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• W2010004911 abstract "In this report we describe acis-acting element within the core promoter of theCD155 gene specifying the polio virus receptor that is bound by the nuclear respiratory factor-1 (NRF-1) transcription factor. DNase I footprint analysis identified a nuclear protein binding site from −282 to −264 nucleotides upstream of the translation initiation codon of the CD155 gene, which we have called foot print IV (FPIV). Linker scanning mutagenesis revealed that a tandem repeat motif, GCGCAGGCGCAG, located within FPIV was essential for the basal activity of the CD155 core promoter. The results of the electrophoretic mobility shift assay experiments suggested that identical FPIV binding activities were present in a variety of nuclear extracts and that the tandem repeat was essential for binding. A one-hybrid screen was then carried out using FPIV as bait to clone the cDNA of the FPIV binding factor. The sequences of the cDNAs that were cloned from the screen were identical to NRF-1, a result that was confirmed by further electrophoretic mobility shift assay experiments. Overexpression of full-length NRF-1 and a dominant-negative form of NRF-1 modulated reporter gene expression driven by the core promoter. Remarkably, CD155 is the first gene shown to be regulated by NRF-1 that possesses an expression profile during embryogenesis correlating with this factor's proposed role in the development of the vertebrate optic system. We propose that NRF-1, which has been shown by others to be expressed during embryogenesis in animal systems, may be involved in regulating the expression of CD155 at specific stages of central nervous system development. In this report we describe acis-acting element within the core promoter of theCD155 gene specifying the polio virus receptor that is bound by the nuclear respiratory factor-1 (NRF-1) transcription factor. DNase I footprint analysis identified a nuclear protein binding site from −282 to −264 nucleotides upstream of the translation initiation codon of the CD155 gene, which we have called foot print IV (FPIV). Linker scanning mutagenesis revealed that a tandem repeat motif, GCGCAGGCGCAG, located within FPIV was essential for the basal activity of the CD155 core promoter. The results of the electrophoretic mobility shift assay experiments suggested that identical FPIV binding activities were present in a variety of nuclear extracts and that the tandem repeat was essential for binding. A one-hybrid screen was then carried out using FPIV as bait to clone the cDNA of the FPIV binding factor. The sequences of the cDNAs that were cloned from the screen were identical to NRF-1, a result that was confirmed by further electrophoretic mobility shift assay experiments. Overexpression of full-length NRF-1 and a dominant-negative form of NRF-1 modulated reporter gene expression driven by the core promoter. Remarkably, CD155 is the first gene shown to be regulated by NRF-1 that possesses an expression profile during embryogenesis correlating with this factor's proposed role in the development of the vertebrate optic system. We propose that NRF-1, which has been shown by others to be expressed during embryogenesis in animal systems, may be involved in regulating the expression of CD155 at specific stages of central nervous system development. central nervous system base pair(s) activator protein nuclear respiratory factor electrophoretic mobility shift assay polymerase chain reaction dithiothreitol reverse transcriptase embryonic day foot print IV Tris/borate/EDTA The human polio virus receptor protein, which has recently been given the designation CD155, is a highly glycosylated 80-kDa type Ia single pass transmembrane cell surface protein that belongs to the immunoglobulin superfamily (1.Mendelsohn C.L. Wimmer E. Racaniello V.R. Cell. 1989; 56: 855-865Abstract Full Text PDF PubMed Scopus (814) Google Scholar, 2.Koike S. Horie H. Ise I. Okitsu A. Yoshida M. Iizuka N. Takeuchi K. Takegami T. Nomoto A. EMBO J. 1990; 9: 3217-3224Crossref PubMed Scopus (270) Google Scholar, 3.Zibert A. Selinka H.C. Elroy-Stein O. Moss B. Wimmer E. Virology. 