Cholesterol Biosynthesis from Lanosterol
2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês
10.1074/jbc.m101661200
ISSN1083-351X
AutoresJai-Hyun Kim, Joon No Lee, Young‐Ki Paik,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThe 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in the pathway of cholesterol biosynthesis. We have previously reported that sterol depletion in vivo caused a significant induction of both liver mRNA and enzyme activity of Dhcr7 (Bae, S.-H., Lee, J. N., Fitzky, B. U., Seong, J., and Paik, Y.-K. (1999) J. Biol. Chem. 274, 14624–14631). In this paper, we also observed liver cell-specific sterol-mediated Dhcr7 gene induction in vitro by sterol depletion in rat hepatoma cells, suggesting the presence of sterol-mediated regulatory elements in the Dhcr7 gene. To understand the mechanisms responsible for regulating Dhcr7 expression, we have isolated the 5′-flanking region of the gene encoding rat Dhcr7 and have characterized the potential regulatory elements of the gene that are responsible for sterol-mediated regulation. The Dhcr7 promoter contains binding sites for Sp1 (at −177, −172, −125, and −20), NF-Y (at −88 and −51), and SREBP-1 or ADD1 (at −33). Deletion analysis of the Dhcr7 gene promoter (−1053/+31), employing a nested series of Dhcr7-luciferase constructs, demonstrated that the −179 upstream region of the gene is necessary and sufficient for optimal efficient sterol-regulated transcription. DNase I footprinting and electrophoretic mobility shift assay showed that the SRE1/E box (−33/−22) involved in sterol response of many sterol-related enzyme genes was protected specifically by the overexpressed recombinant ADD1. Mutational analysis for the functional relationship between the identified cis-elements in this region indicate that one of the binding sites for Sp1 (GC box at −125) and NF-Y (CCAAT box at −88) plays a cooperative role in the sterol-mediated activation, in which the latter site also acts as a co-regulator for SREBP-activated Dhcr7 promoter activity. We believe that Dhcr7 is the first enzyme characterized with a sterol-regulatory function in the post-lanosterol pathway. This may be important for understanding the coordinated control of cholesterol biosynthesis as well as the molecular mechanism of Smith-Lemli-Opitz syndrome-related protein in mammals. The 7-dehydrocholesterol reductase (Dhcr7) is the terminal enzyme in the pathway of cholesterol biosynthesis. We have previously reported that sterol depletion in vivo caused a significant induction of both liver mRNA and enzyme activity of Dhcr7 (Bae, S.-H., Lee, J. N., Fitzky, B. U., Seong, J., and Paik, Y.-K. (1999) J. Biol. Chem. 274, 14624–14631). In this paper, we also observed liver cell-specific sterol-mediated Dhcr7 gene induction in vitro by sterol depletion in rat hepatoma cells, suggesting the presence of sterol-mediated regulatory elements in the Dhcr7 gene. To understand the mechanisms responsible for regulating Dhcr7 expression, we have isolated the 5′-flanking region of the gene encoding rat Dhcr7 and have characterized the potential regulatory elements of the gene that are responsible for sterol-mediated regulation. The Dhcr7 promoter contains binding sites for Sp1 (at −177, −172, −125, and −20), NF-Y (at −88 and −51), and SREBP-1 or ADD1 (at −33). Deletion analysis of the Dhcr7 gene promoter (−1053/+31), employing a nested series of Dhcr7-luciferase constructs, demonstrated that the −179 upstream region of the gene is necessary and sufficient for optimal efficient sterol-regulated transcription. DNase I footprinting and electrophoretic mobility shift assay showed that the SRE1/E box (−33/−22) involved in sterol response of many sterol-related enzyme genes was protected specifically by the overexpressed recombinant ADD1. Mutational analysis for the functional relationship between the identified cis-elements in this region indicate that one of the binding sites for Sp1 (GC box at −125) and NF-Y (CCAAT box at −88) plays a cooperative role in the sterol-mediated activation, in which the latter site also acts as a co-regulator for SREBP-activated Dhcr7 promoter activity. We believe that Dhcr7 is the first enzyme characterized with a sterol-regulatory function in the post-lanosterol pathway. This may be important for understanding the coordinated control of cholesterol biosynthesis as well as the molecular mechanism of Smith-Lemli-Opitz syndrome-related protein in mammals. 