Cloning and Characterization of the Rat Lysyl Oxidase Gene Promoter
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m610108200
ISSN1083-351X
AutoresSong Gao, Yinzhi Zhao, Lingfa Kong, Paul Toselli, Iih‐Nan Chou, Phillip J. Stone, Wande Li,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoLysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5′-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from –78 to –51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5′ Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region –709/–598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment –612/–580, which contains a nuclear factor I (NFI)-binding site at the region –594/–580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at –147/–133, were identified in the LO promoter region –804/–1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation. Lysyl oxidase (LO) stabilizes the extracellular matrix by cross-linking collagen and elastin. To assess the transcriptional regulation of LO, we cloned the 5′-flanking region with 3,979 bp of the rat LO gene. LO transcription started at multiple sites clustered at the region from –78 to –51 upstream of ATG. The downstream core promoter element functionally independent of the initiator predominantly activated the TATA-less LO gene. 5′ Deletion assays illustrated a sequence of 804 bp upstream of ATG sufficient for eliciting the maximal promoter activity and the region –709/–598 exhibiting strongly enhancing effects on the reporter gene expression in transiently transfected RFL6 cells. DNase I footprinting assays showed a protected pattern existing in the fragment –612/–580, which contains a nuclear factor I (NFI)-binding site at the region –594/–580 confirmed by electrophoretic mobility supershift assays. Mutations on this acting site decreased both NFI binding affinity in gel shift assays and stimulation of SV40 promoter activities in cells transfected with the NFI-binding site-SV40 promoter chimeric construct. Furthermore, at least two functional NFI-binding sites, including another one located at –147/–133, were identified in the LO promoter region –804/–1. Only NFI-A and NFI-B were expressed in rat lung fibroblasts, and their interaction with the LO gene was sensitively modulated by exogenous stimuli such as cigarette smoke condensate. In conclusion, the isolated rat LO gene promoter contains functionally independent initiator and downstream core promoter elements, and the conserved NFI-binding sites play a critical role in the LO gene activation. Lysyl oxidase (LO) 2The abbreviations used are:LOlysyl oxidaseDPEdownstream core promoter elementInrinitiatorNFInuclear factor IECMextracellular matrixbFGFbasic fibroblast growth factorGAPDHglyceraldehyde-3-phosphate dehydrogenaseRTreverse transcriptionEMSAelectrophoretic mobility shift assayM-MLVMoloney murine leukemia virusBSAbovine serum albuminRLM-RACERNA ligase-mediated rapid amplification of cDNA endsDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumChIPchromatin immunoprecipitationCSCcigarette smoke condensateFforwardRreverse. 2The abbreviations used are:LOlysyl oxidaseDPEdownstream core promoter elementInrinitiatorNFInuclear factor IECMextracellular matrixbFGFbasic fibroblast growth factorGAPDHglyceraldehyde-3-phosphate dehydrogenaseRTreverse transcriptionEMSAelectrophoretic mobility shift assayM-MLVMoloney murine leukemia virusBSAbovine serum albuminRLM-RACERNA ligase-mediated rapid amplification of cDNA endsDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumChIPchromatin immunoprecipitationCSCcigarette smoke condensateFforwardRreverse. (EC 1.4.3.13) is a copper-dependent enzyme secreted by fibrogenic cells such as fibroblasts (1Kagan H.M. Li W. J. Cell. Biochem. 2003; 88: 660-672Crossref PubMed Scopus (689) Google Scholar). This enzyme catalyzes the initiation of cross-linking of collagen and elastin, major structural components of the extracellular matrix (ECM), by oxidizing peptidyl lysine residues within these proteins to peptidyl α-aminoadipic-δ-semialdehyde, leading to the formation of condensation products stabilizing polymeric collagen or elastin as insoluble fibers. Thus, LO plays a central role in ECM morphogenesis and tissue repair (1Kagan H.M. Li W. J. Cell. Biochem. 