1991; 182: 250-259Crossref PubMed Scopus (13) Google Scholar, 4.Bibb J.A. Bernhardt G. Wimmer E. J. Virol. 1994; 68: 6111-6115Crossref PubMed Google Scholar, 5.Bernhardt G. Bibb J.A. Bradley J. Wimmer E. Virology. 1994; 199: 105-113Crossref PubMed Scopus (54) Google Scholar). CD155 was originally cloned based on its ability to serve as the cellular receptor for polio virus (1.Mendelsohn C.L. Wimmer E. Racaniello V.R. Cell. 1989; 56: 855-865Abstract Full Text PDF PubMed Scopus (814) Google Scholar) (reviewed in Refs. 6.Solecki D. Gromeier M. Harber J. Bernhardt G. Wimmer E. J. Mol. Recognit. 1998; 11: 2-9Crossref PubMed Scopus (11) Google Scholar and 7.Wimmer E. Harber J.J. Bibb J.A. Gromeier M. Lu H.-H. Bernhardt G. Wimmer E. Cellular Receptors for Animal Viruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 101-127Google Scholar). CD155 belongs to a subgroup of genes within the immunoglobulin superfamily; these genes share a V-C2-C2 domain structure as well as primary sequence identity. Thus far, cDNAs have been cloned that encode two new human molecules related to CD155, named PRR1 and PRR2 (polio virus receptor related) (8.Lopez M. Eberle F. Mattei M.G. Gabert J. Birg F. Bardin F. Maroc C. Dubreuil P. Gene (Amst.). 1995; 155: 261-265Crossref PubMed Scopus (146) Google Scholar, 9.Eberle F. Dubreuil P. Mattei M.G. Devilard E. Lopez M. Gene (Amst.). 1995; 159: 267-272Crossref PubMed Scopus (162) Google Scholar). Other CD155-related proteins possessing the V-C2-C2 immunoglobulin domain structure appear to be relatively conserved during evolution (10.Koike S. Ise I. Sato Y. Yonekawa H. Gotoh O. Nomoto A. J. Virol. 1992; 66: 7059-7066Crossref PubMed Google Scholar). Koike et al. (10.Koike S. Ise I. Sato Y. Yonekawa H. Gotoh O. Nomoto A. J. Virol. 1992; 66: 7059-7066Crossref PubMed Google Scholar) have shown that African green monkeys possess polio virus receptors encoded by two related but distinct genes AGMα1 andAGMα2 (African Green Monkey receptor). Two genes,mPRR2, also known as MPH (mouse polio virus receptor homologue), and Tage4 that are related to CD155, also exist in the mouse (11.Morrison M.E. Racaniello V.R. J. Virol. 1992; 66: 2807-2813Crossref PubMed Google Scholar, 12.Chadeneau C. LeMoullac B. Denis M.G. J. Biol. Chem. 1994; 269: 15601-15605Abstract Full Text PDF PubMed Google Scholar, 13.Chadeneau C. LeMoullac B. LeCabellec M. Mattei M. Meflah K. Denis M.G. Mamm. Genome. 1996; 7: 636-637Crossref PubMed Scopus (18) Google Scholar). Members of the new CD155 gene family have emerged as cellular receptors for animal viruses. As pointed out earlier, CD155 is the receptor for all three serotypes of the polio virus. There is no available evidence for an alternative receptor except, perhaps, for mouse-adapted polio viruses (14.Gromeier M. Lu H.H. Wimmer E. Microb. Pathog. 1995; 18: 253-267Crossref PubMed Scopus (19) Google Scholar). hPRR1 has recently been identified as a receptor for the α-herpes viruses (15.Cocchi F. Menotti L. Mirandola P. Lopez M. Campadelli-Fiume G. J. Virol. 1998; 72: 9992-10002Crossref PubMed Google Scholar, 16.Geraghty R.J. Krummenacher C. Cohen G.H. Eisenberg R.J. Spear P.G. Science. 1998; 280: 1618-1620Crossref PubMed Scopus (773) Google Scholar), and hPRR2 and mPRR2 have been identified as receptors for the pseudo-rabies virus (17.Shukla D. Rowe C.L. Dong Y. Racaniello V.R. Spear P.G. J. Virol. 