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• W2022248355 abstract "Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT) encoded by the Pcyt1a and Pcyt1b genes. Previously, we identified an Ets-1-binding site located at -49/-47 in the promoter of Pcyt1a as an important transcriptional element involved in basal CTα transcription (Sugimoto, H., Sugimoto, S., Tatei, K., Obinata, H., Bakovic, M., Izumi, T., and Vance, D. E. (2003) J. Biol. Chem. 278, 19716-19722). In this study, we determined whether or not there were other important elements and binding proteins for basal CTα transcription in the Pcyt1a promoter, and if other Ets family proteins bind to the Ets-1-binding site. The results indicate the formation of a ternary complex with Ets-1 binding at -49/-47 and Sp1 binding at -58/-54 of the Pcyt1a promoter that is important for activating CTα transcription. When nuclear extracts of COS-7 cells expressing various Ets family repressors were incubated with DNA probes, binding of Net to the probes was observed. Net dose-dependently depressed the promoter-luciferase activity by 98%, even when co-expressed with Ets-1. RNA interference targeting Net caused an increase of endogenous CTα mRNA. After synchronizing the cell cycle in NIH3T3 cells, CTα mRNA increased at the S-M phase corresponding to an increase of Ets-1 mRNA and a decrease of Net mRNA. These results indicated that Net is an important endogenous repressor for CTα transcription. Phosphatidylcholine biosynthesis via the CDP-choline pathway is primarily regulated by CTP:phosphocholine cytidylyltransferase (CT) encoded by the Pcyt1a and Pcyt1b genes. Previously, we identified an Ets-1-binding site located at -49/-47 in the promoter of Pcyt1a as an important transcriptional element involved in basal CTα transcription (Sugimoto, H., Sugimoto, S., Tatei, K., Obinata, H., Bakovic, M., Izumi, T., and Vance, D. E. (2003) J. Biol. Chem. 278, 19716-19722). In this study, we determined whether or not there were other important elements and binding proteins for basal CTα transcription in the Pcyt1a promoter, and if other Ets family proteins bind to the Ets-1-binding site. The results indicate the formation of a ternary complex with Ets-1 binding at -49/-47 and Sp1 binding at -58/-54 of the Pcyt1a promoter that is important for activating CTα transcription. When nuclear extracts of COS-7 cells expressing various Ets family repressors were incubated with DNA probes, binding of Net to the probes was observed. Net dose-dependently depressed the promoter-luciferase activity by 98%, even when co-expressed with Ets-1. RNA interference targeting Net caused an increase of endogenous CTα mRNA. After synchronizing the cell cycle in NIH3T3 cells, CTα mRNA increased at the S-M phase corresponding to an increase of Ets-1 mRNA and a decrease of Net mRNA. These results indicated that Net is an important endogenous repressor for CTα transcription. Phosphatidylcholine (PC) 3The abbreviations used are: PCphosphatidylcholineCTαCTP:phosphocholine cytidylyltransferase αCTβCTP:phosphocholine cytidylyltransferase βCMVcytomegalovirusEBSEts-binding siteG3PDHglyceraldehyde-3-phosphate dehydrogenaseLUCluciferaseRTreverse transcriptionTEF-4transcriptional enhancer factor-4SRFserum-response factorSREserum-response elementTCFternary complex factorDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumRNAiRNA interferencedsRNAdouble-stranded RNAGFPgreen fluorescent proteinNLSnuclear localization signal.3The abbreviations used are: PCphosphatidylcholineCTαCTP:phosphocholine cytidylyltransferase αCTβCTP:phosphocholine cytidylyltransferase βCMVcytomegalovirusEBSEts-binding siteG3PDHglyceraldehyde-3-phosphate dehydrogenaseLUCluciferaseRTreverse transcriptionTEF-4transcriptional