Nuclear Factor 1 Interferes with Sp1 Binding through a Composite Element on the Rat Poly(ADP-ribose) Polymerase Promoter to Modulate Its Activity in Vitro
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m010360200
ISSN1083-351X
AutoresMarc‐André Laniel, Guy G. Poirier, Sylvain L. Guérin,
Tópico(s)DNA Repair Mechanisms
ResumoPoly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the rapid and extensive poly(ADP-ribosyl)ation of nuclear proteins in response to DNA strand breaks, and its expression, although ubiquitous, is modulated from tissue to tissue and during cellular differentiation. PARP-1 gene promoters from human, rat, and mouse have been cloned, and they share a structure common to housekeeping genes, as they lack a functional TATA box and contain multiple GC boxes, which bind the transcriptional activator Sp1. We have previously shown that, although Sp1 is important for rat PARP1 (rPARP) promoter activity, its finely tuned modulation is likely dependent on other transcription factors that bind the rPARP proximal promoter in vitro. In this study, we identified one such factor as NF1-L, a rat liver isoform of the nuclear factor 1 family of transcription factors. The NF1-L site on the rPARP promoter overlaps one of the Sp1 binding sites previously identified, and we demonstrated that binding of both factors to this composite element is mutually exclusive. Furthermore, we provide evidence that NF1-L has no effect by itself on rPARP promoter activity, but rather down-regulates the Sp1 activity by interfering with its ability to bind the rPARP promoter in order to modulate transcription of the rPARP gene. Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the rapid and extensive poly(ADP-ribosyl)ation of nuclear proteins in response to DNA strand breaks, and its expression, although ubiquitous, is modulated from tissue to tissue and during cellular differentiation. PARP-1 gene promoters from human, rat, and mouse have been cloned, and they share a structure common to housekeeping genes, as they lack a functional TATA box and contain multiple GC boxes, which bind the transcriptional activator Sp1. We have previously shown that, although Sp1 is important for rat PARP1 (rPARP) promoter activity, its finely tuned modulation is likely dependent on other transcription factors that bind the rPARP proximal promoter in vitro. In this study, we identified one such factor as NF1-L, a rat liver isoform of the nuclear factor 1 family of transcription factors. The NF1-L site on the rPARP promoter overlaps one of the Sp1 binding sites previously identified, and we demonstrated that binding of both factors to this composite element is mutually exclusive. Furthermore, we provide evidence that NF1-L has no effect by itself on rPARP promoter activity, but rather down-regulates the Sp1 activity by interfering with its ability to bind the rPARP promoter in order to modulate transcription of the rPARP gene. poly(ADP-ribose) polymerase-1 base pair(s) chloramphenicol acetyltransferase carboxymethyl-Sepharose dimethylsulfate electrophoretic mobility shift assay ethidium bromide human growth hormone nicotinamide adenine dinucleotide nuclear factor 1 polymerase chain reaction rat PARP-1 Drosophila Schneider line 2 Poly(ADP-ribose) polymerase-1 (PARP-1)1 is a nuclear enzyme which catalyzes the addition of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) onto itself and other nuclear proteins such as histones and topoisomerases (reviewed in Refs. 1De Murcia G. Huletsky A. Poirier G.G. Biochem. Cell Biol. 1988; 66: 626-635Crossref PubMed Google Scholar and2Lautier D. Lagueux J. Thibodeau J. Ménard L. Poirier G.G. Mol. Cell. 