2003; 88: 660-672Crossref PubMed Scopus (689) Google Scholar). lysyl oxidase downstream core promoter element initiator nuclear factor I extracellular matrix basic fibroblast growth factor glyceraldehyde-3-phosphate dehydrogenase reverse transcription electrophoretic mobility shift assay Moloney murine leukemia virus bovine serum albumin RNA ligase-mediated rapid amplification of cDNA ends Dulbecco's modified Eagle's medium fetal bovine serum chromatin immunoprecipitation cigarette smoke condensate forward reverse. lysyl oxidase downstream core promoter element initiator nuclear factor I extracellular matrix basic fibroblast growth factor glyceraldehyde-3-phosphate dehydrogenase reverse transcription electrophoretic mobility shift assay Moloney murine leukemia virus bovine serum albumin RNA ligase-mediated rapid amplification of cDNA ends Dulbecco's modified Eagle's medium fetal bovine serum chromatin immunoprecipitation cigarette smoke condensate forward reverse. In addition to the major function in stabilizing the ECM, LO also exhibits other biological activities. As reported, expression of transfected LO cDNA inhibited Ha-ras-induced cell transformation indicating an anti-tumorigenic effect of LO (2Kenyon K. Contente S. Trackman P.C. Tang J. Kagan H.M. Friedman R.M. Science. 1991; 253: 802Crossref PubMed Scopus (188) Google Scholar). LO can oxidize lysine residues in various globular proteins other than collagen and elastin (1Kagan H.M. Li W. J. Cell. Biochem. 2003; 88: 660-672Crossref PubMed Scopus (689) Google Scholar). Oxidation of basic fibroblast growth factor (bFGF) by LO blocks the proliferation of bFGF-stimulated cells and highly tumorigenic bFGF autocrine-transformed cells (3Li W. Nugent M.A. Zhao Y. Chau A.N. Li S.J. Chou I-N Liu G. Kagan H.M. J. Cell. Biochem. 2003; 88: 152-164Crossref PubMed Scopus (58) Google Scholar). Purified mature bovine LO displays chemotactic activity for monocytes and vascular smooth muscle cells (4Lazarus H.M. Cruikshank W.W. Narasimhan N. Kagan H.M. Center D.M. Matrix Biol. 1994; 14: 727-731Crossref Scopus (76) Google Scholar, 5Li W. Liu G. Chou I-N. Kagan H.M. J. Cell. Biochem. 2000; 78: 550-557Crossref PubMed Scopus (92) Google Scholar). LO and its oxidized substrates exist within the nuclei, potentially using histone H1 as a substrate and modulating the promoter activity of the collagen type III gene (6Li W. Nellaiappan K. Strassmaier T. Graham L. Thomas K.M. Kagan H.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12817-12822Crossref PubMed Scopus (147) Google Scholar, 7Giampuzzi M. Oleggini R. Di Donato A. Biochim. Biophys. Acta. 2003; 1647: 245-251Crossref PubMed Scopus (59) Google Scholar, 8Giampuzzi M. Botti G. Duca M.D. Arata L. Ghiggeri G. Gusmano R. Ravazzolo R. Donato A.D. J. Biol. Chem. 2000; 275: 36341-36349Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Increased LO activity is associated with fibrotic diseases such as lung and liver fibrosis and atherosclerosis (1Kagan H.M. Li W. J. Cell. Biochem. 2003; 88: 660-672Crossref PubMed Scopus (689) Google Scholar), whereas decreased LO activity is associated with lathyritic agent-induced emphysema in animal models (9Snider G.L. Lucey E.C. Stone P.J. Am. Rev. Respir. Dis. 1986; 133: 149-169Crossref PubMed Scopus (243) Google Scholar) and with disorders of copper metabolism like Menkes syndrome (10Harris E.D. Nutr. Rev. 1993; 51: 235-245Crossref PubMed Scopus (12) Google Scholar). LO gene expression is regulated at the mRNA level in response to intraor extracellular agents or conditions. LO transcription is up-regulated by transforming growth factor-β1 and interleukin-1β (11Boak A.M. Roy R. Berk J. Taylor L. Polgar P. Goldstein R.H. Kagan H.M. Am. J. Respir. Cell Mol. Biol. 1994; 11: 751-755Crossref PubMed Scopus (81) Google Scholar, 12Roy R. Polgar P. Wang Y. Goldstein R.H. Taylor L. Kagan H.M. J. Cell. Biochem. 