enhancer factor-4SRFserum-response factorSREserum-response elementTCFternary complex factorDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumRNAiRNA interferencedsRNAdouble-stranded RNAGFPgreen fluorescent proteinNLSnuclear localization signal. is the major membrane phospholipid in mammalian cells and tissues and is produced in all nucleated cells via the CDP-choline pathway (1Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (189) Google Scholar, 2Lykidis A. Jackowski S. Prog. Nucleic Acid Res. Mol. Biol. 2000; 65: 361-393Crossref Google Scholar, 3Johnson J.E. Cornell R.B. Mol. Membr. Biol. 1999; 16: 217-235Crossref PubMed Scopus (235) Google Scholar, 4Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science Publishers B.V., Amsterdam2002: 205-232Google Scholar). An important rate-limiting and regulated enzyme in this pathway is CTP:phosphocholine cytidylyltransferase (CT), and cDNAs of four isoforms for CT have been reported (5Kalmar G.B. Kay R.J. Lachance A. Aebersold R. Cornell R.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6029-6033Crossref PubMed Scopus (129) Google Scholar, 6Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 237: 14022-14029Abstract Full Text Full Text PDF Scopus (111) Google Scholar, 7Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 8Karim M. Jackson P. Jackowski S. Biochim. Biophys. Acta. 2003; 1633: 1-12Crossref PubMed Scopus (76) Google Scholar). The most active and ubiquitous form of CT in cells and tissues is CTα, which was purified (9Weinhold P.A. Rounsifer M.E. Feldman D.A. J. Biol. Chem. 1986; 261: 5104-5110Abstract Full Text PDF PubMed Google Scholar, 10Feldman D.A. Weinhold P.A. J. Biol. Chem. 1987; 262: 9075-9081Abstract Full Text PDF PubMed Google Scholar) and cloned (5Kalmar G.B. Kay R.J. Lachance A. Aebersold R. Cornell R.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6029-6033Crossref PubMed Scopus (129) Google Scholar). cDNAs for three other isoenzymes, CTβ1, CTβ2, and CTβ3, were recently cloned and are encoded by a second gene (6Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 237: 14022-14029Abstract Full Text Full Text PDF Scopus (111) Google Scholar, 7Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 8Karim M. Jackson P. Jackowski S. Biochim. Biophys. Acta. 2003; 1633: 1-12Crossref PubMed Scopus (76) Google Scholar). Regulation of CTα activity has been extensively studied. The helical domain for the binding of specific lipids is important for translocation between the membrane and the cytosol (11Utal A.K. Jamil H. Vance D.E. J. Biol. Chem. 1991; 266: 24084-24094Abstract Full Text PDF PubMed Google Scholar), and a highly phosphorylated domain at the C terminus can modulate its enzyme activity (12Wieprecht M. Wieder T. Paul C. Geilen C.C. Orfanos C.E. J. Biol. Chem. 1996; 271: 9955-9961Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition to post-translational regulating mechanisms, CTα mRNA expression is strictly regulated. Its expression is increased in colony-stimulating factor 1-stimulated macrophages (13Tessner T.G. Rock C.O. Kalmar G.B. Cornell R.B. Jackowski S. J. Biol. Chem. 1991; 266: 16261-16264Abstract Full Text PDF PubMed Google Scholar) during liver development (14Sesca E. Perletti G.P. Binasco V. Chiara M. Tessitore L. Biochem. Biophys. Res. Commun. 1996; 229: 158-162Crossref PubMed Scopus (28) Google Scholar), after partial hepatectomy of rat liver (15Houweling M. Tijburg L.B. Vaartjes W.J. Batenburg J.J. Kalmar G.B. Cornell R.B. Van Golde L.M. Eur. J. Biochem. 