7-dehydrocholesterol reductase adipocyte determination and differentiation-dependent factor 1 lipoprotein-deficient serum fetal bovine serum sterol regulatory element SRE-binding protein nuclear factor Y electrophoretic mobility shift assay sonic hedgehog Smith-Lemi-Opitz syndrome polymerase chain reaction base pair(s) hydroxymethyl glutaryl The 7-dehydrocholesterol reductase (Dhcr7,1 EC1.3.1.21) catalyzes the reduction of the Δ7-double bond of sterol intermediates, which is the terminal reaction, in the pathway of cholesterol biosynthesis from lanosterol (1Gibbon G.F. Goad L.J. Goodwin T.W. Nes W.R. J. Biol. Chem. 1971; 246: 3967-3976Abstract Full Text PDF PubMed Google Scholar, 2Lee J.N. Paik Y.-K. J. Biochem. Mol. Biol. 1997; 30: 370-377Google Scholar, 3Bae S.-H. Paik Y.-K. Biochem. J. 1997; 326: 609-616Crossref PubMed Scopus (74) Google Scholar). Studies of this enzyme have recently drawn our attention due to its implication in both cholesterol metabolism and mammalian developmental biology (4Bae S.-H. Lee J.N. Fitzky B.U. Seong J. Paik Y.-K. J. Biol. Chem. 1999; 274: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 5Porter J.A. Young K.E. Beachy P.A. Science. 1996; 11: 255-259Crossref Scopus (1126) Google Scholar, 6Cooper M.K. Porter J.A. Young K.E. Beachy P.A. Science. 1998; 5: 1603-1607Crossref Scopus (785) Google Scholar). In particular, mutations of Dhcr7 has been involved in genetic disease such as Smith-Lemi-Opitz syndrome (SLOS) (7Fitzky B.U. Witsch-Baumgartner M. Erdel M. Lee J.N. Paik Y.-K. Glossmann H. Utermann G. Moebius F.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8181-8186Crossref PubMed Scopus (334) Google Scholar, 8Waterham H.R. Wijburg F.A. Hennekam R.C. Vreken P. Poll-The B.T. Dorland L. Duran M. Jira P.E. 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This connection between cholesterol and the developmental program in mammals was established based on the critical role of cholesterol in autoprocessing of sonic hedgehog (shh) protein (5Porter J.A. Young K.E. Beachy P.A. Science. 1996; 11: 255-259Crossref Scopus (1126) Google Scholar, 6Cooper M.K. Porter J.A. Young K.E. Beachy P.A. Science. 1998; 5: 1603-1607Crossref Scopus (785) Google Scholar, 19Porter J.A. Ekker S.C. Park W.J. von Kessler D.P. Young K.E. Chen C.H. Ma Y. Woods A.S. Cotter R.J. Koonin E.V. Beachy P.A. Cell. 1996; 12: 21-34Abstract Full Text Full Text PDF Scopus (439) Google Scholar), a morphogen, which binds to, patched protein in developing the central nervous system and limbs. For example, the mouse mutated in shh showed SLOS-like phenotype (20Chiang C. Litingtung Y. Lee E. Young K.E. Corden J.L. Westphal H. Beachy P.A. Nature. 1996; 383: 407-413Crossref PubMed Scopus (2599) Google Scholar,21Lanoue L. Dehart D.B. Hinsdal M.E. Maeda N. Tint G.S. Sulik K.K. Am. J. Med. Genet. 1997; 73: 24-31Crossref PubMed Scopus (105) Google Scholar). Recently, cDNAs encoding Dhcr7 from mouse (22Moebius F.F. Fitzky B.U. Lee J.N. Paik Y.-K. Glossmann H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1899-1902Crossref PubMed Scopus (194) Google Scholar), rat (4Bae S.-H. Lee J.N. Fitzky B.U. Seong J. Paik Y.-K. J. Biol. Chem. 1999; 274: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), human (7Fitzky B.U. Witsch-Baumgartner M. Erdel M. Lee J.N. Paik Y.-K. Glossmann H. Utermann G. Moebius F.