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It has also been shown that a decrease in PARP-1 mRNA levels is associated with cellular differentiation (23Suzuki H. Uchida K. Shima H. Sato T. Okamoto T. Kimura T. Miwa M. Biochem. Biophys. Res. Commun. 1987; 146: 403-409Crossref PubMed Scopus (36) Google Scholar, 24Prasad S.C. Thraves P.J. Bhatia K.G. Smulson M.E. Dritschilo A. Cancer Res. 1990; 50: 38-43PubMed Google Scholar, 25Chabert M.G. Niedergang C.P. Hog F. Partisani M. Mandel P. Biochim. Biophys. Acta. 1992; 1136: 196-202Crossref PubMed Scopus (12) Google Scholar, 26Bhatia M. Kirkland J.B. Meckling-Gill K.A. Biochem. J. 1995; 308: 131-137Crossref PubMed Scopus (61) Google Scholar) and senescence (27Salminen A. Helenius M. Lahtinen T. Korhonen P. Tapiola T. Soininen H. Solovyan V. Biochem. Biophys. Res. Commun. 1997; 238: 712-716Crossref PubMed Scopus (34) Google Scholar), whereas an increase is observed upon activation of lymphocytes (17Menegazzi M. Gerosa F. Tommasi M. Uchida K. Miwa M. Sugimura T. Suzuki H. Biochem. Biophys. Res. Commun. 1988; 156: 995-999Crossref PubMed Scopus (43) Google Scholar, 28McNerney R. Tavasolli M. Shall S. Brazinski A. Johnstone A. Biochim. Biophys. Acta. 1989; 1009: 185-187Crossref PubMed Scopus (36) Google Scholar) or peripheral blood mononuclear cells (18Menegazzi M. Suzuki H. De Prati A.C. Tommasi M. Miwa M. Gandini G. Gerosa F. FEBS Lett. 1992; 297: 59-62Crossref PubMed Scopus (13) Google Scholar). All these studies show that, although PARP-1 is ubiquitously expressed, its modulation, likely through complex transcriptional regulation, is critical to major cellular functions. In order to better understand the transcriptional mechanisms regulating PARP-1 expression, the PARP-1 gene promoter has been identified and cloned from three mammalian species, human (29Ogura T. Nyunoya H. Takahashi-Masutani M. Miwa M. Sugimura T. Esumi H. Biochem. Biophys. Res. Commun. 1990; 167: 701-710Crossref PubMed Scopus (37) Google Scholar, 30Yokoyama Y. Kawamoto T. Mitsuuchi Y. Kurosaki T. Toda K. Ushiro H. Terashima M. Sumimoto H. Kuribayashi I. Yamamoto Y. Maeda T. Ikeda H. Sagara Y. Shizuta Y. Eur. J. Biochem. 1990; 194: 521-526Crossref PubMed Scopus (25) Google Scholar), rat (31Potvin F. Thibodeau J. Kirkland J.B. Dandenault B. Duchaine C. Poirier G.G. FEBS Lett. 1992; 302: 269-273Crossref PubMed Scopus (14) Google Scholar), and mouse. 2T. Ogura, unpublished data. All three mammalian proximal promoters share a similar structure proper to housekeeping genes; they lack a functional consensus TATA box, are GC-rich, and contain a consensus initiator sequence surrounding the transcription start site. 3M. A. Laniel and S. L. Guérin, unpublished observations. The human promoter has been shown to contain binding sites for transcription factors Sp1, AP-2 (30Yokoyama Y. Kawamoto T. Mitsuuchi Y. Kurosaki T. Toda K. Ushiro H. Terashima M. Sumimoto H. Kuribayashi I. Yamamoto Y. Maeda T. Ikeda H. Sagara Y. Shizuta Y. Eur. J. Biochem. 1990; 194: 521-526Crossref PubMed Scopus (25) Google Scholar), YY1 (32Oei S.L. Griesenbeck J. Schweiger M. Babich V. Kropotov A. Tomilin N. Biochem. Biophys. Res. Commun. 1997; 240: 108-111Crossref PubMed Scopus (92) Google Scholar), and Ets (33Soldatenkov V.A. Albor A. Patel B.K.R. Dreszer R. Dritschilo A. Notario V. Oncogene. 1999; 18: 3954-3962Crossref PubMed Scopus (76) Google Scholar), whereas the mouse promoter was recently shown to be down-regulated by a complex of adenovirus E1A protein and pRb (34Pacini A. Quattrone A. Denegri M. Fiorillo C. Nediani C. Ramon y Cajal S. Nassi P. J. Biol. Chem. 1999; 274: 35107-35112Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In the case of the rat PARP-1 (rPARP) proximal promoter, we have shown that it is highly but not completely dependent on five Sp1 binding sites (35Potvin F. Roy R.J. Poirier G.G. Guérin S.L. Eur. J. Biochem. 