1996; 62: 411-417Crossref PubMed Scopus (70) Google Scholar) but down-regulated by bFGF and interferon-γ (13Hong H-H. Trackman P.C. J. Periodontol. 2002; 73: 145-152Crossref PubMed Scopus (34) Google Scholar, 14Song Y.L. Ford J.W. Gordon D. Shanley C.J. Arteroscler. Thromb. Vasc. Biol. 1999; 20: 982-988Crossref Scopus (44) Google Scholar). Steady-state LO mRNA levels were diminished in some cancer and transformed cell lines (1Kagan H.M. Li W. J. Cell. Biochem. 2003; 88: 660-672Crossref PubMed Scopus (689) Google Scholar, 2Kenyon K. Contente S. Trackman P.C. Tang J. Kagan H.M. Friedman R.M. Science. 1991; 253: 802Crossref PubMed Scopus (188) Google Scholar) but elevated in invasive breast cancer cells (15Kirschmann D.A. Seftor E.A. Fong S.F.T. Nieva D.R.C. Sullivan C.M. Edwards E.M. Sommer P. Csiszar K. Hendrix M.J.C. Cancer Res. 2002; 62: 4478-4483PubMed Google Scholar). In addition, environmental agents can also act upon the LO gene resulting in its transcriptional modification. For example, our recent studies indicated that cigarette smoke condensate (CSC), the particulate phase of smoke, inhibited synthesis of nascent LO transcripts leading to reduced levels of LO mRNAs in treated rat fetal lung fibroblasts (16Gao S. Chen K. Zhao Y. Rich C.B. Chen L. Li S.J. Toselli P. Stone P. Li W. Toxicol. Sci. 2005; 87: 197-203Crossref PubMed Scopus (33) Google Scholar). Transcription of the LO gene is driven by proximal promoter sequences, which interact with ubiquitous transcription factors. A region of 1,865 bp of the rat LO promoter upstream of ATG was found to direct luciferase reporter gene expression. CSC suppressed luciferase activity in cells transiently transfected with the LO promoter-luciferase gene chimeric vector consistent with its effects on LO mRNA levels (16Gao S. Chen K. Zhao Y. Rich C.B. Chen L. Li S.J. Toselli P. Stone P. Li W. Toxicol. Sci. 2005; 87: 197-203Crossref PubMed Scopus (33) Google Scholar). Although several cis-elements have been characterized in the LO promoter region for human, mouse, and rat (17Csiszar K. Entersz I. Trackman P.C. Samid D. Boyd C.D. Mol. Biol. Rep. 1996; 23: 97-108Crossref PubMed Scopus (24) Google Scholar, 18Jourdan-Le Saux C. Gleyzal C. Raccurt M. Sommer P. J. Cell. Biochem. 1997; 64: 328-341Crossref PubMed Scopus (15) Google Scholar, 19Contente S. Kenyon K. Sriraman P. Subramanyan S. Friedman R.M. Mol. Cell. Biochem. 1999; 194: 79-91Crossref PubMed Scopus (39) Google Scholar), the precise mechanisms for their activation remain to be understood. Because the rat LO gene is widely used as a model for studies on the expression and regulation of the LO gene, it is important and necessary to further define the rat LO gene promoter. Nuclear factor I (NFI) was originally described as a factor required for the replication of adenovirus DNA and then shown as a transcription factor widely involved in the regulation of constitutive or inducible gene expression, including both transactivation and repression (20Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (420) Google Scholar). NFI encoded by four different genes (nfi-A, nfi-B, nfi-C/CTF, and nfi-X) binds to the consensus sequence TTGGC(N5)GCCAA (N indicates any nucleotide) on duplex DNA as a dimer. Notably, NFI can also bind to the individual half-site, i.e. TTGGC or GCCAA, with a some-what reduced affinity. The highly conserved N-terminal residues contain the DNA binding domain, whereas the proline-rich C-terminal residues constitute the transcriptional regulation domain (20Gronostajski R.M. Gene (Amst.). 