1999; 73: 4493-4497Crossref PubMed Google Scholar). The biological importance of CD155 and its relatives has slowly begun to be elucidated. CD155, as well as mPRR2, are expressed during embryogenesis. 1M. Gromeier, D. Solecki, and E. Wimmer, submitted for publication.1M. Gromeier, D. Solecki, and E. Wimmer, submitted for publication. Recently, immunohistochemistry was used to determine that CD155 is expressed in regions of the developing human central nervous system, such as the notochord, floor plate, neural tube and the optic system.1Surprisingly, in situ hybridization analysis of mPRR2 mRNA indicated that this gene was also expressed in these structures of embryonic mice. 2M. Gromeier and E. Wimmer, unpublished results.2M. Gromeier and E. Wimmer, unpublished results. Many cell adhesion molecules belonging to the immunoglobulin superfamily important for the development of the CNS3 are expressed during embryogenesis in the floor plate and optic system (19.Burns F.R. von Kannen S. Guy L. Raper J.A. Kamholz J. Chang S. Neuron. 1991; 7: 209-220Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 20.Mohajeri M.H. Bartsch U. van der Putten H. Sansig G. Mucke L. Schachner M. Eur. J. Neurosci. 1996; 8: 1085-1097Crossref PubMed Scopus (38) Google Scholar, 21.Morales G. Sanchez-Puelles J.M. Schwarz U. de la Rosa E.J. Eur. J. Neurosci. 1996; 8: 1098-1105Crossref PubMed Scopus (14) Google Scholar). Recently, it has been reported that both mPRR2 (22.Aoki J. Koike S. Asou H. Ise I. Suwa H. Tanaka T. Miyasaka M. Nomoto A. Exp. Cell Res. 1997; 235: 374-384Crossref PubMed Scopus (115) Google Scholar) and hPRR2 (23.Lopez M. Aoubala M. Jordier F. Isnardon D. Gomez S. Dubreuil P. Blood. 1998; 92: 4602-4611Crossref PubMed Google Scholar) possess activity as homotypic adhesion molecules. In addition, cell adhesion activity has also has been demonstrated for hPRR1 (24.Takahashi K. Nakanishi H. Miyahara M. Mandai K. Satoh K. Satoh A. Nishioka H. Aoki J. Nomoto A. Mizoguchi A. Takai Y. J. Cell Biol. 1999; 145: 539-549Crossref PubMed Scopus (446) Google Scholar). This may suggest that some of the members of the CD155 gene family may be adhesion molecules involved in the development of the CNS. We have cloned the promoter region of the CD155 gene (25.Solecki D. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1999; 274: 1791-1800Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Our initial analyses have mapped a 280-bp core promoter fragment that is high in GC nucleotide content, lacks TATA and CAAT boxes, harbors a region of multiple transcriptional start sites, and appears to contain determinants required for cell type-specific promoter activity (25.Solecki D. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1999; 274: 1791-1800Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar,26.Solecki D. Schwarz S. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1997; 272: 5579-5586Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Indeed, we have used a transgenic mouse system to determine that the 3.0-kilobase CD155 promoter is capable of directing reporter gene expression to the appropriate anatomical structures where CD155 is expressed during embryogenesis.1 We have been interested in identifying the cis-acting elements andtrans-acting factors that could potentially play a role in the tissue-specific activity of the CD155 promoter. Moreover, we have hoped that our studies would identify target elements or factors that could be involved in orchestrating the activity of theCD155 promoter during embryonic development. We have recently reported the presence of three cis-acting elements within the CD155 core promoter region referred to as FPI, FPII, and FPIII (25.Solecki D. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1999; 274: 1791-1800Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Members of the activator protein-2 (AP-2) transcription factor have been found to bind to FPI and FPII and are able to activate reporter gene expression driven by theCD155 core promoter. Here, we report the characterization of a fourth cis-acting element (FPIV) within theCD155 core promoter that is essential for basal promoter activity. This element is bound by a nuclear protein that is present in the nuclear extracts prepared from murine embryos of gestational stages where the CD155 promoter is active in vivo. We have determined that the transcription factor is nuclear respiratory factor-1 (NRF-1). NRF-1, a regulatory protein present in the nuclear extracts of murine embryos and many established cell lines, binds to FPIV and functions as a potent transcriptional activator of theCD155 core promoter. Interestingly, NRF-1 belongs to a family of developmentally expressed transcription factors (27.Virbasius C.A. Virbasius J.V. Scarpulla R.C. Genes Dev. 1993; 7: 2431-2445Crossref PubMed Scopus (278) Google Scholar). In view of these observations, we will discuss the potential significance of the NRF-1/FPIV interaction relative to the in vivo activity of the CD155 promoter. The HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), HEK293 (embryonic kidney), SK-N-MC (neuroblastoma), HTB15 (glioblastoma), and Ntera-2/clone D1 (teratocarcinoma) cell lines were grown in Dulbecco's modified Eagle's medium, 10% fetal bovine serum. Mouse embryonic nuclear extracts were prepared using a modification of the method of Tamura et al. (28.Tamura T. Konishi Y. Makino Y. Mikoshiba K. Methods (Orlando). 1996; 10: 312-319Crossref Scopus (3) Google Scholar). Briefly, embryos of defined gestational age were harvested and homogenized in a solution containing 10 mm Hepes, 15 mm KCl, 1 mm EDTA, 2.2 m sucrose, and 5% glycerol. The homogenate was layered over a solution containing 10 mmHepes, 15 mm KCl, 1 mm EDTA, 2.0 msucrose, and 10% glycerol and centrifuged at 24,000 rpm for 1 h. The resulting pellet of nuclei was harvested and resuspended in a solution containing 10 mm Hepes, 550 mm KCl, 0.1 mm EDTA, 3 mm MgCl2, and 10% glycerol to lyse the nuclei and strip the chromatin of DNA-binding proteins. After the chromatin was pelleted by centrifugation at 40,000 rpm for 1 h, the supernatant containing the nuclear proteins was harvested and concentrated by ammonium sulfate precipitation. Nuclear extracts from human cell lines for use in EMSA experiments were prepared according to the procedure by Schreiber et al.(29.Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3916) Google Scholar). DNase I footprinting was performed as described previously (25.Solecki D. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1999; 274: 1791-1800Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Briefly, end-labeled DNA probes were generated via the polymerase chain reaction (PCR), using an oligonucleotide primer carrying a 5′-terminal [γ-32P]ATP label. PCR was performed under standard conditions using 10 ng of pGL2-H template, 25 pmol of labeled and unlabeled primers, and 1.2 units Taq polymerase. Radiolabeled PCR products were subjected to electrophoresis on a 10% native polyacrylamide gel; the bands were visualized by autoradiography, and a selected band was excised from the gel and passively eluted. DNase I protection assays were performed using 100,000 cpm of labeled probe which was incubated in a 50 μl binding reaction containing 2 μg of poly[d(I-C)] and nuclear protein. After a 30-min incubation on ice, 50 μl of a solution (room temperature) of 5 mm CaCl2 and 10 mmMgCl2 was added to each reaction and incubated for 1 min at room temperature. One μl of DNase I (100 ng/ml) was then added and incubated another minute at room temperature. The