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8181-8186Crossref PubMed Scopus (334) Google Scholar), and Arabidopsis thaliana (23Lecain E. Chenivesse X. Spagnoli R. Pompon D. J. Biol. Chem. 1996; 271: 10866-10873Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) have been isolated. Dhcr7 mRNA was expressed mainly in liver and its regulation appeared to be controlled by tissue sterol contents as well as some cholesterogenic inhibitors (2Lee J.N. Paik Y.-K. J. Biochem. Mol. Biol. 1997; 30: 370-377Google Scholar, 4Bae S.-H. Lee J.N. Fitzky B.U. Seong J. Paik Y.-K. J. Biol. Chem. 1999; 274: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). However, molecular cloning and the nature of sterol-mediated transcriptional control of the Dhcr7 gene in any species have not been reported previously. Furthermore, the relative importance of multiple motifs at the 5′-flanking region in transcriptional regulation of the Dhcr7 gene is also not known. In order to characterize the 5′-flanking region and the response of multiple regulatory elements to sterols, we isolated the 5′-flanking region of the rat Dhcr7 gene and the complete genomic sequence (GenBankTM accession numberAF279892). 2J. N. Lee, S.-H. Bae, and Y.-K. Paik, submitted for publication. In this paper, we describe the promoter activity and the sterol-mediated regulation of this gene by various mutation analyses, with emphasis on the proximal promoter (−179 to +1), which controls transcriptional induction of the gene in response to starvation of cellular sterols. A rat Promoter FinderTM DNA Walking Kit was purchased from CLONTECH (Palo Alto, CA). The following materials were purchased from manufacturers, as indicated. Hyper x-ray film, [α-32P]dCTP (3000 Ci/mmol), [γ-32P]ATP (3000 Ci/mmol), poly(dI-dC) ·poly(dI-dC), Amersham Pharmacia Biotech; DNA sequencing reagents, PerkinElmer Life Sciences; restriction endonucleases, T4-DNA ligase, T4-polynucleotide kinase, and Klenow fragment of DNA polymerase I, New England Biolabs; RNasin ribonuclease inhibitor and avian myeloblastosis virus-reverse transcriptase,Promega; lipoprotein-deficient serum (LPDS), Sigma; Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), gentamycin, and l-glutamine, Life Technologies; sterols including cholesterol and various oxysterols including 25-, 20-, 19-, 4-, 7α-, 7β-hydroxycholesterols, 6-keto, and 7-keto-sterols, Steraloid. The sources of the following drugs or agents are also indicated. AY-9944, Dr. D. Dvornik at Wyth-Ayerst, Princeton; and LovastatinTM, Dr. Y-K. Sim at Choongwae Pharmaceutical Co., Korea. RNA was isolated by phenol-chloroform extraction using TRI reagent Kit (Molecular Research Center Inc.). Total RNA (20 μg) was electrophoresed in a 1% formaldehyde-agarose gel and vacuum-transferred to Hybond N+ membrane (Amersham Pharmacia Biotech) with 20 × SSC (150 mm NaCl, 15 mm sodium citrate, pH 7.0). After prehybridization of the membranes at 42 °C for 5 h in 5 × SSC, 10 × Denhardt's solution, 100 μg/ml denatured herring sperm DNA, the membranes were hybridized to32P-labeled rat Dhcr7 cDNA probe for 18 h. Then the membranes were washed with 0.1% SSC, 0.1% SDS at 65 °C. Hybridization signals were visualized by autoradiography using Hyper x-ray film with an intensifying screen at −70 °C. The signals that appeared in the membranes were quantitatively analyzed with a BAS 2500 system (Fuji Photo Film Co.) and normalized to the quantity of β-actin mRNA expression. At the initial stage of this work, rat Dhcr7 promoter was amplified from DNA provided in the rat Promoter FinderTMDNA walking kit (CLONTECH) according to the manufacturer's instructions. The primer r7R-9 (+49 ∼ +28) 5′-GCGTGCGGGATCCGGGCGGTTG-3′, corresponding to the cDNA for rat Dhcr7, and anchor primer AP1 of the promoter finder kit were used in the primary PCR. The primary PCR products were diluted and used for the secondary PCR with nested primer r7R-48 (+31 ∼ +7) 5′-GTTGATTCCAAGCTCCAGCAGCGCC-3′ in the 5′ region of Dhcr7 and anchor primer AP2. Secondary PCR products were cloned into pT7Blue(R)-T vector and sequenced with an ABI automated DNA sequencing system. The cloned Dhcr7 promoter region was also