1993; 214: 927-933Crossref PubMed Scopus (28) Google Scholar, 16Houweling M. Cui Z. Tessitore L. Vance D.E. Biochim. Biophys. Acta. 1997; 1346: 1-9Crossref PubMed Scopus (56) Google Scholar), and during S phase of the cell cycle (17Golfman L.S. Bakovic M. Vance D.E. J. Biol. Chem. 2001; 276: 43688-43692Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). phosphatidylcholine CTP:phosphocholine cytidylyltransferase α CTP:phosphocholine cytidylyltransferase β cytomegalovirus Ets-binding site glyceraldehyde-3-phosphate dehydrogenase luciferase reverse transcription transcriptional enhancer factor-4 serum-response factor serum-response element ternary complex factor Dulbecco's modified Eagle's medium fetal bovine serum RNA interference double-stranded RNA green fluorescent protein nuclear localization signal. phosphatidylcholine CTP:phosphocholine cytidylyltransferase α CTP:phosphocholine cytidylyltransferase β cytomegalovirus Ets-binding site glyceraldehyde-3-phosphate dehydrogenase luciferase reverse transcription transcriptional enhancer factor-4 serum-response factor serum-response element ternary complex factor Dulbecco's modified Eagle's medium fetal bovine serum RNA interference double-stranded RNA green fluorescent protein nuclear localization signal. The murine CTα gene (Pcyt1a) was isolated, and its exon/intron organization resembles the functional domains of the enzyme (18Tang W. Keesler G.A. Tabas I. J. Biol. Chem. 1997; 272: 13146-13151Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The gene is transcribed from two transcriptional start sites and lacks a TATA box but contains a GC-rich region that is characteristic of many other TATA-less promoters (19Bakovic M. Waite K. Tang W. Tabas I. Vance D.E. Biochim. Biophys. Acta. 1999; 1438: 147-165Crossref PubMed Scopus (39) Google Scholar). The binding sites for Sp1, Sp3 (19Bakovic M. Waite K. Tang W. Tabas I. Vance D.E. Biochim. Biophys. Acta. 1999; 1438: 147-165Crossref PubMed Scopus (39) Google Scholar, 20Bakovic M. Waite K. Vance D.E. J. Lipid Res. 2000; 41: 583-594Abstract Full Text Full Text PDF PubMed Google Scholar), transcriptional enhancer factor-4 (TEF-4) (21Sugimoto H. Bakovic M. Yamashita S. Vance D.E. J. Biol. Chem. 2001; 276: 12338-12344Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar), Ets-1 (22Sugimoto H. Sugimoto S. Tatei K. Obinata H. Bakovic M. Izumi T. Vance D.E. J. Biol. Chem. 2003; 278: 19716-19722Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), and sterol-response element-binding protein (23Ridgway N.D. Lagace T.A. Biochem. J. 2003; 372: 811-819Crossref PubMed Scopus (31) Google Scholar, 24Kast H.R. Nguyen C.M. Ainsfeld A.M. Ericsson J. Edwards P.A. J. Lipid. Res. 2001; 42: 1266-1272Abstract Full Text Full Text PDF PubMed Google Scholar) were reported in the 200-bp proximal promoter. Three Sp1-binding core elements, -139/-136, -67/-62, and -19/-14, have been identified, and they are involved in basal, activator, and suppressor activities. Sp1, Sp2, and Sp3 can competitively bind to these elements and regulate the expression of the CTα gene (20Bakovic M. Waite K. Vance D.E. J. Lipid Res. 2000; 41: 583-594Abstract Full Text Full Text PDF PubMed Google Scholar). When C3H10T1/2 fibroblasts were stably transfected with a cDNA encoding Ras, there was increased binding of Sp3 to the -67/-62 region. Thus, Sp3 is an important factor for CTα expression in these fibroblasts (25Bakovic M. Waite K. Vance D.E. J. Biol. Chem. 2003; 278: 14753-14761Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Banchio et al. (26Banchio C. Schang L.M. Vance D.E. J. Biol. Chem. 2003; 278: 32457-32464Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) recently reported that CTα expression during the S phase of the cell cycle is mediated by Sp1 phosphorylation by cyclin-dependent kinase 2 and binding of Sp1 to the -67/-62 region. Bakovic et al. (19Bakovic M. Waite K. Tang W. Tabas I. Vance D.E. Biochim. Biophys. Acta. 1999; 1438: 