FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1972978588> ?p ?o ?g. }

• W1972978588 endingPage "27997" @default.
• W1972978588 startingPage "27988" @default.
• W1972978588 abstract "Heterodimers of the peroxisome proliferator-activated receptors (PPAR) and the retinoid X receptors (RXR) recognize response elements (PPREs) that exhibit the consensus sequence 5′-A(A/T)CT(A/G)GGNCAAAG(G/T)TCA-3′. The consensus PPRE includes both a 5′-extension and a direct repeat (DR1) comprised of two canonical core recognition sequences (underlined) for nuclear receptor zinc fingers separated by a single nucleotide spacer. The extended binding site recognized by PPARs is very similar to sites that bind monomers of the nuclear receptors Rev-ErbA and ROR suggesting that the latter could bind to PPREs and affect gene transcription. However, Rev-ErbA and ROR bind weakly to naturally occurring PPREs relative to the consensus binding site, and significant effects on PPARα transactivation of a CYP4A6-Z reporter were not observed. In contrast, PPAR/RXR heterodimers bind to a DR2 element containing the conserved 5′-extended sequence that is recognized by dimers of RORα or Rev-ErbA. PPARα/RXRα positively regulate transcription from this element, and co-expression of Rev-ErbA blocks this effect. The nuclear receptors NGFI-B and ROR utilize a carboxyl-terminal extension (CTE) of the zinc finger DNA binding domain in their interactions with the 5′-extension of a single zinc finger-binding site. DNA binding domains (DBD) of PPARs α, δ, and γ that contain the zinc finger motif and a CTE display binding to core recognition sequences that is dependent on the 5′-extended sequence found in PPREs. Unlike DBDs of other nuclear receptors that form heterodimers with RXR, the PPAR-DBDs did not exhibit cooperative binding with the DBD of RXR and exhibit the opposite polarity for binding to the direct repeat motif. In contrast to the corresponding DBD of RXR, the PPAR-DBDs bind as monomers to a single extended binding site as well as to the consensus PPRE. A chimera linking the zinc finger domain of RXRα to the CTE from PPARα bound to a single extended binding site indicating a functional role for the CTE of PPARs in extended binding site recognition. Heterodimers of the peroxisome proliferator-activated receptors (PPAR) and the retinoid X receptors (RXR) recognize response elements (PPREs) that exhibit the consensus sequence 5′-A(A/T)CT(A/G)GGNCAAAG(G/T)TCA-3′. The consensus PPRE includes both a 5′-extension and a direct repeat (DR1) comprised of two canonical core recognition sequences (underlined) for nuclear receptor zinc fingers separated by a single nucleotide spacer. The extended binding site recognized by PPARs is very similar to sites that bind monomers of the nuclear receptors Rev-ErbA and ROR suggesting that the latter could bind to PPREs and affect gene transcription. However, Rev-ErbA and ROR bind weakly to naturally occurring PPREs relative to the consensus binding site, and significant effects on PPARα transactivation of a CYP4A6-Z reporter were not observed. In contrast, PPAR/RXR heterodimers bind to a DR2 element containing the conserved 5′-extended sequence that is recognized by dimers of RORα or Rev-ErbA. PPARα/RXRα positively regulate transcription from this element, and co-expression of Rev-ErbA blocks this effect. The nuclear receptors NGFI-B and ROR utilize a carboxyl-terminal extension (CTE) of the zinc finger DNA binding domain in their interactions with the 5′-extension of a single zinc finger-binding site. DNA binding domains (DBD) of PPARs α, δ, and γ that contain the zinc finger motif and a CTE display binding to core recognition sequences that is dependent on the 5′-extended sequence found in PPREs. Unlike DBDs of other nuclear receptors that form heterodimers with RXR, the PPAR-DBDs did not exhibit cooperative binding with the DBD of RXR and exhibit the opposite polarity for binding to the direct repeat motif. In contrast to the corresponding DBD of RXR, the PPAR-DBDs bind as monomers to a single extended binding site as well as to the consensus PPRE. A chimera linking the zinc finger domain of RXRα to