confirmed by data obtained from genomic clone sequencing. To analyze Dhcr7 promoter, the 5′-flanking region of the gene was ligated into the luciferase reporter vector as follows. The parent plasmid, p7R1084, was constructed by Klenow enzyme treatment of a DNA fragment (NdeI/EcoRI) spanning −1053 to +31 nucleotides, followed by insertion into the SmaI site of the luciferase vector pGL3-Basic (Promega). Plasmid p7R1084 (−1053/+31) was digested with NheI and PvuII to remove the 5′-fragment (NheI/PvuII) of the Dhcr7 insert, and religated after treatment with T4 DNA polymerase to produce p7R537 (−506/+31). In the same manner, p7R1084 was digested with PstI,BstXI, and ApaI, and religated to produce p7R318 (−287/+31), p7R210 (−179/+31), and p7R148 (−117/+31), respectively. Plasmid p7R164 (−133/+31) and p7R72 (−41/+31) were generated by cloning PCR products obtained by using appropriate synthetic oligonucleotides into a SmaI-digested pGL3-Basic vector. The truncated chimeric plasmid p7R287-117 was prepared by inserting a 171-bp PstI/ApaI fragement of p7R1084 into aSmaI-digested pGL3-Basic vector. All luciferase reporter genes except p7R287-117 had the same 3′ end in a SmaI site of pGL3-Basic and varying 5′ ends. All plasmids were verified by DNA sequencing. The various mutant constructs were generated by PCR using the wild type Dhcr7 promoter DNA as a template with Pfu polymerase and mutagenic oligonucleotides designed to introduce each specific multibase point mutation. The mutated Dhcr7 promoter constructs were designated as M1 to M14. The sequences of the oligonucleotides used for the mutation of the different parts of the Dhcr7 promoter are shown below, with the mutated sequences in bold: SRE/m (−46/−18); 5′-GTAGTCTGTGACCAGTAATCACCTGGGGG-3′, E box/m (−46/−18); 5′-GTAGTCTGTGACCTCACGTCACTACGGGG-3′, SRE·E box/m (−46/−18); 5′-GTAGTCTGTGACCAGTAATCACTACGGGG-3′, ADD/m (−46/−18); 5′-GTAGTCTGTTCCCAGTAATTCGTACGGGG-3′, NFY/m (−64/−33); 5′-GAAATCCACTTCGGAGATCTAGTCTGTGACCT-3′, GC1·2/m (−191/−156); 5′-GTCCCCAGGGGCGTGGGATGGTTGGGGCTGGCCCTG-3′, CCAAT/m (−100/−69); 5′-GCTCACTGCCTTCAGATCTCACAGGGCGCGG-3′, GC3/m (−135/−109); 5′-GCCGCCGACTGTATTGCCCGGTGGGCT-3′, GC4/m (−29/−3); 5′-GTCACCTGGGTTGAGTGCTTCAGGCAG-3′, SRE/m1 (−46/−18); 5′-GTAGTCTGTGACCTCACGTTCGCTGGGGG-3′, SRE/m2 (−46/−18); 5′-GTAGTCTGTGACCAGTAATTCGCTGGGGG-3′. The PCR reaction products were digested with restriction enzymes and inserted into the luciferase vector pGL3-Basic. The DNA sequence of each mutant clone was confirmed by sequencing. HepG2, CHO-K1, and H4IIE cells were grown in RPMI medium 1640 (Life Technologies, Inc.) supplemented with 5% (v/v) FBS, 1 mm glutamine, and 10 μg/ml gentamycin in a 5% CO2 incubator at 37 °C. Cells plated onto 6-well plates were grown to 50–70% confluence before transfection. Two μg of test constructs were co-transfected into HepG2 cells with 0.2 μg of Renilla luciferase control vector, pRL-SV40 (Promega). Each plasmid containing Dhcr7 promoter-luciferase fusion gene and 2.0 μl of Lipofectin reagent (Life Technologies) was diluted into 80 μl of Opti-MEM I (Life Technologies). Plasmid DNA and Lipofectin reagent were then combined and added to each plate according to the Lipofectin manufacturer recommendations. The cells were transfected on serum-free media for 18 h and then were switched to a medium with 5% LPDS plus 10 μm Lovastatin (designated as sterol starvation condition) or 1.5 μg/ml 25-hydroxycholesterol (designated as sterol rich condition). After incubation for 48 h, with the appropriate medium, cells were harvested, and extracts were assayed for luciferase activity (see below for details). Following transfection and drug treatment, the cells were washed with phosphate-buffered saline and lysed in 0.5 ml of 1 × passive lysis buffer (Promega). Cell extracts were assayed forFirefly and Renilla luciferase activities using the Dual-luciferase Reporter Assay SystemTM according to the manufacturer's recommendations (Promega). Amounts of lysates employed for the Firefly luciferase activity assays of test constructs were normalized to the Renilla luciferase activities. 