2000; 249: 31-45Crossref PubMed Scopus (420) Google Scholar). Four cysteine residues are conserved in all DNA binding domains of NFI isoforms, but only three cysteines are required for the DNA binding activity. The fourth cysteine nonessential for DNA binding makes NFI proteins sensitive to oxidative damage. Exposure of cells to H2O2 inhibited the binding of NFI to its DNA consensus sequence (21Morel Y. Barouki R. J. Biol. Chem. 1998; 273: 26969-26976Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Mutation of the fourth cysteine induced NFI resistance to oxidative inactivation (22Bandyopadhyay S. Gronostajski R.M. J. Biol. Chem. 1994; 269: 29949-29955Abstract Full Text PDF PubMed Google Scholar). The feature of oxidation sensitivity of NFI may play a critical role in the cellular response to oxidative stress (23Bandyopadhyay S. Starke D.W. Mieyal J.J. Gronostajski R.M. J. Biol. Chem. 1998; 273: 392-397Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). NFI has been reported to regulate the expression of a wide range of cellular and viral genes such as collagen (24Rossi P. Karsenty G. Roberts A.B. Roche N.S. Sporn M.B. de Crombrugghe B. Cell. 1988; 52: 405-414Abstract Full Text PDF PubMed Scopus (440) Google Scholar), cytochrome P450 1A1 (21Morel Y. Barouki R. J. Biol. Chem. 1998; 273: 26969-26976Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 25Morel Y. Mermod N. Barouki R. Mol. Cell. Biol. 1999; 19: 6825-6832Crossref PubMed Google Scholar), metallothionein-I (26Majumder S. Ghoshal K. Gronostajski R.M. Jacob S.T. Gene Expr. 2001; 9: 203-215Crossref PubMed Scopus (26) Google Scholar), mouse mammary tumor virus, etc. (27Mink S. Hartig E. Jennewein P. Doppler W. Cato A.C. Mol. Cell. Biol. 1992; 12: 4906-4918Crossref PubMed Scopus (95) Google Scholar). To further understand the control of LO gene transcription, we have cloned the 5′-flanking region of the rat LO gene, mapped its transcription start sites and core promoter elements, characterized the regulatory activities in transcription of its different subsegments, and demonstrated NFI as a critical transactivator interacting with the cognate cis-element in the promoter region for LO gene activation. Cell Culture—The rat fetal lung fibroblasts (RFL6) obtained from the ATCC were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2 and 95% air incubator as described previously (28Chen L.J. Zhao Y. Gao S. Chou I-N. Toselli P. Stone P. Li W. Toxicol. Sci. 2005; 83: 372-379Crossref PubMed Scopus (35) Google Scholar). Stock cultures were derived from the frozen cell line and passaged every 4 days. A total of six passages was used for experiments. Primer Extension Analysis—To identify start sites of the LO transcription, we first carried out a primer extension assay as described (29Kozak M. Nucleic Acids Res. 1998; 26: 4853-4859Crossref PubMed Scopus (60) Google Scholar). Total RNA was isolated from RFL6 cells with TRIzol reagent (Invitrogen). An antisense oligodeoxynucleotide, 5′-ATGATGCTCCCGGCTCGTCCCTGCT-3′ (Integrated DNA Technologies, Coralville, IA), corresponding to positions from –23 to +2 in the rat LO sequence using the first nucleotide preceding the ATG codon as –1, was labeled with [γ-32P]ATP (PerkinElmer Life Sciences) by T4 polynucleotide kinase (New England Biolabs). Approximately 20,000–40,000 cpm of labeled oligonucleotides were annealed with 20 μgof total RNA in 5× M-MLV first strand buffer (Invitrogen) and adjusted to a final volume of 19 μl with diethyl pyrocarbonate treated double distilled H2O. Samples were heated at 95 °C for 1 min and subsequently incubated at 48 °C for 45 min. Then 2 μl of 0.1 m dithiothreitol, 8 μl of 2.5 mm dNTP, and 40 units of M-MLV RT enzyme (Invitrogen) were added into the reaction mixture. After incubation at 37 °C for 30 min, samples were mixed with 8 μl of a formamide sequencing gel loading buffer, heated at 90 °C for 1 min, and then chilled rapidly on ice. Aliquots of samples were analyzed on a 6% urea/acrylamide sequencing gel. The transcription start sites were identified by the sequencing ladders directly parallel to the run-off reverse transcripts (29Kozak M. Nucleic Acids Res. 1998; 26: 4853-4859Crossref PubMed Scopus (60) Google Scholar). 5′-RLM-RACE—The assay for 5′-RNA ligase-mediated rapid amplification of cDNA ends (5′-RLM-RACE) was performed to confirm the rat LO transcription start sites with the First Choice™ RLM-RACE kit (Ambion, Austin, TX) following the manufacturer's instructions (30Husain A. Zhang X. Doll M.A. States J.C. Barker D.F. Hein D.W. Drug Metab. Dispos. 