the CTE from PPARα bound to a single extended binding site indicating a functional role for the CTE of PPARs in extended binding site recognition. peroxisome proliferator activated receptor acyl-CoA oxidase dimethyl sulfoxide a direct repeat with a spacing of one nucleotide electrophoretic mobility-shift assay glutathione S-transferase maltose-binding protein peroxisome proliferator response element a generic term for a cytochrome P-450 monooxygenase, individual P450s are designated according to a uniform system of nomenclature (49Nelson D.R. Kamataki T. Waxman D.J. Guengerich F.P. Estabrook R.W. Feyereisen R. Gonzalez F.J. Coon M.J. Gunsalus I.C. Gotoh O. Okuda K. Nebert D.W. DNA Cell Biol. 1993; 12: 1-51Crossref PubMed Scopus (1655) Google Scholar) and the gene designations are preceded by the letters CYP polymerase chain reaction retinoic acid receptor retinoid X receptor α thyroid hormone receptor 643, pirinixic acid ([4-chloro-6-(2,3-xylindino)-2-pyrimidinylthio]acetic acid) DNA binding domain carboxyl-terminal extension. The peroxisome proliferator activated receptor α (PPARα)1 mediates the transcriptional regulation of several genes encoding enzymes involved in lipid metabolism in response to peroxisome proliferators and fatty acids. Responsive genes include the microsomal cytochrome P450 fatty acid ω-hydroxylases (1Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar, 2Aldridge T.C. Tugwood J.D. Green S. Biochem. J. 1995; 306: 473-479Crossref PubMed Scopus (178) Google Scholar), the peroxisomal fatty acyl-CoA oxidase (3Tugwood J.D. Issemann I. Anderson R.G. Bundell K.R. McPheat W.L. Green S. EMBO J. 1992; 11: 433-439Crossref PubMed Scopus (804) Google Scholar, 4Osumi T. Wen J.-K. Hashimoto T. Biochem. Biophys. Res. Commun. 1991; 175: 866-871Crossref PubMed Scopus (184) Google Scholar), the peroxisomal bifunctional enzyme (5Zhang B. Marcus S.L. Sajjadi F.G. Alvares K. Reddy J.K. Subramani S. Rachubinski R.A. Capone J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7541-7545Crossref PubMed Scopus (235) Google Scholar, 6Bardot O. Aldridge T.C. Latruffe N. Green S. Biochem. Biophys. Res. Commun. 1993; 192: 37-45Crossref PubMed Scopus (233) Google Scholar), and the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthetase (7Rodrı́guez J.C. Gil-Gómez G. Hegardt F.G. Haro D. J. Biol. Chem. 1994; 269: 18767-18772Abstract Full Text PDF PubMed Google Scholar). PPARα binds to response elements (PPREs) within the 5′-flanking regions of these genes as a heterodimer with the retinoid X receptor, RXR (8Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1521) Google Scholar, 9Gearing K.L. Göttlicher M. Teboul M. Widmark E. Gustafsson J.-Å. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1440-1444Crossref PubMed Scopus (345) Google Scholar, 10Palmer C.N.A. Hsu M.-H. Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1994; 269: 18083-18089Abstract Full Text PDF PubMed Google Scholar). PPREs contain a DR1 motif consisting of imperfect direct repeats of the nuclear receptor core recognition sequence (AGGTCA) separated by a single nucleotide (8Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1521) Google Scholar). The repeated core recognition sequences interact directly with the amino-terminal zinc finger of each nuclear receptor (11Lee M.S. Kliewer S.A. Provencal J. Wright P.E. Evans R.M. Science. 1993; 260: 1117-1121Crossref PubMed Scopus (252) Google Scholar). The four nucleotides immediately 5′ of the DR1 motif are also highly conserved among known PPREs and exhibit a consensus of A(A/T)CT (12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 13Johnson E.F. Palmer C.N. Griffin K.J. Hsu M.H. FASEB J. 1996; 10: 1241-1248Crossref PubMed Scopus (169) Google Scholar). These nucleotides are essential for the function of the principal PPRE (Z element) of the gene encoding P450 4A6, a microsomal fatty acid ω-hydroxylase (CYP4A6), and are required for the binding of PPAR/RXR heterodimers to the Z element in vitro (12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The contribution of the extended binding site is less obvious for PPAR/RXR heterodimer binding to elements containing perfect