32P-Labeled DNA probes (−258/+41) were prepared from Dhcr7 gene and incubated with 0.2 or 2 μg of purified ADD1 protein in Buffer A (10 mm Tris (pH 7.6), 100 mm KCl, 10 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, and 4 μg of poly(dI-dC)·poly(dI-dC)) at room temperature for 30 min in a total volume of 20 μl. At the end of the reaction, 80 μl of a solution containing 3 mm CaCl2 and 6 mmMgCl2 was added the mixture. After digestion with DNase I, the DNA samples were extracted by a phenol/chloroform solution and electrophoresed on a 6% sequencing gel with a same end-labeled sequencing ladder. Recombinant ADD1 (amino acid residues 284 to 403 including the bHLH-LZ domain) was overexpressed in Escherichia coli and purified from the culture extracts as described previously (24Kim J.B. Spotts G.D. Halvorsen Y.D. Shih H.M. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (298) Google Scholar). Rat hepatoma H4IIE cell nuclear extracts were prepared according to the method of Dignamet al. (25Dignam J.D. Levovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (10033) Google Scholar). Nuclear extracts were quantified by the Bradford assay (Bio-Rad) and stored at −70 °C. For EMSA, annealed oligonucleotide probes were 5′-end labeled using [γ-32P]ATP by T4-ploynucleotide kinase. Binding reactions were carried out in Buffer A in the presence of 1 mg/ml bovine serum albumin. Approximately 0.1 pmol of the labeled probe was mixed with 2–6 μg of nuclear extract or 10–100 ng of recombinant ADD1 in 20 μl, then incubated for 30 min at room temperature. EMSA was performed on a 5% polyacrylamide gel with 0.5 × TBE buffer (40 mm Tris borate, 1 mm EDTA) and processed for autoradiography. For binding competition assays, 100-fold molar excess of unlabeled oligonucleotides were added before the addition of labeled probe. The DNA sequence of oligonucleotides used were as follows: ADD (ADD1-binding site of Dhcr7 promoter), 5′-GTAGTCTGTGACCTCACGTCACCTGGGGG-3′; GC3 (Sp1-binding site of Dhcr7 promoter), 5′-CGCCGACTGGCGGGCCCGGTGG-3′; CCAAT1 (NF-Y-binding site of Dhcr7 promoter), 5′-CTGCCTTCACCAATCACAGGGC-3′; GC-box (Sp1-binding site), 5′-ATTCGATCGGGGCGGGGCGAGC-3′; CCAAT box (NF-Y binding site), 5′-GTGATCAGCCAATCAGAGCGAG-3′; ABS (ADD1-binding site: E box), 5′-GATCCTGATCACGTGATCGAGGAG-3′; and SRE-1, 5′-GATCCTGATCACCCCACTGAGGAG-3′. We have previously reported (4Bae S.-H. Lee J.N. Fitzky B.U. Seong J. Paik Y.-K. J. Biol. Chem. 1999; 274: 14624-14631Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) that when rats were maintained on a sterol starvation condition by feeding CL-diet (5% (w/w) cholestyramine plus 0.1% (w/w) Lovastatin in chow) for 14 days, both hepatic mRNA expression and the specific enzyme activity of Dhcr7 were increasedin vivo about 3- and 5-fold, respectively. To examine if this type of sterol-mediated Dhcr7 induction can also be seen in cultured cells in vitro, Northern blot analysis was performed using Dhcr7 mRNA obtained from H4IIE cells and CHO-K1 cells that had been maintained in either FBS or LPDS medium. As shown in Fig. 1 A, mRNA from the Dhcr7 gene was specifically induced more than 3-fold in H4IIE cells (hepatic cells). However, this induction by sterol depletion was seen only slightly in CHO-K1 cells (non-hepatic cells). Dhcr7 mRNA was highly inducible in a dose-dependent manner when cells were grown under sterol starvation conditions, where cells were treated with the two cholesterogenic enzyme inhibitors, AY-9944 (Dhcr7 inhibitor) and Lovastatin (HMG-CoA reductase inhibitor) (Fig. 1, B and C). In contrast to this observation, the Dhcr7 gene was suppressed by treatment of cells with 25-hydroxycholesterol (Fig. 1 D). These results predicted sterol-mediated regulatory sequences in the Dhcr7 gene. To test this possibility, we isolated and analyzed the 5′-flanking region of this gene in detail. A 1.1-kilobase upstream