1993; 215: 73-80Crossref PubMed Scopus (19) Google Scholar, 36Bergeron M.J. Leclerc S. Laniel M.A. Poirier G.G. Guérin S.L. Eur. J. Biochem. 1997; 250: 342-353Crossref PubMed Scopus (22) Google Scholar), and that other nuclear proteins, including one likely belonging to the nuclear factor 1 (NF1) family of transcription factors (37Laniel M.A. Bergeron M.J. Poirier G.G. Guérin S.L. Biochem. Cell Biol. 1997; 75: 427-434Crossref PubMed Scopus (25) Google Scholar), bind to and potentially regulate rPARP promoter transcription. In this study, we further characterize the rPARP promoter and demonstrate that it binds the rat liver form of NF1 known as NF1-L (38Paonessa G. Gounari F. Frank R. Cortese R. EMBO J. 1988; 7: 3115-3123Crossref PubMed Scopus (173) Google Scholar). Furthermore, we provide evidence that NF1-L does not actively modulate rPARP transcriptional activity but rather prevents Sp1 from binding the rPARP promoter in vitro and thus causes a concentration-dependent down-regulation of rPARP promoter activity. The rPARP promoter fragment pCR3 (Fig. 1), spanning region −103 to +13 relative to the mRNA start site, and its Sp1 triple-mutant derivative pCR3/F2-F3-F4m, have been described previously (36Bergeron M.J. Leclerc S. Laniel M.A. Poirier G.G. Guérin S.L. Eur. J. Biochem. 1997; 250: 342-353Crossref PubMed Scopus (22) Google Scholar). The site-directed mutant pCR3-L3m was produced by the polymerase chain reaction (PCR), using pCR3 as template and the synthetic oligomers L3m (5′-GCATGCCTGCAGTCGCGCTAAAAAAAAACCCCG-3′, mutated nucleotides are shown in boldface) and pCAT-3′ (5′-CTCAGATCCTCTAGAGTCG-3′). Unique PstI andXbaI sites (underlined in the primer sequences) were included for cloning purposes. Amplifications were performed in a final volume of 50 μl, and reaction mixtures contained 100 ng of DNA, 250 μm each dNTP (Amersham Pharmacia Biotech, Baie-d'Urfé, Quebec, Canada), 50 mm KCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 9.0, 2 μm each synthetic primer, and 3.5 units of TaqDNA polymerase (Amersham Pharmacia Biotech). Using a GeneAmp 2400 thermal cycler (PE Biosystems, Streetsville, Ontario, Canada), the samples were subjected to an initial denaturation at 94 °C for 180 s and were then processed through 35 cycles of denaturation at 94 °C for 20 s, annealing at 61 °C for 20 s, and elongation at 72 °C for 40 s, followed by a final elongation of 120 s at 72 °C. The resulting PCR products were run through a 1.5% agarose gel in 1× TAE (40 mm Tris acetate, 1 mm EDTA) containing 0.1 μg/ml ethidium bromide (EtBr) and were isolated using the QIAEX II gel extraction kit (Qiagen, Mississauga, Ontario, Canada). The purified DNA fragments were then digested with PstI (Amersham Pharmacia Biotech) andXbaI (Amersham Pharmacia Biotech) and ligated upstream of the chloramphenicol acetyltransferase (CAT) reporter gene in thePstI-XbaI-linearized vector pCATbasic (Promega, Madison, WI). The DNA insert of each recombinant plasmid was sequenced by chain-termination dideoxy sequencing (39Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52771) Google Scholar) to confirm the mutations. The expression plasmid pPacNF1-L was constructed by amplifying the NF1-L insert from vector NF1–21/pBS (kindly provided by Dr Paolo Monaci, Istituto di Ricerche di Biologia Molecolare, Rome, Italy), using the synthetic oligonucleotides 5′-NF1 (5′-GAATTCCTCGAGGCAGTTATGTATTC-3′) and 3′-NF1 (5′-GAATTCCTCGAGGGTGGTCTGTCTGG-3′). A uniqueXhoI site (underlined in the primer sequences) was included for cloning purposes. The PCR reaction was