AGGTCA motifs. Rather, it appears to facilitate the binding of PPAR/RXR heterodimers to elements containing divergent core binding sites (12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 14IJpenberg A. Jeannin E. Wahli W. Desvergne B. J. Biol. Chem. 1997; 272: 20108-20117Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). These imperfect DR1 motifs bind other nuclear receptors poorly. As a result, the deviations of the core sites from the consensus recognition sequence and the presence of a suitable extended binding site sequence may attenuate the extent of interference imposed upon peroxisome proliferator signaling by other signaling pathways that occur through DR1 containing response elements. The 5′-extensions conserved in PPREs and the adjacent core site of the DR1 are similar to the extended binding sites, (A/T)A(A/T)NT(A/G)GGTCA, utilized by monomers of the nuclear receptors Rev-ErbA (15Harding H.P. Lazar M.A. Mol. Cell. Biol. 1993; 13: 3113-3121Crossref PubMed Google Scholar), BD73 (16Dumas B. Harding H.P. Choi H.S. Lehmann K.A. Chung M. Lazar M.A. Moore D.D. Mol. Endocrinol. 1994; 8: 996-1005PubMed Google Scholar,17Retnakaran R. Flock G. Giguere V. Mol. Endocrinol. 1994; 8: 1234-1244Crossref PubMed Scopus (84) Google Scholar), and ROR (18Giguère V. Tini M. Flock G. Ong E. Evans R.M. Otulakowski G. Genes Dev. 1994; 8: 538-553Crossref PubMed Scopus (451) Google Scholar, 19Forman B.M. Chen J. Blumberg B. Kliewer S.A. Henshaw R. Ong E.S. Evans R.M. Mol. Endocrinol. 1994; 8: 1253-1261Crossref PubMed Scopus (183) Google Scholar, 20Giguere V. McBroom L.D. Flock G. Mol. Cell. Biol. 1995; 15: 2517-2526Crossref PubMed Google Scholar, 21McBroom L.D. Flock G. Giguere V. Mol. Cell. Biol. 1995; 15: 796-808Crossref PubMed Google Scholar). This suggests that the binding mechanism of PPAR to PPREs may resemble that of receptor monomers to extended binding sites. The carboxyl-terminal extension (CTE) of the zinc finger domains of NGFI-B (22Wilson T.E. Paulsen R.E. Padgett K.A. Milbrandt J. Science. 1992; 256: 107-110Crossref PubMed Scopus (280) Google Scholar) and RORα (20Giguere V. McBroom L.D. Flock G. Mol. Cell. Biol. 1995; 15: 2517-2526Crossref PubMed Google Scholar) appears to recognize 5′-extensions of the core binding site and contribute to the binding of each to a single extended recognition site. In this report, we demonstrate that PPARs contain a functionally similar CTE to that of Rev-ErbA and of RORα that contributes to the recognition of the 5′-upstream sequence conserved in PPREs. An extended DNA binding domain (DBD) of mPPARα containing both the zinc finger motif and the CTE displays monomeric binding that is dependent on the sequence 5′ of the upstream core site, as no binding is observed when this upstream sequence deviates substantially from the consensus derived from PPREs. Although the RXR-DBD does not bind to a single extended core site, a chimeric protein linking the RXR zinc finger domain with the CTE of PPARα does bind as a monomer to a single core binding site with a 5′ consensus extended sequence. These results indicate that the interaction of the CTE of PPARα with the 5′ sequence flanking the DR1 provides a primary mechanism for PPARα/RXR heterodimers to compete effectively with other receptor dimers for binding to PPREs. PCR was utilized to generate truncated receptor cDNAs. The upstream oligonucleotide primers contained a codon for the initiator methionine followed by a codon for alanine, GCT, to facilitate expression in E. coli. The lower primers included a stop codon. Both primers contained suitable restriction sites at the 5′ ends to simplify construction of expression plasmids. Each of the constructs derived from the mouse PPARα (1Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar), mouse PPARγ1 (23Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1280) Google Scholar), human PPARδ (Nuc1) (24Schmidt A. Endo N. Rutledge S.J. Vogel R. Shinar D. Rodan G.A. Mol. Endocrinol. 