147-165Crossref PubMed Scopus (39) Google Scholar) reported that the murine CTα gene promoter is positively regulated by an enhancer element (Eb) located between -103 and -83 bp, and there was a protein bound to Eb in nuclear extracts of HeLa cells. We identified TEF-4 as that binding protein (21Sugimoto H. Bakovic M. Yamashita S. Vance D.E. J. Biol. Chem. 2001; 276: 12338-12344Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). It was interesting that TEF-4 enhanced the basal transcription of CTα in COS-7 cells by interactions with elements shorter than -71 that do not contain TEF-4-binding site. When we determined the main transcriptional region involved in basal CTα transcription, Ets-binding core element -49/-47 (EBS) was identified as the binding site of Ets-1, and its stimulating activity was enhanced by TEF-4 (22Sugimoto H. Sugimoto S. Tatei K. Obinata H. Bakovic M. Izumi T. Vance D.E. J. Biol. Chem. 2003; 278: 19716-19722Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). In this study, we determined whether or not there were other important elements and binding proteins for basal CTα transcription, and whether other Ets family proteins bind to the EBS. We identified a newly recognized important site for basal CTα transcription located at -58/-54, which was clarified as an additional binding site for Sp1. We demonstrate that that Sp1 binding at -58/-54 interacts with Ets-1 binding at the EBS and promotes CTα transcription. We also identify Net, an Ets-related protein, as a potent inhibitor of CTα transcription. Materials—The luciferase vector pGL3-basic that contains the cDNA for Photinus pyraris luciferase, the control pRL-CMV or pRL-TK vector that contains the cDNA for Renilla reniformis luciferase, and the dual-luciferase reporter assay system were obtained from Promega (Madison, WI). FuGENE 6™ transfection reagents, Dulbecco's modified Eagle's medium (DMEM) (high glucose), and fetal bovine serum (FBS) were from Roche Applied Science, Sigma, and Invitrogen, respectively. COS-7 and NIH3T3 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). Construction of Ets-1, Elk-1, Net, Mutated Net, ERF, Fli-1, Tel, and Net Fused with GFP Expression Vectors—The mouse full-length cDNA coding murine Ets-1 was obtained as reported before (22Sugimoto H. Sugimoto S. Tatei K. Obinata H. Bakovic M. Izumi T. Vance D.E. J. Biol. Chem. 2003; 278: 19716-19722Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The mouse full-length cDNAs coding murine Elk-1, Net, ERF, Fli-1, and Tel were obtained through PCR of a reverse-transcribed product of mRNA from NIH3T3 cells using Superscript II (Invitrogen). PCR was performed with the complementary primers, 5′-CTAGTGATGGACCCATCTGTGACGC-3′ and 5′-TGGCTTCTGGGGCCCTGGGGAGAGC-3′ for Elk-1; 5′-CTGGGTATGGAGAGTGCAATCACGC-3′ and 5′-GGATTTCTGAGAGCTGGGGGACAGC-3′ for Net; 5′-CCCAGCATGAAGACCCCGGCGGACAC-3′ and 5′-GGAATCTCGGTGCTCCAGGGAGAGTTG-3′ for ERF; 5′-GGCCAAATGGACGGGACTATTAAGG-3′ and 5′-GTAGTAGCTGCCTAAGTGTGAAGGC-3′ for Fli-1; and 5′-GTGAGACATGTCTGAGACTCCTGCTC-3′ and 5′-TTCCCGGGTCTCTTCCTTTACAGCC-3′ for Tel with Platinum Taq DNA polymerase high fidelity (Invitrogen). The 1.25-1.66-kb PCR products were cloned into pcDNA3.1/V5 (Invitrogen) in-frame with a V5 tag. The expression constructs were named as pcEts-1, pcElk-1, pcNet, pcERF, pcFli-1, and pcTel, and the sequences were confirmed. cDNA encoding Sp1 (pcSp1) was kindly obtained from Dr. Norman Wong, University of Calgary. A construct (pcNetm1) for a mutated Net (Netm1), in which two lysine residues (Lys-49 and Lys-51 in the nuclear localization signal (NLS) of Net) were replaced with asparagine residues, was created from pcNet by PCR using QuikChange™ site-directed mutagenesis kits (Stratagene, La Jolla, CA) with