reactions were then terminated by the addition of 90 μl of stop solution (0.2m NaCl, 0.03 m EDTA, 1% SDS, and linear polyacrylamide as a carrier for ethanol precipitation); the mixture was then phenol/chloroform extracted twice, and the DNA was ethanol-precipitated. The samples were then electrophoresed on 10% polyacrylamide sequencing gels with a sequencing reaction as a marker. Site-directed mutagenesis of the BE CD155 promoter fragment, the CD155 core promoter spanning nucleotides −343 to −58 upstream of the CD155 translation initiating ATG codon (Fig.1 A; (26.Solecki D. Schwarz S. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1997; 272: 5579-5586Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar)), was carried out using the megaprimer mutagenesis technique of Picard et al. (30.Picard V. Ersdal-Badju E. Aiqin L. Bock S.C. Nucleic Acids Res. 1994; 22: 2587-2591Crossref PubMed Scopus (216) Google Scholar). To generate a megaprimer for each mutant construct, 100 ng of pGL2-BE plasmid was amplified in a reaction containing 50 pmol of the 4532 flanking primer, 50 pmol of mutagenic primer, 5 μl of 10x buffer, 2 μl of 10 mmdNTP mix, and 0.5 μl of Taq-polymerase (2.5 unit, Stratagene) in a total reaction volume of 50 μl. PCR amplification conditions were 94 °C, 30 s; 55 °C, 45 s; 72 °C, 45 s; for 35 cycles. All megaprimers were then gel-purified. To extend a megaprimer to generate to a full-length 280-bp BE fragment, 100 ng of pGL2-BE plasmid was amplified in a reaction containing 50 pmol of either 4529 flanking primer, 1–2 μg of megaprimer, 5 μl of 10x buffer, 2 μl of 10 mm dNTP mix, and 0.5 μl ofPfu-polymerase (2.5 unit, Stratagene). The primers used for megaprimer PCR reactions were: 4532, 5′-GGCGCTAGCGCCGCCTCTTCTAGTG-3′; 4529, 5′-GCCAGATCTGCTCGCTCTGCCGCGG-3′; IV (1.Mendelsohn C.L. Wimmer E. Racaniello V.R. Cell. 1989; 56: 855-865Abstract Full Text PDF PubMed Scopus (814) Google Scholar), 5′-CGCCTGCGCAGGACTAGTCCGGCGCTCAGT-3′; IV (2.Koike S. Horie H. Ise I. Okitsu A. Yoshida M. Iizuka N. Takeuchi K. Takegami T. Nomoto A. EMBO J. 1990; 9: 3217-3224Crossref PubMed Scopus (270) Google Scholar), 5′-GCGCTGCGCCTGACTAGTTCTCTCCCGGCG-3′; IV (3.Zibert A. Selinka H.C. Elroy-Stein O. Moss B. Wimmer E. Virology. 1991; 182: 250-259Crossref PubMed Scopus (13) Google Scholar), 5′-CCCCGCGCGCTGACTAGTCGCAGGTCTCTC-3′; and ΔTR, 5′-GGCCCTCCCCGCGCGAGGTCTCTCCCGGCG-3′. All cell lines were transfected by the calcium phosphate procedure. Each transfection mixture for the linker scan series of constructs was composed of 18 μg of wild type or mutant BE reporter constructs and 1 μg of pRL-TK (standard to the measure of efficiency of transfection). The compositions of the co-transfection experiments was 9.0 μg of the BE or BE ΔTR, mixed with up to 1.5 μg of pcDNA3(NRF-1). Co-transfections were supplemented with empty pcDNA3 to keep the amount of backbone plasmid constant for each experiment. 50 μl of 2.5 m CaCl2 was added to the DNA mixtures that were subsequently diluted to a total volume of 500 μl with Tris/EDTA buffer. These solutions were then separately combined dropwise with 500 μl of ice-cold 2x HBSS and incubated ten minutes at room temperature. Half of the precipitates were then added to a separate 6-cm plate of tissue culture cells (∼105cells), and the plates were incubated at 37 °C. 4 h later the medium was removed, and a solution of 20% glycerol in HBSS added. Following a 3-min incubation at 37 °C, 3 ml of medium was added, and the supernatant was removed again and replaced by fresh medium with serum. All transfected cells were harvested 18 h post-transfection, and cell extracts (usually 200–400 μl) were made using the reporter lysis buffer from Promega. The oligodeoxynucleotides used for EMSA were: FPIVs, 5′-GAGAGACCTGCGCAGGCGCAGCG-3′; FPIVas, 5′-GCGCGCTGCGCCTGCGCAGGTCT-3′; IV (3.Zibert A. Selinka H.C. Elroy-Stein O. Moss B. Wimmer E. Virology. 