1992; 6: 1634-1641Crossref PubMed Scopus (366) Google Scholar), human RXRα (25Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1257) Google Scholar), or human Rev-ErbA (15Harding H.P. Lazar M.A. Mol. Cell. Biol. 1993; 13: 3113-3121Crossref PubMed Google Scholar) cDNAs were placed under the control of the lac promoter in the vector, pSPORT1 (Life Technologies, Inc.), modified to include anNdeI restriction site at the initiation codon located at an optimal distance from the promoter (26Barnes H.J. Methods Enzymol. 1996; 275: 3-14Crossref PubMed Google Scholar). The DNA binding domain (DBD) of RXR was expressed as a fusion protein with glutathioneS-transferase using the vector pGEX2T (Amersham Pharmacia Biotech). All PCR amplifications were performed in a Perkin-Elmer 9600 thermal cycler using 30 cycles of denaturation at 90 °C for 15 s and of annealing and extension at 60 °C for 30 s. The segment encoding amino acids 88–210 of PPAR (PPAR-DBD-L) was amplified using primers PPARDBDU, cgtggatcccat atg Gct AGC ACG GAC GAG TCC CCC, and PPARDBDL, gggggatcc tca GAT TCT CTT GCC CAG AGA TTT GAG. Lowercase letters indicate nucleotides that are not found in the cDNA. The resulting product was digested with NdeI and BamHI and then ligated intoNdeI/BamHI-digested, modified pSPORT. The segment encoding amino acids 88–195, PPAR-DBD, was generated as described above using a different lower primer, PPARDBDL3, gggggatcc tca CAG GTC GTG TTC ACA GGT AAG. The segment encoding amino acids 88–186, PPAR-DBD-S, and the segment encoding amino acids 88–178, PPAR-DBD-VS, were generated as described above using the following lower primers, PPARDBDL4 (for PPAR-DBD-S), gggggatcc tca TGC TTT CAG TTT TGC TTT TTC AGA TC and PPARDBDL5 (for PPAR-DBD-VS), gggggatcc tca TCT TCC AAA GCG AAT TGC ATT GTG. The insert for the Nuc1-DBD (amino acids 60–167 of human PPARδ) was generated using the following oligonucleotides: NUC1DBDU, cgtggatcccat ATG Gct TGT GAC GGG GCC TCA; and NUC1DBDL, gggggatcc tca CTG GCT CCC CTC GTT TGC AG. The insert for the Rev-ErbA DBD (amino acids 121–227) was obtained using EAR1DBDU, cgtggatcccat atg gct AAC ATC ACC AAG CTG AAT GGC; and EAR1DBDL, gggggatcc tca GGC CAG GTT CAT GGC ACT CTG. The insert for the PPARγ-DBD (amino acids 105–202) was generated using PPARγDBDU, gggaattcat ATG Gct ATT GAG Tgt CGA GTC TG; and PPARγDBDL, gggtctagagcatgc tca CAG CTG GTC GAT ATC ACT GGA GAT C. The resulting PCR products were digested withNdeI and SphI prior to being ligated intoNdeI/SphI-digested pSPORT. The segment encoding the RXR-DBD (amino acids 127–235) was generated using RXRDBDU, cgtggatcccat ATG GCT TCC TTC ACC AAG CAC; and RXRDBDL, gggggatcc tca CTT GGG CTC CAC GGC CAG CTC. The resulting PCR product was digested with BamHI and ligated into BamHI-digested pGEX2T to generate the protein domain fused to the carboxyl terminus of glutathione S-transferase (GST). The identity and integrity of each construct was confirmed by sequence determination. Lysates of cultures of E. coli XL-1 Blue cells (Stratagene, La Jolla, CA) expressing the individual DNA binding domains were prepared as described previously (12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). A chimera containing the zinc finger domain of human RXRα (amino acids 127–200) and the CTE of the zinc finger domain from murine PPARα (amino acids 167–195) was generated by PCR. Two initial amplification reactions were employed and performed as described above. One reaction used RZPATU and PPARDBDL3 and the other utilized RZPATL and RXRDBDU. The sequence of the RZPATU primer is TGC CTG GCC ATG GGC ATG TCA CAC AAT GCA ATT CGC TTT. The RZPATL primer is the reverse complement of RZPATU. The products of the two amplification reactions were mixed and used as the template for an amplification reaction containing the outer primers (PPARDBDL3 and RXRDBDU). The resulting product was digested withNdeI and BamHI prior to being ligated intoNdeI/BamHI-digested, modified pSPORT. The construct was verified by sequencing, and the chimera was expressed in E. coli XL-1 Blue (Stratagene, La Jolla, CA) as described (12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). cDNAs encoding mouse PPARα (1Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar), mouse PPARγ1 (23Kliewer S.A. Forman B.M. Blumberg B. Ong E.S. Borgmeyer U. Mangelsdorf D.J. Umesono K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7355-7359Crossref PubMed Scopus (1280) Google Scholar), Nuc1 (24Schmidt A. Endo N. Rutledge S.J. Vogel R. Shinar D. Rodan G.A. Mol. Endocrinol. 