the complementary primer 5′-GGCCTCCGCAACAACAACACCAACATGAAC-3′. The cDNAs in pcNet and pcNetm1 were subcloned into pEGFP-C2 (Clontech) to fuse the recombinant proteins at the C terminus of GFP, and the resultant expression vectors were named pEGFP-Net (for the expression of GFP-Net) and pEGFP-Netm1 (for GFP-Netm1), respectively. Preparation of Deleted and Mutated CTα Promoter-Luciferase Reporters—Various 5′-deleted CTα promoter regions, LUC.C7 (-1268/+38), LUC.C8 (-201/+38), LUC.D2 (-130/+38), LUC.D1.5 (-71/+38), and LUC.D3 (-52/+38), inserted into the promoter-less luciferase vector pGL3-basic (Promega), were prepared as described previously (19Bakovic M. Waite K. Tang W. Tabas I. Vance D.E. Biochim. Biophys. Acta. 1999; 1438: 147-165Crossref PubMed Scopus (39) Google Scholar). To prepare mutated promoters, GGCGG (-58/-54) was mutated to AACAA and named LUC.mC7-c or LUC.mD1.5-c, TCC (-49/-47) was converted to AAA and named LUC.mC7-b or LUC.mD1.5-b, and both GGCGG (-58/-54) and TCC (-49/-47) were changed to AACAA and AAA, respectively, and named LUC.mC7-cb or LUC.mD1.5-cb. These mutations were created from LUC.C7 or LUC.D1.5 by PCR using QuikChange™ site-directed mutagenesis kits. The mutated complementary primers were used for making each mutated construct, and the resulting plasmids were used for transformation of Escherichia coli strain JM109 cells. The sequences of all deleted and mutated constructs were confirmed. Tissue Culture, Transfection, and Luciferase Assays—COS-7 or NIH3T3 cells were grown in DMEM supplemented with 10% fetal bovine serum. Cells (2.5 × 105) were plated on 60-mm plates (Discovery Labware) and grown overnight. Seven and a half μl of FuGENE 6™ was suspended in 250 μl of serum-free medium and mixed with 0.125, 0.4, or 1.25 μg of pcEts-1, pcNet, pcERF, pcFli-1, pcTel, or pcDNA for preparation of nuclear extracts to perform gel-shift analyses, immunoblot analysis, or total mRNA for RT-PCR. For luciferase assays, cells (1 × 105) were plated on 35-mm plates and grown overnight. Three μl of FuGENE 6™ was suspended in 100 μl of serum-free medium and mixed with 0.5 μg of the CTα promoter-luciferase constructs (see above), 0.001 μg of pRL-CMV or pRL-TK Renilla vector as a transfection control, and 0.005-0.5 μg of either pcEts-1, pcSp1, pcElk-1, pcNet, pcERF, pcFli-1, pcTel, pcNetm1, or pcDNA. Transfection was initiated by dropwise addition of DNA suspension to the cell culture. Twenty four or 48 h later, cells were harvested and lysed in 200 μl of Passive lysis buffer (Promega), and 5 μl of the cell lysate was used for the dual-luciferase assay according to the manufacturer's instructions. Luciferase activity was normalized for transfection efficiency by using the ratio of the activities obtained with the CTα promoter mutated or deletion constructs (see above) and the pRL-CMV or pRL-TK construct carrying the cytomegalovirus or thymidine kinase promoter-luciferase fusion. When the effects of Sp1 on CTα promoter were examined, luciferase values were normalized against total protein concentration determined by protein assay (Bio-Rad) as the Renilla luciferase activity was affected by the transfection of pcSp1. Synchronization of the Cell Cycle—NIH3T3 cells (1 × 105) were plated on 60-mm plates and grown overnight. Twenty four h later, DMEM with 10% FBS was changed to DMEM with 0.5% FBS. After 3 days of serum starvation, the medium was changed to DMEM with 10% FBS. To analyze the phase of the cell cycle at the indicated time after 10% FBS supplementation, cells were fixed with 70% ethanol and then stained with 50 μg/ml propidium iodide (Sigma). After cells were stained, flow cytometric analysis was performed using Epics XL (Beckman Coulter, Inc., Fullerton, CA). To analyze