1991; 182: 250-259Crossref PubMed Scopus (13) Google Scholar)s, 5′-GAGAGACCTGCGACTAGTAGCG-3′; and IV (3.Zibert A. Selinka H.C. Elroy-Stein O. Moss B. Wimmer E. Virology. 1991; 182: 250-259Crossref PubMed Scopus (13) Google Scholar)as, 5′-GCGCGCTGACTAGTCGCAGGTCT-3′. One nmol of each the coding and noncoding oligodeoxynucleotide were reassociated in a volume of 50 μl using a thermocycler. Settings were 5 min at 95 °C and 1 h each at 65 °C, 60 °C, 55 °C, 50 °C, 45 °C, and 40 °C. The oligodeoxynucleotides were designed to possess a G as the 5′-protruding nucleotide. Ten pmol of reassociated oligodeoxynucleotide was end-labeled by a fill in reaction using CombiPol Polymerase (InViTek). In a volume of 20 μl, the buffer, 0.5 μl of enzyme, 50 μCi of [α-32P]dCTP, 1 μl of 25 mmMgCl2, and the oligodeoxynucleotide were incubated at 40 °C for 10 min, 45 °C for 10 min, and 50 °C for 20 min. The labeled oligodeoxynucleotide was purified by Sephadex G50 chromatography (Nick columns, Amersham Pharmacia Biotech). Usually more than 50% of label were found to be incorporated into the oligodeoxynucleotide. A binding reaction containing 1 μl of 10× incubation buffer (50 mm Tris-HCl, pH 7.5, 250 mm NaCl, 5 mm EDTA, 5 mm DTT, 25% glycerol), 1.5 μg of poly(dI-dC)(dI-dC), various concentrations of competitor or antibody (goat anti-NRF-1 antiserum was the kind gift of Richard Scarpulla), and 4 μl of cell extract was prepared and preincubated for 10 min at room temperature. After preincubation 1 μl of labeled oligodeoxynucleotide corresponding to 100 fmol were added, and the incubation continued for another 20 min. Samples were loaded onto a 6% 1/2× TBE polyacrylamide gel. After the electrophoresis (200 V) the gel was fixed in 10% acetic acid/30% methanol for 30 min and dried. The Matchmaker one-hybrid system kit (CLONTECH) was used to screen an E9/E10.5 mouse embryo library for the cDNAs of FPIV-binding proteins. Oligonucleotides consisting of four tandem repeats of the FPIV sequence were associated and then were cloned into pcDNA3. The identity of the cloned insert was verified by sequence analysis. The four tandem repeats were then subcloned into the yeast integration vectors pHISi-1 and pLacZi. These resulting vectors, p4xFPIV-HISi-1 and p4xFPIV-LacZi, were then linearized, transformed sequentially into the YM4271 yeast strain, and grown on the proper medium to select for colonies harboring the plasmids that had recombined in their proper genetic loci. The resulting doubly integrated yeast strain, YM4271(His, LacZ 4xFPIV), was found to possess very low background expression of the integrated HIS3 and LacZ reporter genes and was suitable for use in a library screen (data not shown). Library Screen—Competent YM4271(His, LacZ 4xFPIV) cells were transformed with 20 μg of an E9/E10.5 mouse embryo library using the procedure outlined by the Matchmaker one-hybrid system protocol (CLONTECH). The cells that were transformed with the library were then plated onto SD/-His/-Leu/+15 mm 3-aminotriazole selection plates and incubated at 30°C for 3 days. Colonies that grew under these selection conditions were then streaked onto fresh SD/-His/-Leu/+15 mm3-aminotriazole selection plates and were then subjected to colony to assay the expression of the LacZ reporter gene. Colonies that were His and LacZ positive were candidates for expressing library clones that encoded proteins that could bind to the 4xFPIV-target element. Therefore, the library plasmids from the His+ and LacZ+ colonies obtained from the library screen were isolated from the yeast, transformed into Escherichia coli DH5α cells, and subjected to sequence analysis. Total cellular RNA for use in RT-PCR was isolated