performed in a final volume of 50 μl, and the mixture contained 200 ng DNA, 250 μmeach dNTP, 50 mm KCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 9.0, 2.5 μm each synthetic primer, and 2.5 units of Pfu DNA polymerase (Stratagene, La Jolla, CA). Using a GeneAmp 2400 thermal cycler, the samples were subjected to an initial denaturation at 94 °C for 30 s and were then processed through 32 cycles of denaturation at 94 °C for 30 s, annealing at 47 °C for 30 s, and elongation at 72 °C for 120 s, followed by a final elongation of 420 s at 72 °C. The resulting PCR product was run through a 1% agarose gel in 1× TAE, 0.1 μg/ml EtBr, and was isolated using the QIAEX II gel extraction kit. The purified DNA fragment was then digested withXhoI (Amersham Pharmacia Biotech) and ligated into theXhoI-linearized vector pPac, which was obtained by removing the XhoI-inserted Sp1 cDNA from vector pPacSp1 (kindly provided by Dr. Guntram Suske, Institut für Tumorforschung, Philipps Universität, Marburg, Germany). The recombinant plasmid was then sequenced by chain-termination dideoxy sequencing (39Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52771) Google Scholar) to confirm the reading frame. Crude nuclear extracts from rat pituitary GH4C1 cells (kindly provided by Dr David D. Moore, Massachusetts General Hospital, Boston, MA) were prepared essentially as described previously (36Bergeron M.J. Leclerc S. Laniel M.A. Poirier G.G. Guérin S.L. Eur. J. Biochem. 1997; 250: 342-353Crossref PubMed Scopus (22) Google Scholar, 40Roy R.J. Gosselin P. Guérin S.L. BioTechniques. 1991; 11: 770-777PubMed Google Scholar) and kept frozen in small aliquots at −80 °C until use. The NF1-l-containing rat liver carboxymethyl (CM)-Sepharose fraction has been described previously (41Roy R.J. Guérin S.L. Eur. J. Biochem. 1994; 219: 799-806Crossref PubMed Scopus (30) Google Scholar). For EMSA, the 140-bp HindIII-XbaI pCR3 or pCR3/F2-F3-F4m fragments or double-stranded synthetic oligonucleotides bearing either the L3 sequence from the rPARP promoter (5′-CACAGCAGTCGCGCTGGAACCCAACCCCGCCATG-3′; Ref. 37Laniel M.A. Bergeron M.J. Poirier G.G. Guérin S.L. Biochem. Cell Biol. 1997; 75: 427-434Crossref PubMed Scopus (25) Google Scholar) or the target sequence for human HeLa CTF/NF-I (5′-GATCTTATTTTGGATGAAGCCAATATGAG-3′; Ref. 42de Vries E. van Driel W. van den Heuvel S.J.L. van der Vliet P.C. EMBO J. 1987; 6: 161-168Crossref PubMed Scopus (106) Google Scholar) were 5′- 32P-end-labeled as described previously (43Laniel M.A. Béliveau A. Guérin S.L. Moss T. Methods in Molecular Biology. 148. Human Press Inc., Totowa, NJ2001: 13-30Google Scholar) and used as probes. Approximately 2 × 104 cpm (except in Fig. 2, where 4 × 104cpm were used) of labeled DNA probe was incubated for ∼8 min at room temperature with GH4C1 nuclear extract or NF1-l-enriched CM-Sepharose fraction in the presence of 1 μg or 200 ng, respectively, of poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech) and 50 mm KCl in buffer D (10 mm HEPES, 10% v/v glycerol, 0.1 mm EDTA, 0.25 mmphenylmethylsulfonyl fluoride). The DNA-protein complexes were separated by electrophoresis on native polyacrylamide gels (4% for the pCR3/F2-F3-F4m probe, 6% for the pCR3 probe, 8% for the oligonucleotide probes) run against Tris/glycine buffer at 4 °C, as described previously (43Laniel M.A. Béliveau A. Guérin S.L. Moss T. Methods in Molecular Biology. 