the cellular localization of Net, transfection was initiated by dropwise addition of pEGFP, pEGFP-Net, or pEGFP-Netm1 (0.5 μg) and FuGENE 6™ suspension to the cell culture 2 h after 10% FBS supplementation. Twenty four h after transfection, GFP images were obtained with a Leica DM IRBE fluorescent microscopy (Leica microsystems, Wetzlar, Germany) with a filter set for GFP. Preparation of Nuclear Extracts and Electromobility Gel-shift Assays—COS-7 cells were cultured and transfected with plasmids as described above. After 48 h, nuclear extracts from COS-7 cells were prepared according to Andrews and Faller (27Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2207) Google Scholar) with minor modifications (19Bakovic M. Waite K. Tang W. Tabas I. Vance D.E. Biochim. Biophys. Acta. 1999; 1438: 147-165Crossref PubMed Scopus (39) Google Scholar). Five hundred pmol of the opposite strands of pS (5′-GGGCGGGCGGGAGGCGGGACTTCCGGTCC-3′), pmS-c (5′-GGGCGGGCGGGAaacaaGACTTCCGGTCC-3′), pmS-b (5′-GGGCGGGCGGGAGGCGGGACTaaaGGTCC-3′), pE (5′-GGCGGGAGGCGGGACTTCCGGTCCGCAGTC-3′), pmE-c (5′-GGCGGGAaacaaGACTTCCGGTCCGCAGTC-3′), pmE-b (5′-GGCGGGAGGCGGGACTaaaGGTCCGCAGTC-3′), and pmE-cb (5′-GGCGGGAaacaaGACTaaaGGTCCGCAGTC-3′) were annealed (70 °C, 10 min) in 100 μl of 25 mm Tris-HCl, pH 8.0, 0.5 mm MgCl2, and 25 mm NaCl and then cooled to room temperature. An aliquot (10 pmol) of the double-stranded oligonucleotides was 5′-end-labeled with [32P]ATP (Amersham Biosciences) and T4 polynucleotide kinase and purified on a Sephadex G-50 column (Amersham Biosciences). A DNA protein-binding reaction was performed for 30 min at room temperature in 40 μl of 1× binding buffer (40 mm Tris-HCl, pH 7.9, 4 mm MgCl2, 2 mm EDTA, 100 mm NaCl, 2 mm dithiothreitol, 200 μg/ml bovine serum albumin, 20% glycerol, and 0.2% Nonidet P-40) containing 1 μg of poly(dI-dC) (Amersham Biosciences), 1 μl of the radiolabeled probe (50,000-80,000 cpm), and nuclear extracts of COS-7 cells. In some cases, unlabeled double-stranded pS (100-fold molar excess), anti-Ets-1/-2 antibody, anti-Sp1 antibody, anti-NF-κB antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-V5 antibody (Invitrogen) was included in the incubation mixture. The labeled probe was separated from DNA-protein complexes by electrophoresis on 6% nondenaturing polyacrylamide gels in Tris borate/EDTA buffer (44.5 mm Tris-HCl, pH 8.3, 44.5 mm boric acid, and 1 mm EDTA) at 4 °C until the xylene cyanol dye reached 5 cm from the bottom of the gel. Autoradiography was performed by exposure of the gel to an imaging plate for 15-30 min, and the images were analyzed by Fuji BAS-2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). RNAi Analysis—dsRNAs named Random (5′-CGAUUCGCUAGACCGGCUUCAUUGC-3′) as a control and Net-878 (5′-GCAAAAAACCCAAAGGCUUGGAAAU-3′) from 878 to 903 nucleotide sequence from ATG of Net cDNA were obtained. NIH3T3 cells (1 × 105) were plated on 35-mm dishes, and medium was changed to 0.5 ml of Opti-MEM (Invitrogen) after 24 h. Five μl of Lipofectamine™ 2000 (Invitrogen) was suspended in 250 μl of Opti-MEM and then mixed with 100 pmol of dsRNA. Transfection was initiated by dropwise addition of dsRNA suspension to the cell culture to a final concentration of 50 nm in the medium. Opti-MEM containing 30% FBS (625 μl) was added to the medium 4 h later, and total RNA was obtained after 24, 48, and 72 h. RT-PCR—COS-7 and NIH3T3 cells were cultured and transfected as described above. After cells were cultured for the indicated times, total RNA was obtained with RNA extraction kits (Qiagen, Valencia, CA) according to the manufacturer's instructions. For quantification of the amounts of mRNA of CTα, Ets-1, Net, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) in COS-7 or NIH3T3 