fragment of the putative transcription initiation site from the rat genomic clone was isolated, sequenced, and analyzed to identify several cis-acting regulatory elements. The transcription initiation site was previously determined.2 The nucleotide sequence (1,053 bp), including exon 1, its 5′-flanking region, and part of intron 1, is shown (Fig. 2). The proximal region (−179 to +1) is 67% G + C and contains several well known transcription factor binding sites (Fig. 2). These are four GC boxes binding to Sp1 (−177/−172 (GC1), −172/−167 (GC2), −125/−121 (GC3), and −20/−15(GC4)), two CCAAT boxes binding to NF-Y (−88/−81 (CCAAT1) and −51/−45 (inverted, CCAAT2)) and an SRE1/E box binding to SREBP-1 or ADD1 (−33/−22). These regulatory sites are regarded as typical motifs seen in several genes involved in cholesterol homeostasis (26Bennett M.K. Ngo T.T. Athanikar J.N. Rosenfeld J.M. Osborne T.F. J. Biol. Chem. 1999; 274: 13025-13032Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 27Dooley K.A. Millinder S. Osborne T.F. J. Biol. Chem. 1998; 273: 1349-1356Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 28Ericsson J. Jackson S.M. Edwards P.A. J. Biol. Chem. 1996; 271: 24359-24364Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 29Guan G. Jiang G. Koch R.L. Shechter I. J. Biol. Chem. 1995; 270: 21958-21965Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Jackson S.M. Ericsson J. Mantovani R. Edwards P.A. J. Lipid Res. 1998; 39: 767-776Abstract Full Text Full Text PDF PubMed Google Scholar, 31Natarajan R. Ghosh S. Grogan W.M. Biochem. Biophys. Res. Commun. 1998; 243: 349-355Crossref PubMed Scopus (20) Google Scholar, 32Sanchez H.B. Yieh L. Osborne T.F. J. Biol. Chem. 1995; 270: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 33Spear D.H. Kutsunai S.Y. Correll C.C. Edwards P.A. J. Biol. Chem. 1992; 267: 14462-14469Abstract Full Text PDF PubMed Google Scholar, 34Xiong S. Chirala S.S. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3948-3953Crossref PubMed Scopus (72) Google Scholar). There was no TATA box within the promoter region of the Dhcr7, implying the presence of multiple transcription start sites as previously demonstrated.2 The transcription start site by the longest 5′-rapid amplification of cDNA ends product is located at an adenine (A) residue, which is 307 bp away from the ATG codon in Dhcr7 cDNA2 (Fig. 2). Therefore, Dhcr7 gene transcription appears to be driven by a TATA-less GC-rich promoter. This 5′-flanking region of the Dhcr7 promoter (−1053 to +1) also contains other well characterized regulatory elements that may control expression of this gene. These include two AP-1 (−454/−449 and −130/−125), four AP-4 (−510/−505, −290/−284, −167/−162, and −103/−98), NF-1 (−422/−417), NFAT (−555/−550), and SRY (−521/−516) binding sites. There are many IK2 and MZF-1-binding sites in the Dhcr7 promoter and part of intron 1. With regard to sterol-mediated regulation, we have focused on the proximal region (−179/+1) for further analysis on Dhcr7 gene transcription. To identify the transcriptional regulatory regions in rat Dhcr7 gene and to determine whether this region is regulated by exogenous sterols, a nested series of fragments containing various lengths of the 5′-flanking region, together with a part of the first exon (+31 out of 52 bp) were generated by either PCR or restriction endonuclease digestion. The parental plasmid used for this study was designated as p7R1084, spanning nucleotides −1053 to +31. Each deletion construct was prepared by inserting DNA fragments of various lengths into the luciferase gene and then analyzed for promoter activity using a transient assay system, as described under "Experimental Procedures." These deletion constructs were transfected into HepG2 cells and their luciferase activities measured following treatment with sterols (Fig.3). To normalize activity in different transfected cells, we co-transfected the cells with pRL-SV40, a non sterol-regulated Renilla luciferase