2007; 35: 721-727Crossref PubMed Scopus (78) Google Scholar). Briefly, 10 μg of total RNA extracted from RFL6 cells was treated with calf intestinal phosphatase to remove the free 5′-phosphate group. Tobacco acid pyrophosphatase was then used to specifically remove the cap structure from the full-length mRNA, leaving a 5′-monophosphate. Note that non-tobacco acid pyrophosphatase-treated RNAs were also included in experiments as negative controls. An RNA oligonucleotide adaptor was next ligated to the newly decapped mRNA by T4 RNA ligase. With the ligated RNA as a template, LO cDNA was synthesized by reverse transcription using M-MLV reverse transcriptase and (dT)15 primers. The resulting cDNA was then amplified by nested PCR using Platinum Taq High Fidelity DNA polymerase (Invitrogen) as well as the rat LO gene primers (reverse) and the adaptor primers (forward) provided by the manufacturer. The gene-specific antisense inner primer 5′-GACTCTCGAGGGTTGTCACGCAGCAGCAGAATGG-3′ and the nested PCR outer primer 5′-CAGATGGGCTTGGAGTCCTC-3′ were designed for the RLM-RACE assay based on the sequence of rat LO cDNA (16Gao S. Chen K. Zhao Y. Rich C.B. Chen L. Li S.J. Toselli P. Stone P. Li W. Toxicol. Sci. 2005; 87: 197-203Crossref PubMed Scopus (33) Google Scholar, 31Consortium Rat Genome Sequencing Nature. 2004; 428: 493-521Crossref PubMed Scopus (1632) Google Scholar). The 5′-RLM-RACE PCR products were analyzed on agarose gels and cloned into the pBluescript II SK (–) vector for sequencing as described (30Husain A. Zhang X. Doll M.A. States J.C. Barker D.F. Hein D.W. Drug Metab. Dispos. 2007; 35: 721-727Crossref PubMed Scopus (78) Google Scholar). Construction of Reporter Plasmids and Cell Transfection—The full length of the rat LO gene promoter and various subregions thereof was obtained by PCR using the rat genomic DNA extracted from RFL6 cells as a template. One common reverse primer and eight different forward primers were used for generation by PCR of progressive deletions of the 5′-flanking region of the LO gene, which were subsequently cloned into the SacI/XmaI sites of the promoterless and enhancerless firefly luciferase reporter vector pGL3-Basic. Each of the 5′ primers contained four nucleotides, GTCA, a restriction site, i.e. SacI or XmaI (labeled with underline), and a specific primer sequence, followed by a number in parentheses indicating the 5′ end position of each primer in the LO promoter region as follows: reverse, 5′-GTCACCCGGGATGATGCTCCCGGCTCGTCCCTGCT-3′, corresponding to positions from –23 to +2 in the rat LO gene sequence using the first nucleotide preceding the ATG codon as –1; forward 1, 5′-GTCAGAGCTCTGTTTGCCCTGCACATCTTTAGTAA-3′ (–3,979); forward 2, 5′-GTCAGAGCTCACTTTGGAGGAAATGAAAGAGGGAC-3′ (–3,237); forward 3, 5′-GTCAGAGCTCGAATGCACTAGGAAAGTCTGGAGGA-3′ (–1,865); forward 4, 5′-GTCAGAGCTCAGTCACAACCTCCCCCATCCCAACG-3′ (–709); forward 5, 5′-GTCAGAGCTCTATTTTGGCTTGGGCCCATGGCCTG-3′ (–598); forward 6, 5′-GTCAGAGCTCGAGTGTGTCTCAGGATGTGTGTTCC-3′ (–517); forward 7, 5′-GTCAGAGCTCGAGTGGTGGAGCTGTCCGCCTTGC-3′ (–410); and forward 8, 5′-GTCAGAGCTCAGACACTGTGCGCTCTCCCGGAC-3′ (–277). LO gene-specific sequences for the primer design were based on the Rattus norvegicus chromosome 18 WGS supercontig NW_047513 (GenBank™ accession number) (16Gao S. Chen K. Zhao Y. Rich C.B. Chen L. Li S.J. Toselli P. Stone P. Li W. Toxicol. Sci. 2005; 87: 197-203Crossref PubMed Scopus (33) Google Scholar, 31Consortium Rat Genome Sequencing Nature. 2004; 428: 493-521Crossref PubMed Scopus (1632) Google Scholar). LO promoter fragments obtained were restricted with SacI and XmaI and ligated into similarly restricted plasmid pGL3-Basic (Promega, Madison, WI) upstream of the firefly luciferase gene as described (16Gao S. Chen K. Zhao Y. Rich C.B. Chen L. Li S.J. Toselli P. Stone P. Li W. Toxicol. Sci. 2005; 87: 197-203Crossref PubMed Scopus (33) Google Scholar). The resulting LO promoter constructs are referred to as the Prom with a number(s) corresponding to the distance in nucleotides from the 5′ end of the promoter sequence to the translation start codon ATG, including Prom–3,979, Prom–3,237, Prom–1,865, Prom–709, Prom–598, Prom–517, Prom–410, and