cells, 0.5 μg of total RNA was reverse-transcribed at 50 °C for 30 min and then subjected to 31 cycles of amplification (94 °C for 30 s, 56 °C for 30 s, and 72 °C for 30 s) using one-step RT-PCR kit (Qiagen) and DNA Engine Opticon 2 system (MJ Research, MA). The primers used for CTα were 5′-ATGCACAGTGTTCAGCCAA-3′ for COS-7 cells or 5′-ATGCACAGAGTTCAGCTAAAG-3′ for NIH3T3 cells (sense) and 5′-GGGCTTACTAAAGTCAACTTCAA-3′ (antisense), and they produced an ∼200-bp CTα fragment. The primers of RT-PCR analysis for Ets-1 were 5′-GGCACCATGAAGGCGGCCGTCGATC-3′ (sense) and 5′-GGTCTTTGGGGATTCCCAGTCG-3′ (antisense), for Net were 5′-GTACACAAACTTCTGCCCGATC-3′ (sense) and 5′-CTGGGTATGGAGAGTGCAATCACGC-3′ (antisense), and for G3PDH were 5′-TCCACCACCCTGTTGCTGTA-3′ (sense) and 5′-ACCACAGTCCATGCCATCAC-3′ (antisense). The contents of CTα, Ets-1, or Net mRNAs were normalized to those of the G3PDH mRNA using DNA Engine Opticon 2 system. Statistical Analysis—All values are expressed as means ± S.D. Group means were compared by Student's t test or Cochran-Cox test after analysis of variance to determine the significance of difference between the individual means. Statistical significance was assumed at p < 0.05. Gel-shift Analysis of Nuclear Extracts of COS-7 Cells with pS and pE Promoter Elements—As we reported before (21Sugimoto H. Bakovic M. Yamashita S. Vance D.E. J. Biol. Chem. 2001; 276: 12338-12344Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 22Sugimoto H. Sugimoto S. Tatei K. Obinata H. Bakovic M. Izumi T. Vance D.E. J. Biol. Chem. 2003; 278: 19716-19722Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), the important promoter element for basal CTα transcription in COS-7 cells is from -71 to -43, and we identified Ets-1 binding to -53/-44 as one of the important activators. When we searched the CTα promoter region using the TRANSFAC transcription factor data base, several consensus elements for the binding sites of transcription factors were identified (22Sugimoto H. Sugimoto S. Tatei K. Obinata H. Bakovic M. Izumi T. Vance D.E. J. Biol. Chem. 2003; 278: 19716-19722Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Sp1 was suggested to bind to -62/-50 regions (Fig. 1A), and NF-κB was also suggested to bind to -55/-46. To test whether or not there were other proteins that bind to the Pcyt1a promoter elements from -70 to -36 in nuclear extracts, we prepared pS (-70/-42) and pE (-65/-36) probes (Fig. 1, B and C). After transfection with pcEts-1 into COS-7 cells for 48 h, nuclear extracts of COS-7 cells were incubated with pS. There were slow moving bands indicated by arrow b as shown in Fig. 1B, lanes 1 and 2. The band disappeared when Sp1 binding consensus GGCGG (-58/-54) was mutated to AACAA as in pmS-c (Fig. 1B, lanes 9 and 10). To confirm what kind of nuclear proteins bind to pS, nuclear extracts were incubated with anti-Sp1 (Fig. 1B, lanes 5 and 6) or anti-NF-κB (lanes 7 and 8). After incubation with anti-Sp1, the band intensity was much decreased (Fig. 1B, lanes 5 and 6), and supershifts indicated by arrow a appeared in Fig. 1B, lanes 5 and 6. However, when nuclear extracts were incubated with anti-NF-κB (Fig. 1B, lanes 7 and 8), the intensity of the band did not change compared with lanes 1 and 2, respectively. These results indicated that the promoter element from -58 to -54 is an important site for Sp1 binding. When nuclear extracts were incubated with anti-Sp1, the upper slow moving band indicated in Fig. 1B, arrow b, clearly decreased its density, and weak supershifts indicated by arrow a were detected in lanes 5 and 6. Therefore, the anti-Sp1 antibody was thought to have two effects on Sp1 binding to the DNA probe: inhibition of binding and formation of a larger complex detected as