148. Human Press Inc., Totowa, NJ2001: 13-30Google Scholar). Competition experiments were performed using EMSA conditions similar to those described above, except that the protein extracts were incubated with the pCR3-labeled probe in the presence of molar excesses (as specified in the caption to Figs. 2 and 5 A) of unlabeled double-stranded oligonucleotides bearing the target sequence of CTF/NF-I (see above), the high affinity binding site for Sp1 (5′-GATCATATCTGCGGGGCGGGGCAGACACAG-3′) (44Dynan W.S. Tjian R. Cell. 1983; 35: 79-87Abstract Full Text PDF PubMed Scopus (912) Google Scholar), the PARP-1 promoter initiator sequence (5′-GATCGCGCCGCCAGGCATCAGCAATCTATCCTG-3′),3 L3, or L3m (see above). In the case of the pCR3/F2-F3-F4m fragment, the GH4C1 protein extract was incubated with the labeled probe in the presence of molar excesses (as specified in the caption to Fig. 6) of theHindIII-XbaI pCR3 fragment (which was isolated from a 1.5% agarose gel in 1× TAE, 0.1 μg/ml EtBr and purified using the QIAEX II gel extraction kit), or the unlabeled oligonucleotides CTF/NF-I or Sp1 (see above).Figure 6Co-transfection experiments in rat GH4C1 andDrosophila SL2 cells. A, both the wild-type rPARP promoter-bearing plasmid pCR3 (black columns) and its mutated derivative pCR3-L3m (white columns) were transiently transfected into GH4C1 cells along with the NF1-L expression plasmid (0.5 or 1 μg). As a negative control, pCR3 was also cotransfected along with the empty vector pSI (which was used for constructing the NF1-L expression plasmid). CAT activities were measured and normalized as detailed under "Experimental Procedures." Values are expressed as percentage of CAT activity relative to the level directed by either pCR3 and pCR3-L3m when cotransfected with the control plasmid pSI. Standard deviation is provided for each value shown. B, the rPARP promoter-bearing plasmid pCR3 was transfected into Drosophila SL2 cells along with the empty vector pPac, or with expression plasmids encoding either NF1-L (0.2, 0.5, or 1 μg) or Sp1 (1 μg), either individually or in combination. CAT activities were measured and normalized to β-galactosidase as described under "Experimental Procedures." Standard deviation is provided for each selected condition.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For supershift experiments, similar EMSA conditions as described above were used, except that following formation of the DNA-protein complexes, 1.5 μl of pre-immune rabbit serum, anti-NF1-L rabbit serum (kindly provided by Prof. P. C. van der Vliet, Laboratory of Physiological Chemistry, Utrecht University, Utrecht, The Netherlands) or anti-Sp1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubation allowed to proceed for another 8 min. Deoxyribonuclease I (DNase I) footprinting analysis of the rPARP promoter fragment pCR5 (36Bergeron M.J. Leclerc S. Laniel M.A. Poirier G.G. Guérin S.L. Eur. J. Biochem. 1997; 250: 342-353Crossref PubMed Scopus (22) Google Scholar), which encompasses the pCR3 region, was performed using 5 μl of the NF1-l-containing CM-Sepharose fraction. Briefly, after pCR5 was 5′-end-labeled on the top strand, 3 × 104 cpm labeled probe was incubated for 10 min at room temperature with the protein extract and treated with DNase I (Worthington, Freehold, NJ) as described previously (45Guérin S.L. Moore D.D. Mol. Endocrinol. 1988; 2: 1101-1107Crossref PubMed Scopus (14) Google Scholar). Digestion products were resolved by electrophoresis on a 8% polyacrylamide sequencing gel. For dimethylsulfate (DMS) methylation interference, the 140-bpHindIII-XbaI pCR3 fragment was 5′-end-labeled on the top strand and partially methylated with DMS essentially as described (46Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 2nd Ed. John Wiley & Sons, New York1992: 12.8-12.10Google Scholar). Once methylated, 6 × 104 cpm labeled probe was incubated with 10 μl of the NF1-l-containing CM-Sepharose fraction in buffer D and DNA-protein complexes were separated by native polyacrylamide gel electrophoresis as described above. The NF1-l-DNA complex was then visualized by autoradiography and isolated by electroelution (47Harvey M. Brisson I. Guérin S.L. BioTechniques. 