Prom–277. In addition, the deletion constructs Prom–1,336, Prom–804, Prom–1,865/–1,335 (containing the sequence from –1,865 to –1,335), Prom–1865/–804 (containing the sequence from –1,865 to –804), and Prom–1,865/–911 (containing the sequence from –1,856 to –911) were derived from the construct Prom–1,865, which was digested with restriction enzymes KpnI/PvuII, KpnI/EcoRV, SmaI/PvuII, NotI/XhoI and HindIII alone, respectively, followed by self-ligation or treatment with mungbean nuclease and then self-ligation. Thus, a total of 13 LO promoter constructs were prepared (Fig. 4A). Each construct was sequenced from both ends to ensure the correct orientation and fidelity. To evaluate functionalities of the initiator (Inr), the downstream core promoter element (DPE) and the NFI-binding sites in the LO gene activation, we also isolated two other LO promoter 5′ deletions such as –80/–1 and –160/–1 containing Inr and DPE core promoter elements and NFI-binding sites, respectively. The reverse primer used for PCR amplification was the same as described above corresponding to the region from –23 to +2 of the rat LO gene sequence. The forward primers for the LO promoter –80/–1 and –160/–1 were 5′-GATCGAGCTCTCCTTCGCGGGATCTGAGTC-3′ and 5′-GATCGAGCTCCGGCCGCTCGCCCTTGGCAC-3′, respectively. LO promoter-luciferase reporter constructs were created with the pGL3-Basic vector. Using these newly and previously prepared LO promoter-reporter gene constructs compassing sequences –80/–1, –160/–1, and –804/–1 (see Fig. 3A and Fig. 8C) as DNA templates, mutations of the Inr, the DPE, or the NFI-binding sites were performed according to the QuikChange mutagenesis protocol (Stratagene, La Jolla, CA). Mutagenic primer pairs used for the PCR amplification were 5′-GATCTGAGTCCCTGTCTTGGTGTTTCTCCTAGCCACGTCC-3′ for the Inr mutagenesis, 5′-CGTCCCTCCCCGAGAAGCCCCGAGCCGGGAGCATC-3′ for the DPE mutagenesis, 5′-CTGCCGCTCGCCCTGAACACCAGTCCCTGCGACC-3′ for the NFI-binding site 1 mutagenesis, and 5′-CTTCATGCATATTTGAACTTGGGCCCATGGCCTGGCTG-3′ for the NFI-binding site 2 mutagenesis (complementary reverse primers not shown, mutated nucleotides labeled with underlines). Each mutation was verified by direct sequencing.FIGURE 8Determination of functional NFI-binding sites in the LO promoter region –804/–1. A, sequences of synthetic LO promoter fragments with putative NFI-binding sites (labeled with underlines). B, evaluation by EMSA. 32P-Labeled synthetic LO promoter fragments containing putative NFI-binding sites as shown in A and 5 μg of nuclear extracts (NE) prepared from RFL6 cells or BSA, an internal control, were incubated in the reaction mixture in the absence or presence of the specific antibody against NFI. After the reaction, samples were analyzed on the native 6% polyacrylamide gel followed by autoradiography. Reaction products are shown in lanes 1–3 with the probe NFI-BS1 (–158/–123), lanes 4–6 with the probe NFI-BS2 (–604/570), and lanes 7–9 with the probe NFI-BS3 (–686/–652). C, schematic representation of LO promoter-reporter chimeras with or without NFI-binding site mutations. The site-directed mutations of NFI-binding sites were performed with the QuikChange mutagenesis kit. The mutated site is labeled with shaded circle with ×, and the nonmutated site is marked with shaded circle. D, relative luciferase activities of LO promoter-reporter constructs. The LO promoter-reporter constructs each as shown in C, and the pRL-TK vector, an internal control, were transiently cotransfected into RFL6 cells. Twenty four hours after transfection, cells were growth-arrested in 0.3% FBS/DMEM for an additional 24 h and then harvested for assaying the reporter gene expression. Firefly luciferase activities elicited by the LO promoter with or without mutations of NFI-binding sites were normalized to Renilla luciferase activities derived from the pRL-TK vector. Data shown are the mean ± S.D. of three experiments, each determined with triplicate dishes. *, p < 0.05; **, p < 0.01 relative to wild type controls.