1992; 6: 1634-1641Crossref PubMed Scopus (366) Google Scholar), human RXRα (25Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1257) Google Scholar), human Rev-ErbA (15Harding H.P. Lazar M.A. Mol. Cell. Biol. 1993; 13: 3113-3121Crossref PubMed Google Scholar), or human RORα1 (19Forman B.M. Chen J. Blumberg B. Kliewer S.A. Henshaw R. Ong E.S. Evans R.M. Mol. Endocrinol. 1994; 8: 1253-1261Crossref PubMed Scopus (183) Google Scholar) were in vitrotranscribed and translated in a TNT-coupled rabbit reticulocyte lysate system (Promega, Madison, WI) at 30 °C for 90 min using the manufacturer's protocol. E. coli lysates containing expressed DBDs and rabbit reticulocyte lysates containing in vitro transcribed/translated full-length receptors were analyzed for binding to32P-labeled double-stranded oligonucleotides by EMSA as described previously (12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The reticulocyte lysates containing PPAR and RXR proteins were pre-mixed in 10 mm Tris, pH 8.0, buffer containing 150 mm KCl, 6% glycerol, 0.05% Nonidet P-40, 1 mm dithiothreitol, 1 μm phenylmethylsulfonyl fluoride, 1 μm 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 100 ng/μl poly[d(I-C)] and 40 ng/μl sonicated sperm DNA (Promega, Madison, WI). For the reticulocyte lysates containing Rev-ErbA or RORα1, the incubation buffer contained 10 mm Tris, pH 8.0, 50 mm KCl, 6% glycerol, 0.05% Nonidet P-40, 1 μm phenylmethylsulfonyl fluoride, 1 μm 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 mm dithiothreitol, and 20 ng/μl poly[d(I-C)]. When supershift assays were performed, 1 μl of anti-PPARα antibody or anti-Rev-ErbA monoclonal antibody (clone 17A9) was added to the reaction. After a 15-min preincubation on ice, 10 fmol of 32P-labeled double-stranded oligonucleotides were added and incubated at room temperature for another 20 min prior to electrophoresis through a 4% polyacrylamide (37.5:1), 0.5× TBE, 1.25% glycerol gel at 130 V for 100 min at room temperature. The gel was analyzed using a Molecular Dynamics PhosphorImager, model SI (Sunnyvale, CA). COS-1, RK-13, and JEG-3 cell lines were obtained from the American Type Culture Collection. COS-1 cells were maintained in Dulbecco's modified Eagle's medium containing high glucose (4 g/liter) (BioWhittaker, Walkersville, MD). RK-13 cells were cultured in minimum essential medium with Earle's balanced salts. JEG-3 cells were grown in Dulbecco's modified Eagle's medium with 1 g/liter glucose (Sigma). For each cell line, the medium was supplemented with 10% fetal calf serum (Summit, Ft. Collins, CO). The luciferase reporter plasmid pLuc-4A6–880 as well as the expression constructs for pCMV-PPARα (1Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar), pCMV-PPARα-G (1Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar), pCDM-Rev-ErbA(15), pCMX-RORα1 (19Forman B.M. Chen J. Blumberg B. Kliewer S.A. Henshaw R. Ong E.S. Evans R.M. Mol. Endocrinol. 1994; 8: 1253-1261Crossref PubMed Scopus (183) Google Scholar), pSVβGal (Promega, Madison, WI), and pCMVβGal (CLONTECH, Palo Alto, CA) have been previously described. The expression vector for PPARα-CDEF was constructed by inserting the PPARα-CDEF region into pCMV5. The luciferase reporter plasmids pLuc-TK-Z and pLuc-TK-RevDR2 were generated by inserting the CYP4A6-Z PPRE (1Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar) or the RevDR2 element (27Harding H.P. Lazar M.A. Mol. Cell. Biol. 1995; 15: 4791-4802Crossref PubMed Scopus (172) Google Scholar, 28Harding H.P. Atkins G.B. Jaffe A.B. Seo W.J. Lazar M.A. Mol. Endocrinol. 1997; 11: 1737-1746PubMed Google Scholar) into the luciferase reporter vector harboring the thymidine kinase promoter from herpes simplex virus as described (29Hsu M. Palmer C.N.A. Griffin K.J. Johnson E.F. Mol. Pharmacol. 1995; 48: 559-567PubMed Google Scholar). All reporter and expression constructs were introduced into cultured cells by a modified calcium phosphate coprecipitation procedure (10Palmer C.N.A. Hsu M.