1993; 14: 943-945Google Scholar). The isolated labeled DNA was finally treated with piperidine (46Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology. 2nd Ed. John Wiley & Sons, New York1992: 12.8-12.10Google Scholar) and further analyzed on a 8% sequencing gel. Rat GH4C1 cells were grown in Ham's F-10 medium (Sigma-Aldrich, Oakville, Ontario, Canada) supplemented with 10% fetal bovine serum (Life Technologies, Inc., Burlington, Ontario, Canada) and 20 μg/ml gentamycin, under 5% CO2 at 37 °C.Drosophila Schneider line 2 cells (SL2, ATCC CRL-1963) were grown in Schneider insect medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum and 20 μg/ml gentamycin at 27 °C without CO2. The GH4C1 cells were transiently transfected with either pCR3 or its mutant derivative pCR3-L3m by the calcium phosphate precipitation method as described (48Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6499) Google Scholar), using 15 μg of test plasmid and 5 μg of the human growth hormone (hGH) gene-encoding plasmid pXGH5 (49Selden R.F. Burke-Howie K. Rowe M.E. Goodman H.M. Moore D.D. Mol. Cell. Biol. 1986; 6: 3173-3179Crossref PubMed Scopus (472) Google Scholar). In the case where the expression vectors NF1-L/pSI (kindly provided by Dr Winnie Eskild, Institute of Medical Biochemistry, University of Oslo, Oslo, Norway) or NF1-X/pcDNA3 (kindly provided by Dr. Bin Gao, Medical College of Virginia, Richmond, VA) were added, GH4C1 cells were transfected with GeneSHUTTLE-20 cationic liposome (Quantum Biotechnologies, Montréal, Quebec, Canada), using 1 μg of test plasmid, 0.5 μg of pXGH5, and the expression vectors as described in the caption to Fig. 5 B. The SL2 cells were transfected by the calcium phosphate precipitation method, using 13 μg of test plasmid, 5 μg of the β-galactosidase (lacZ) gene-encoding plasmid pAc5/V5-His/LacZ (Invitrogen, Carlsbad, CA), and the pPacSp1 or pPacNF1-L expression vectors as described in the legend to Fig. 6 A. CAT activities were measured as described previously (50Pothier F. Ouellet M. Julien J.P. Guérin S.L. DNA Cell Biol. 1992; 11: 83-90Crossref PubMed Scopus (150) Google Scholar) and normalized to either hGH secreted into the culture medium (for GH4C1), which was assayed using a radioimmunoassay kit (Medicorp, Montréal, Quebec, Canada), or lacZ produced in the cells (for SL2), which was measured as described (51Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Each single value is expressed as either 100× (% CAT in 4 h)/ng of hGH for GH4C1, or (% CAT in 4 h)/(lacZ/min/ml) for SL2, and corresponds to the mean of at least two individual transfection experiments performed in triplicate. Standard deviation for the CAT assay is provided for each transfected plasmid and for each cell type. Only values 3 times greater than the background level caused by the reaction mixture alone (usually corresponding to ∼0.15% chloramphenicol conversion) were considered significant. We have previously demonstrated that, although Sp1 specifically binds to (Fig. 2, A andB) and strongly transactivates the GC-rich rPARP promoter, it is not sufficient to account for all of its transcriptional activity (36Bergeron M.J. Leclerc S. Laniel M.A. Poirier G.G. Guérin S.L. Eur. J. Biochem. 