from 3 × 107 SK-N-MC or HeLa cells according to the TRIzol protocol (Life Technologies, Inc.). Reverse transcription was done with Superscript reverse transcriptase from Life Technologies, Inc. using 10 μg of RNA as template and 1 pmol of gene-specific 3′-primer (NRF RT) in a reaction volume of 12 μl. The mixture was heated to 80 °C for 5 min and then allowed to cool to 42 °C. 4 μl of 5× reaction buffer, 2 μl of 0.1 m DTT, 1 μl of 10 mmdNTP mixture, 1 μl of RNAsin (25 unit), and 1 μl of reverse transcriptase were added and the reaction incubated for 90 min. at 42 °C. The reaction was stopped by adding 20 μl of 0.4m NaOH. Following 10 min at 42 °C, 20 μl of 1m TrisHCl pH 7.5 were added and the RT stocks frozen at −20 °C. The oligodeoxynucleotides used were: NRF RT, 5′-CATTTGATTGCACCTCTGCAAACG-3′; NRF 5′-NTR, 5′-GGATATTTGTTTAATGAATGTGGTATGC-3′; NRF ATG, 5′-TAGAAGCTTATGGAGGAACACGGAGTGACCC-3′; NRF TGA, 5′-GGCTCTAGATCACTGTTCCAATGTCACCACC-3′; ΔNRF, 5′-CTGTCTAGATCACTGTGATGGTACAAGATGAGCTATACTATG-3′. The full-length NRF-1 cDNA was amplified by nested PCR. For the first PCR reaction, 1 μl of RT stock was taken as template and mixed with 2 μl of 10 mm dNTP, 0.5 μl of primers each (50 pmol of NRF 5′-NTR and NRF TGA), 5 μl of 10× buffer, 10 μl of 5× optimizer buffer, 1 μl of CombiPol Polymerase (InViTek), and water to a total volume of 50 μl. Following a denaturation step at 94 °C, 2 min, 35 cycles were done at 94 °C, 45 s; 55 °C, 45 s; and 72 °C, 90 s. For the second PCR reaction, 1 μl of the first PCR reaction was amplified with 50 pmol each of the NRF ATG and NRF TGA primers with the above amplification conditions. Products were separated on a 1.0% agarose gel. The full-length product was observed when both HeLa and SK-N-MC total RNA were used as template for RT. The SK-N-MC full-length product was cut out and gel-purified. Following digestion withHindIII and XbaI restriction enzymes, the fragment was cloned into the pcDNA3 expression vector. The full-length insert was sequenced and found to be identical to the published open reading frame. A putative dominant-negative form of NRF-1 was cloned by amplifying the full-length cDNA with the NRF ATG and ΔNRF primers. The resulting product encoding the first 304 amino acids of NRF-1 was cloned into the HindIII andXbaI sites of pcDNA3. In vitro translation of the NRF-1 cDNA was carried out using the TNT-coupled rabbit reticulocyte system as per the manufacturer's instructions (Promega). Our previous studies identified three functional cis-acting elements (FPI–III) within the CD155 core promoter (for schematic locations see Fig. 1 A) (25.Solecki D. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1999; 274: 1791-1800Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). All three of these cis-acting elements are located within a 280-bp genomic DNA fragment, named BE (see the borders of the BE fragment, Fig. 1 A), that harbors full ability to direct the expression of a reporter gene when transfected into tissue culture cell lines that naturally express CD155 (25.Solecki D. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1999; 274: 1791-1800Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 26.Solecki D. Schwarz S. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1997; 272: 5579-5586Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Serial deletion analysis of theCD155 promoter was used in our original experiments to map the functional boundaries of the BE fragment (26.Solecki D. Schwarz S. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1997; 272: 5579-5586Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The exact 5′-borders of two of the serial deletion constructs, named B and C, are shown in Fig. 1, A and B. The promoter activity of the C construct has been found to be greatly reduced when compared with that of B (26.Solecki D. Schwarz S. Wimmer E. Lipp M. Bernhardt G. J. Biol. Chem. 1997; 272: 5579-5586Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), an observation suggesting the existence of a fourthcis-acting element within this area of the CD155promoter. To investigate this possibility further, we utilized twoBssHII restriction sites that flank the B–C region to generate a fine scale deletion within this area (for location of theBssHII restriction sites see Fig. 1 B). The ΔBssHII construct was transiently transfected into the HeLa and HEp-2 cell lines, and its ability to direct the expression of the luciferase reporter gene was determined. Strikingly, an 80% reduction of promoter activity was observed when the 50 bp in the vicinity the C construct border were removed (Fig. 1 C) or replaced in an inverted orientation (data not shown). These results suggest that a strong cis-element resides within this area. To map the location of any potential nuclear protein binding sites we subjected the 5′-portion of the CD155 core promoter to DNase I footprint analysis. The choice of the nuclear extract was guided by the following observation. Analysis of the CD155promoter/LacZ transgene indicated that murine embryos of the E10.5–E14.5 post conception stages express the LacZ reporter gene in a cell type-specific manner within the developing CNS.1Therefore, nuclear extracts of embryos from the E10.5–E14.5 gestational stages could conceivably contain the full complement oftrans-acting factors required to produce this expression pattern. Because our main aim was to identify transcription factors that could potentially contribute toward the in vivoactivity of the CD155 promoter, we used nuclear extracts prepared from E10.5 murine embryos for DNase I footprint experiments. When footprint analysis was performed with 75 μg of extract, a single protected region, called FPIV, was observed that was located from −282 to −264 base pairs upstream of the initiator ATG of theCD155 gene (see Fig. 2, for relative locations see Fig. 1, A and B). Interestingly, FPIV is located within the boundaries of theBssHII restriction fragment that we had discovered to be essential for core promoter activity. When footprinting experiments were carried out using nuclear extracts prepared from human cell lines, a partial footprint was also observed overlapping with that produced from E10.5 extract (data not shown). These results suggest that a nuclear protein binding site exists within the 5′ of theCD155 core promoter. Several data base searches for transcription factor binding motifs using the FPIV region yielded no significant homology to known transcription factor binding sites. However, a 12-base pair tandem repeat sequence, GCGCAGGCGCAG, showed significant homology to identical motifs found in a variety of other human promoters (data not shown). The DNase I footprint experiment (Fig. 2) identified a segment of theCD155 upstream sequence that is likely to harbor acis-element required for promoter function. To more precisely map this element, a series of linker scan mutations was generated throughout this protected sequence. Each mutant promoter construct contained 6 base pairs of wild type CD155 promoter sequ" @default.
• W2010004911 created "2016-06-24" @default.
• W2010004911 creator A5033777669 @default.
• W2010004911 creator A5067529822 @default.
• W2010004911 creator A5073909769 @default.
• W2010004911 creator A5089051856 @default.
• W2010004911 date "2000-04-01" @default.
• W2010004911 modified "2023-09-27" @default.
• W2010004911 title "Identification of a Nuclear Respiratory Factor-1 Binding Site within the Core Promoter of the human polio virus receptor/CD155 Gene" @default.
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