully K.M. Kalla K.A. Lin S.-C. Yu V.C. Rosenfeld M.G. Genes Dev. 1993; 7: 913-932Crossref PubMed Scopus (207) Google Scholar), served as the prototype RE for the other elements. The highest amount of complex formation was observed on the DR4-type RE, followed by the DR5-type RE. In contrast, less than 20% of maximal CAR-RXR complex formation was observed on the DRs with 1–3 and 6 nucleotide spacing. On ER-type REs CAR-RXR heterodimers exhibited a less stringent spacer specificity with the most efficient binding being observed on the ER8-type RE (∼75% of that on DR4(T/T)). This was followed by the ER7-type RE (60%), the ER6- and ER9-type REs (both 50%), the ER10-type RE (40%), and the ER5-type RE (20%). ER-type REs with a spacer of four or less nucleotides displayed below 10% of maximal CAR-RXR heterodimer complex formation. Interestingly, even in the presence of RXR a small amount of human CAR protein was found to bind as a monomer to each of the DR- and ER-type REs. To analyze the competence of human and mouse CAR in complex formation with DNA both as a heterodimer (with RXR) and as a monomer, further gel shift experiments were performed (Fig. 2). 10 or 20 ng of in vitro translated human and mouse CAR were incubated in the presence of 0, 2.5, and 10 ng RXR. These mixtures were probed with dimeric DR4-type and monomeric REs carrying either AGTTCA (DR4(T/T) and monomer(T)AG) or AGGTCA (DR4(G/G) and monomer(G)AG) hexameric core sequences. On DR4(T/T) only human CAR was found to bind as a monomer in the presence of RXR (Fig. 2A). In contrast, in the absence of the heterodimeric partner both human and mouse CAR showed reasonable amounts of binding to both monomeric and dimeric REs. Monomers as well as heterodimers also formed on REs containing AGGTCA motifs (Fig. 2B). These CAR monomer complexes were ∼10-fold weaker than on REs with AGTTCA motifs (Fig. 2A). Neither CAR homodimer formation nor the independent binding of two CAR monomers was observed on the DR4-type REs. The latter statement was confirmed further by supershift experiments (see Fig. 4B). Taken together, both human and mouse CAR were able to bind as a monomer to DNA and this was dependent on RXR concentration and hexameric motif sequence. To compare the monomer and heterodimer formation ability of CAR with other members of the NR superfamily, gel shift experiments were performed with equal amounts of in vitro translated human CAR and chicken T3Rα in the presence or absence of human RXRα on DR4-, ER7-, and ER8-type REs carrying either AGTTCA (Fig. 3A) or AGGTCA motifs (Fig. 3B). The complex formation of CAR-RXR heterodimers on the REs DR4(T/T), ER7(T/T), and ER8(T/T) was more effective than that of T3R-RXR heterodimers. Furthermore, CAR monomers but not T3R monomers were observed on these REs (Fig. 3A). In contrast, on the REs DR4(G/G), ER7(G/G), and ER8(G/G) the comple" @default.
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