-H. Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1994; 269: 18083-18089Abstract Full Text PDF PubMed Google Scholar). After a 16-h exposure to the DNA-containing culture medium, the cells were washed twice with medium without serum and then placed in medium containing either WY-14,643 (50 μm) or the equivalent volume of solvent (Me2SO, 0.25% v/v final concentration). This medium was replaced after 24 h with fresh medium, and after an additional 24 h, the cells were harvested and washed with Dulbecco's phosphate-buffered saline without calcium and magnesium (Irvine Scientific, Santa Ana, CA). Cells were then lysed by suspension in 0.1m potassium phosphate buffer, pH 7.8, containing 1 mm dithiothreitol and 0.05% Triton X-100 followed by 3 cycles of freezing and thawing. Insoluble material was removed by centrifugation, and luciferase activity was determined using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, Ann Arbor, MI). β-Galactosidase activities were determined using the Galacto-Light Kit (Tropix, Bedford, MA). The luciferase activity obtained for individual cultures was expressed relative to the β-galactosidase activity obtained for the same preparation of lysate. Previous results from this laboratory identified a 4-base pair conserved sequence extending 5′ of the DR1 motif in PPREs (Table I) that is required for PPARα/RXR heterodimer binding to the CYP4A6-Z element (12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). The striking similarity between the 5′-extended site recognized by PPAR and the binding sites for monomers of the orphan nuclear receptors Rev-ErbA and ROR (Table I) suggested that receptors such as RORα and Rev-ErbA might bind to natural PPREs leading to cross-talk and/or competitive inhibition of signal transduction. To examine this possibility, the cDNAs for RXR, PPARα, PPARγ, Rev-ErbA, and RORα1 werein vitro transcribed/translated and examined in EMSA experiments (Fig. 1, A–D). Strong binding is evident for each receptor with the DR1P oligonucleotide that conforms to the consensus PPRE (Table I). As expected, Rev-ErbA and RORα1 also bind to the SHSP oligonucleotide that corresponds closely to the single 5′ consensus binding sites recognized by monomers of these receptors (Table I). PPARα or PPARγ heterodimers with RXR did not exhibit detectable binding to the SHSP oligonucleotide. This result, together with the inability of the full-length mPPARα alone to bind PPREs, suggests that PPARα, either alone or as a heterodimer with RXR, would not impact monomeric receptor regulatory pathways mediated through a single binding site for Rev-ErbA and RORα.Table IOligonucleotide probes used in EMSA and reported consensus recognition sequences for PPAR/RXR, Rev-ErbA, and RORαOligonucleotide probes1-aThe nucleotide sequences of the Z element found in theCYP4A6 gene (1), a shorter version of the Z element (CYP4A6-Zs) (12), the A element in the rat ACO gene (4), and ARE7, which is one of the PPREs in the adipocyte fatty acid-binding protein (aP2) gene (51), are aligned above with DR1P, which contains two consensus core sites, and SHSP, which contains a single consensus core site. Both DR1P and SHSP contain a consensus 5′-extended binding site found for PPREs. The Rev-DR2 element contains two consensus core sites separated by two nucleotides and a 5′-extended binding site (27, 28). The HD-DR2B element, in which mutations were introduced into the rat bifunctional PPRE to disrupt the DR1 motifs but preserve the DR2 motif (31), are aligned with RevDR2 oligonucleotides. Mutations (DR1M and SHSM) introduced to disrupt the 5′-extended binding site of DR1P and SHSP are indicated in lowercase bold letters (10, 12). SHSPCAAAACT AGGTCA A AGG SHSMgcgcgCc AGGTCA A AGG DR1PCAAAACT AGGTCA A AGGTCA GGG DR1MgcgcgCc AGGTCA A AGGTCA GGG CYP4A6-ZCGCAAACACTGAACT AGGGCA A AGTTGA GGGCAG CYP4A6-ZsCACTGAACT AGGGCA A AGTTGA GGGCAGTG ACO-AAGGGGACC AGGACA A AGGTCA CGTTCGGGA ARE7CTTCTTACT GGATCA G AGTTCA CAGATC RevDR2TCCAACT AGGTCA CT AGGTCA AAG HDDR2BCCAGATT AGTTCA AT AGGTCA AAGConsensus recognition sites1-bThe 5′-extended binding site (underlined) of the PPRE consensus sequence (13) is highly similar to the consensus binding site determined for monomers of Rev-ErbA (15) and ROR (18). The core sites in the consensus sequences are double underlined. PPAR/RXRNNNAACT AGGNCA A AGGTCA NGN T G T T Rev-ErbA AAANT AGGTCA T T G RORα TATCA AGGTCA ATA T G1-a The nucleotide sequences of the Z element found in theCYP4A6 gene (1Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1992; 267: 19051-19053Abstract Full Text PDF PubMed Google Scholar), a shorter version of the Z element (CYP4A6-Zs) (12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), the A element in the rat ACO gene (4Osumi T. Wen J.