expression vector. Results were expressed as "-fold" activation by taking the ratio of the luciferase activities after sterol starvation (−sterol) or sterol supplemented (+sterol) conditions. For the analysis of the regulation of the Dhcr7 promoter, HepG2 cells were chosen due to their relatively high transfection efficiency (>30-fold) as compared with that of H4IIE cells (data not shown). The promoter activity of the longest fragment, beginning at −1053 (p7R1084), was set at 100% for the results obtained from HepG2 cells in 5% LPDS (plus 10 μmLovastatin; sterol starvation condition). The basal promoter activity was set at the luciferase activity of p7R72 (−41/+31). As shown in Fig. 3, the promoter activity of the parent construct (p7R1084) was decreased more than 11-fold when sterol (1.5 μg/ml 25-hydroxycholesterol) was added into the LPDS media, confirming the presence of sterol-mediated regulatory elements such as the SRE1/E box located at −33/−22 in the 5′-flanking region (Fig. 2). Deletion of regions spanning nucleotides −1053 and −133 (p7R537, p7R318, p7R210, and p7R164) caused no significant change in either promoter activity or sterol-mediated activation of Dhcr7 promoter activity. However, deletion of nucleotides between −133 and −117 caused almost a 3.5-fold reduction in both the promoter activity relative to the −133 region and the sterol-mediated activation. That is, there was a change in the magnitude of sterol-mediated activation between these two constructs (−133/+31 (13.6-fold) versus −117/+31 (4.0-fold)). This result suggests the presence of positivecis-acting sequences in this region (−133 and −117), which seems sensitive to the absence of sterol. This is further confirmed by the fact that the deletion constructs, p7R148 (−117/+31) and p7R72 (−41/+31), almost abolished a sterol-mediated activation (∼4.0 to ∼2.9-fold). Therefore, it appears that the two tandem repeat GC boxes at −177 (GC1) and −172 (GC2) are less important than other proximal GC boxes (e.g. GC3) for Dhcr7 promoter activity in response to sterols. Addition of the fragment containing sterol-regulated regions which enhanced activity (−287/−117) of the reporter gene itself (pGL3-Basic vector; e.g. p7R287–117), exhibited much lower luciferase activity in the absence of sterol than that of basal promoter construct, suggesting the absence of promoter activity in this sequence. A similar pattern of expression was also observed for these constructs when they were transfected into either CHO-K1 cells or H4IIE cells (Fig. 4). Therefore, we have focused on nucleotides spanning −287 to +31 (p7R318 showed the highest activity on sterol depletion conditions) in the 5′-flanking region of the rat Dhcr7 gene for further analysis of sterol response.Figure 4Comparison of promoter activities of fusion genes containing varying lengths of the Dhcr7 promoter in HepG2, H4IIE, and CHO-K1. Cells were co-transfected with one of the chimeric genes p7R1084, p7R537, p7R318, p7R210, p7R164, p7R148, p7R72, or p7R287-117 and with a pRL-SV40 plasmid for an internal control. Cells were maintained in LPDS-containing media in the presence of 10 μm Lovastatin. Relative luciferase activities were determined as a ratio of luciferase activity of each sample to the activity of Renilla luciferase and were normalized to 100 for p7R1084 in all cells. The relative activity of p7R1084 in HepG2 was 1.4 and 3.8 times higher than in H4IIE and CHO-K1 cells. Values shown here are the mean from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine which oxysterol may be most effective in suppression of the Dhcr7 promoter, luciferase activities were measured for p7R318 (−287/+31) in the presence of different oxysterols. As shown in Fig.5, 25-hydroxycholesterol was found to be the strongest regulator among the seven oxysterols examined. However, the addition of Dhcr7 inhibitor (AY-9944, 2 μm) or HMG-CoA reductase inhibitor (lovastatin, 20 μm) to
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