a super gel-shift. When EBS core element (-49/-47) was mutated to AAA as in pmS-b, the intensity of the bands for Sp1 binding did not change in Fig. 1B, lanes 11 and 12, compared with lanes 1 and 2, respectively. Therefore, the pS element is sufficient for Sp1 binding, and EBS (-49/-47) is not necessary for direct Sp1 binding to pS. After transfection with pcEts-1 into COS-7 cells for 48 h, nuclear extracts were prepared and incubated with the pE probe. There were slow moving bands indicated by arrow a in Fig. 1C, lanes 1 and 2, and the band intensities of the DNA-protein complexes were increased after transfection of pcEts-1. There were faster moving bands indicated by arrow b after pcEts-1 transfection in Fig. 1C, lanes 2, 4, and 6. The intensity of both upper and lower bands was clearly decreased after incubation of nuclear extracts with anti-Sp1, and weak shifts indicated by arrows c and d in Fig. 1C were recognized in lanes 3 and 4, respectively. After incubation of nuclear extracts with anti-Ets-1/2, the intensities of both upper and lower bands were also decreased (Fig. 1C, lanes 5 and 6 compared with lanes 1 and 2, respectively). These results indicate that the upper and lower bands indicated by arrows a and b in Fig. 1C consist of DNA-protein complexes of Ets-1, Sp1, and pE DNA probe with or without other protein(s), respectively. To confirm the binding elements for Ets-1 and Sp1 in pE, we prepared three mutated probes, pmE-c mutated at Sp1-binding consensus core (-58/-54) (Fig. 2B, lanes 3-10), pmE-b mutated at EBS core element (-49/-47) (Fig. 1C, lanes 7 and 8), and pmE-cb mutated at both sites (Fig. 1C, lanes 9 and 10). As shown in Fig. 1C, when nuclear extracts were incubated with pmE-b, the slow moving bands in lanes 7 and 8 indicated by arrow e were weaker than the band at the same position in lanes 1 and 2. When nuclear extracts were incubated with pmE-cb, the slow moving bands in Fig. 1C, lanes 7 and 8, completely disappeared. These results indicate that both -58/-54 and -49/-47 elements are important for Ets-1 and Sp1 binding. Sp1 and Ets-1 Co-operatively Bind to pE—To confirm the above hypothesis, nuclear extracts from COS-7 cells transfected with pcEts-1 were incubated with anti-V5 antibody to recognize V5-tagged Ets-1. There was a fast moving band indicated by arrow b in lane 2, Fig. 2A. The supershift in Fig. 2A, lane 4, indicated by arrow c appeared after incubation with anti-V5 antibody, and the lower band in lane 2 disappeared. When anti-Sp1 antibody was incubated with nuclear extracts, two bands indicated by arrows a and b in Fig. 2A decreased their intensities, and a shift indicated by arrow d was newly recognized (lane 6). When both anti-Sp1 and anti-V5 antibodies were incubated with nuclear extracts, the slow moving band in Fig. 2A, lane 4 Fig. 2A indicated by arrow c and the fast moving band in lane 6 indicated by arrow d decreased (lane 8). These results strongly suggest that the faster moving band indicated from arrow d (Fig. 2A, lane 6) is the complex of Ets-1 and pE DNA probe, and these results strengthen the possibility that the faster moving band indicated by arrow b (lane 2) is the ternary complex of Ets-1, Sp1, and pE DNA p" @default.
• W2022248355 created "2016-06-24" @default.
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• W2022248355 date "2005-12-01" @default.
• W2022248355 modified "2023-09-27" @default.
• W2022248355 title "Sp1 Is a Co-activator with Ets-1, and Net Is an Important Repressor of the Transcription of CTP:Phosphocholine Cytidylyltransferase α" @default.
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• W2022248355 doi "https://doi.org/10.1074/jbc.m503578200" @default.
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