-K. Hashimoto T. Biochem. Biophys. Res. Commun. 1991; 175: 866-871Crossref PubMed Scopus (184) Google Scholar), and ARE7, which is one of the PPREs in the adipocyte fatty acid-binding protein (aP2) gene (51Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2000) Google Scholar), are aligned above with DR1P, which contains two consensus core sites, and SHSP, which contains a single consensus core site. Both DR1P and SHSP contain a consensus 5′-extended binding site found for PPREs. The Rev-DR2 element contains two consensus core sites separated by two nucleotides and a 5′-extended binding site (27Harding H.P. Lazar M.A. Mol. Cell. Biol. 1995; 15: 4791-4802Crossref PubMed Scopus (172) Google Scholar, 28Harding H.P. Atkins G.B. Jaffe A.B. Seo W.J. Lazar M.A. Mol. Endocrinol. 1997; 11: 1737-1746PubMed Google Scholar). The HD-DR2B element, in which mutations were introduced into the rat bifunctional PPRE to disrupt the DR1 motifs but preserve the DR2 motif (31Bardot O. Clemencet M.C. Passilly P. Latruffe N. FEBS Lett. 1995; 360: 183-186Crossref PubMed Scopus (16) Google Scholar), are aligned with RevDR2 oligonucleotides. Mutations (DR1M and SHSM) introduced to disrupt the 5′-extended binding site of DR1P and SHSP are indicated in lowercase bold letters (10Palmer C.N.A. Hsu M.-H. Muerhoff A.S. Griffin K.J. Johnson E.F. J. Biol. Chem. 1994; 269: 18083-18089Abstract Full Text PDF PubMed Google Scholar, 12Palmer C.N.A. Hsu M. Griffin K.J. Johnson E.F. J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar).1-b The 5′-extended binding site (underlined) of the PPRE consensus sequence (13Johnson E.F. Palmer C.N. Griffin K.J. Hsu M.H. FASEB J. 1996; 10: 1241-1248Crossref PubMed Scopus (169) Google Scholar) is highly similar to the consensus binding site determined for monomers of Rev-ErbA (15Harding H.P. Lazar M.A. Mol. Cell. Biol. 1993; 13: 3113-3121Crossref PubMed Google Scholar) and ROR (18Giguère V. Tini M. Flock G. Ong E. Evans R.M. Otulakowski G. Genes Dev. 1994; 8: 538-553Crossref PubMed Scopus (451) Google Scholar). The core sites in the consensus sequences are double underlined. Open table in a new tab The binding of each of the receptors to the CYP4A6-Zs, ACO-A, and ARE7 PPREs (shown in Table I) was characterized relative to the extent of binding observed for each receptor with the DR1P oligonucleotide. The values observed for the other oligonucleotides are expressed as a percentage relative to the binding obtained for that receptor with DR1P, Fig. 1. PPAR/RXR heterodimers display highly similar binding intensities for the naturally occurring PPREs, although the ARE7 element displays preferential complex formation with PPARγ relative to PPARα. In contrast, these elements are bound relatively weakly by Rev-ErbA and RORα1 compared with their binding to DR" @default.
• W1972978588 created "2016-06-24" @default.
• W1972978588 creator A5012375830 @default.
• W1972978588 creator A5024733044 @default.
• W1972978588 creator A5063025028 @default.
• W1972978588 creator A5069441634 @default.
• W1972978588 creator A5090868883 @default.
• W1972978588 date "1998-10-01" @default.
• W1972978588 modified "2023-10-18" @default.
• W1972978588 title "A Carboxyl-terminal Extension of the Zinc Finger Domain Contributes to the Specificity and Polarity of Peroxisome Proliferator-activated Receptor DNA Binding" @default.
• W1972978588 cites W1485053222 @default.
• W1972978588 cites W1526348057 @default.
• W1972978588 cites W1535106643 @default.
• W1972978588 cites W1542767024 @default.
• W1972978588 cites W1552728236 @default.