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further characterize the properties of specific regions of the LO promoter, synthetic oligonucleotides (Integrated DNA Technologies, Coralville, IA) of LO promoter fragments –709/–676, –682/–641, –654/–609, and –612/–580, as shown in Fig. 5A, and the fragment –609/–573 containing the NFI-binding motif or various mutants as well as the NFI-binding consensus sequences, as shown in Fig. 6B, were annealed each to its complementary sequence, phosphorylated with T4 polynucleotide kinase, and ligated into the SmaI-treated pGL3-Promoter reporter vector (Promega, Madison, WI) upstream of the SV40 promoter sequence.FIGURE 6Identification of the NFI-binding site in the subregion –612/–580 of the LO promoter. A, electrophoretic mobility supershift assay of oligonucleotide –612/–580-transcription factor complexes in the presence of different antisera. 1st lane shows the binding reaction of the 32P-end-labeled –612/–580 probe with BSA, an internal control. 2nd to 6th lanes show the binding reactions of the end-labeled –612/–580 probe with RFL6 nuclear extract (NE) in the absence (2nd lane) or presence of antibodies against Oct1 (3rd lane), CdxA (4th lane), Pit-1A (5th lane), or NFI (6th lane). Reaction products were analyzed on the native 6% polyacrylamide gel followed by autoradiography. B, sequences of LO promoter probes. Sequences of LO promoter probes containing the NFI-binding site wild type (NFI-BS-WT) and mutants (NFI-BS-Mut1, NFI-BS-Mut2, NFI-BS-Mut3, and NFI-BS-Mut4), and three positive controls, i.e. one NFI-binding site high affinity probe (NFI-BS-High) and two NFI-binding site consensus probes (NFI-BS-Con1 and NFI-BS-Con2), and a negative control probe (NFI-BS-Mut) (36Corden J. Wasylyk B. Buchwalder A. Sassone-Corsi P. Kedinger C. Chambon P. Science. 1980; 209: 1406-1414Crossref PubMed Scopus (541) Google Scholar). The LO promoter fragment –599/–573 is labeled with uppercase letters. The NFI-binding site –594/–580 is underlined, and the mutated nucleotides in the NFI-binding site are marked with a boldface letter. C, NFI binding to the subregion –609/–573 of the LO promoter determined by EMSA competition assays. 32P-Labeled synthetic LO promoter oligonucleotide probe –609/–573 containing NFI-BS-WT and 5 μg of nuclear extracts (NE) prepared from RFL6 cells or BSA, an internal control, were incubated in the reaction mixture in the absence or presence of 100-fold molecular excess of competitors (Comp) as indicated. After the reaction, samples were analyzed on the native 6% polyacrylamide gel followed by autoradiography. D, NFI binding to the subregion –609/–573 of the LO promoter determined by EMSA supershift competition assays. Experiments were performed as described in C, and competitors were added into the reaction mixture before addition of the antibody against NFI (Ab-NFI).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Each resulting recombinant construct and the pSV-β-galactosidase plasmid (Promega, Madison, WI) or pRL-TK, an internal control for monitoring the transfection efficiency, were transiently cotransfected into RFL6 cells by Lipofectamine reagents (32Ausubel F. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Albright L.M. Coen D.M. Varki A. Janssen K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995: 9.4.1-9.4.3Google Scholar). After a 24-h post-transfection, cells were growth-arrested by incubation with 0.3% FBS/DMEM for an additional 24 h. In some experiments, cells were growth-arrested for 6 h and then exposed for 24 h to agents such as CSC at various doses in the FBS-free medium. Note that cells cotransfected with pGL3-Basic or pGL3-Promoter containing the luciferase gene without the LO promoter and β-galactosidase vectors were always included in any experiment to evaluate the background. Luciferase and β-galactosidase activities in cell lysates were measured by luminometry and spectrophotometry, respectively, as described by manufacturers. LO promoter-luciferase activities in transfected cells were normalized to the transfection efficiency as revealed
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