1997; 250: 342-353Crossref PubMed Scopus (22) Google Scholar). Furthermore, a nuclear protein likely belonging to the NF1 family of transcription factors was shown to bind to the rPARP minimal promoter region, which extends from −103 to +13 (pCR3, see Fig.1 and Ref. 37Laniel M.A. Bergeron M.J. Poirier G.G. Guérin S.L. Biochem. Cell Biol. 1997; 75: 427-434Crossref PubMed Scopus (25) Google Scholar). In fact, EMSA analysis, using a heparin-Sepharose- and CM-Sepharose-enriched rat liver nuclear extract, revealed the specific binding of a protein that is clearly distinct from Sp1 (Fig. 2 A). The interaction of this rat liver DNA-binding protein with the rPARP promoter likely occurs through a putative NF1 binding site (L3) previously identified on the pCR3 fragment (36Bergeron M.J. Leclerc S. Laniel M.A. Poirier G.G. Guérin S.L. Eur. J. Biochem. 1997; 250: 342-353Crossref PubMed Scopus (22) Google Scholar, 37Laniel M.A. Bergeron M.J. Poirier G.G. Guérin S.L. Biochem. Cell Biol. 1997; 75: 427-434Crossref PubMed Scopus (25) Google Scholar), since formation of the DNA-protein complex was completely abolished by competition with an excess of unlabeled oligonucleotide bearing the target sequence for human CTF/NF-I (42de Vries E. van Driel W. van den Heuvel S.J.L. van der Vliet P.C. EMBO J. 1987; 6: 161-168Crossref PubMed Scopus (106) Google Scholar) (Fig. 2 A). In contrast, competition using unlabeled oligonucleotides bearing unrelated sequences such as that for Sp1 or the initiator sequence from the PARP promoter3 had no effect on pCR3-rat liver protein complex formation (Fig. 2 A). However, when using a crude nuclear protein extract from rat pituitary GH4C1 cells, only Sp1 forms a complex with the pCR3 fragment, since its formation is prevented by an Sp1-specific antiserum but not by a pre-immune or an NF1-l-specific antiserum (Fig. 2 B). Using DNase I and DMS methylation interference footprinting, we precisely mapped the binding site of the NF1-like protein to the L3 region of the rPARP proximal promoter (Fig.3, A and B), which we had previously identified (37Laniel M.A. Bergeron M.J. Poirier G.G. Guérin S.L. Biochem. Cell Biol. 1997; 75: 427-434Crossref PubMed Scopus (25) Google Scholar) as a potential NF1 target site due to its similarity both to the consensus sequence recognized by members of the NF1 family (42de Vries E. van Driel W. van den Heuvel S.J.L. van der Vliet P.C. EMBO J. 1987; 6: 161-168Crossref PubMed Scopus (106) Google Scholar) and to the proximal silencer-1 element from the rat growth hormone gene (52Roy R.J. Gosselin P. Anzivino M.J. Moore D.D. Guérin S.L. Nucleic Acids Res. 1992; 20: 401-408Crossref PubMed Scopus (33) Google Scholar). Interestingly, the footprinted L3 region completely overlaps the F2 region (see Figs. 1 and 3), which was shown to bind Sp1 (36Bergeron M.J. Leclerc S. Laniel M.A. Poirier G.G. Guérin S.L. Eur. J. Biochem. 1997; 250: 342-353Crossref PubMed Scopus (22) Google Scholar). In order to unequivocally identify the rat liver L3-binding protein as a genuine member of the NF1 family, we performed supershift experiments using an antiserum specific to the rat liver form of NF1 known as NF1-L (38Paonessa G. Gounari F. Frank R. Cortese R. EMBO J. 1988; 7: 3115-3123Crossref PubMed Scopus (173) Google Scholar). As shown in Fig. 4 A, the rat liver DNA-binding protein formed a complex (NF1-L) of similar electrophoretic mobility using either the NF1 consensus binding site or the L3 sequence as the labeled probe, and this complex was efficiently supershifted by the anti-NF1-L antiserum (C1). As shown in Fig. 4 B, the same rat liver protein bound the pCR3 DNA fragment to form a specific complex (NF1-L), which was supershifted by the anti-NF1-L antiserum (C1), but not by the pre-immune rabbit serum (PS) or by an anti-Sp1 antibody (Sp1Ab). We therefore conclude that the rat liver protein that binds to the rPARP proximal promoter region is NF1
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