• W1972978588 cites W1566896910 @default.
• W1972978588 cites W1607714362 @default.
• W1972978588 cites W1766657157 @default.
• W1972978588 cites W1854481398 @default.
• W1972978588 cites W1906247903 @default.
• W1972978588 cites W1979761686 @default.
• W1972978588 cites W1981101682 @default.
• W1972978588 cites W1981946836 @default.
• W1972978588 cites W1985247182 @default.
• W1972978588 cites W1985865142 @default.
• W1972978588 cites W1987783037 @default.
• W1972978588 cites W1990353159 @default.
• W1972978588 cites W2002519246 @default.
• W1972978588 cites W2030588343 @default.
• W1972978588 cites W2034607609 @default.
• W1972978588 cites W2041781038 @default.
• W1972978588 cites W2042901562 @default.
• W1972978588 cites W2048501185 @default.
• W1972978588 cites W2065485581 @default.
• W1972978588 cites W2075245841 @default.
• W1972978588 cites W2079071895 @default.
• W1972978588 cites W2081070477 @default.
• W1972978588 cites W2082288944 @default.
• W1972978588 cites W2083436346 @default.
• W1972978588 cites W2093197345 @default.
• W1972978588 cites W2093336561 @default.
• W1972978588 cites W2107752044 @default.
• W1972978588 cites W2123444655 @default.
• W1972978588 cites W2127550817 @default.
• W1972978588 cites W2128825094 @default.
• W1972978588 cites W2128840922 @default.
• W1972978588 cites W2142073712 @default.
• W1972978588 cites W2150946709 @default.
• W1972978588 cites W2164015929 @default.
• W1972978588 cites W2292736786 @default.
• W1972978588 cites W98247596 @default.
• W1972978588 doi "https://doi.org/10.1074/jbc.273.43.27988" @default.
• W1972978588 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9774413" @default.
• W1972978588 hasPublicationYear "1998" @default.
• W1972978588 type Work @default.
• W1972978588 sameAs 1972978588 @default.
• W1972978588 citedByCount "98" @default.
• W1972978588 countsByYear W19729785882012 @default.
• W1972978588 countsByYear W19729785882014 @default.
• W1972978588 countsByYear W19729785882016 @default.
• W1972978588 countsByYear W19729785882017 @default.
• W1972978588 countsByYear W19729785882018 @default.
• W1972978588 countsByYear W19729785882019 @default.
• W1972978588 countsByYear W19729785882020 @default.
• W1972978588 countsByYear W19729785882022 @default.
• W1972978588 countsByYear W19729785882023 @default.
• W1972978588 crossrefType "journal-article" @default.
• W1972978588 hasAuthorship W1972978588A5012375830 @default.
• W1972978588 hasAuthorship W1972978588A5024733044 @default.
• W1972978588 hasAuthorship W1972978588A5063025028 @default.
• W1972978588 hasAuthorship W1972978588A5069441634 @default.
• W1972978588 hasAuthorship W1972978588A5090868883 @default.
• W1972978588 hasBestOaLocation W19729785881 @default.
• W1972978588 hasConcept C104317684 @default.
• W1972978588 hasConcept C12554922 @default.
• W1972978588 hasConcept C127078168 @default.
• W1972978588 hasConcept C1491633281 @default.
• W1972978588 hasConcept C154945302 @default.
• W1972978588 hasConcept C170493617 @default.
• W1972978588 hasConcept C185592680 @default.
• W1972978588 hasConcept C186055190 @default.
• W1972978588 hasConcept C187345961 @default.
• W1972978588 hasConcept C24284526 @default.
• W1972978588 hasConcept C2777361361 @default.
• W1972978588 hasConcept C2779664074 @default.
• W1972978588 hasConcept C3019039443 @default.
• W1972978588 hasConcept C41008148 @default.
• W1972978588 hasConcept C5432534 @default.
• W1972978588 hasConcept C552990157 @default.
• W1972978588 hasConcept C55493867 @default.
• W1972978588 hasConcept C76155785 @default.
• W1972978588 hasConcept C86339819 @default.
• W1972978588 hasConcept C86803240 @default.
• W1972978588 hasConcept C94966510 @default.
• W1972978588 hasConcept C95444343 @default.
• W1972978588 hasConceptScore W1972978588C104317684 @default.
• W1972978588 hasConceptScore W1972978588C12554922 @default.
• W1972978588 hasConceptScore W1972978588C127078168 @default.

• next