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• W1998878958 abstract "Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hRFC) gene, involving multiple promoters and noncoding exons. We localized promoter activity to a 452-bp GC-rich region upstream of noncoding exon A, including a 47-bp basal promoter with a CRE/AP-1-like consensus element that bound the bZip family of DNA-binding proteins (e.g.CREB-1 and c-Jun). We now report that three nearly identical tandem repeats (49–61 bp) in the hRFC-A upstream region are involved in regulating promoter activity. By in vitro binding assays, multiple transcription factors (e.g. AP2 and Sp1/Sp3) bound this region. When AP2 was cotransfected with the hRFC-A reporter construct into HT1080 cells, promoter activity increased 3-fold. In Drosophila SL2 cells, Sp1 transactivated promoter A and showed synergism with CREB-1. However, c-Jun was antagonistic to the effects of Sp1. A sequence variant in the hRFC-A repeated region was identified, involving an exact duplication of a 61-bp sequence. This variant had an allelic frequency of 78% in 72 genomic DNAs and resulted in a 63% increase in promoter activity. These results identify important regions in the hRFC-A promoter and critical roles for AP2 and Sp1, in combination with the bZip transcription factors. Moreover, they document a functionally novel polymorphism that increases promoter activity and may contribute to interpatient variations in hRFC expression and effects on tissue folate homeostasis and antitumor response to antifolates. Recently, our laboratory reported an intricate regulation of the human reduced folate carrier (hRFC) gene, involving multiple promoters and noncoding exons. We localized promoter activity to a 452-bp GC-rich region upstream of noncoding exon A, including a 47-bp basal promoter with a CRE/AP-1-like consensus element that bound the bZip family of DNA-binding proteins (e.g.CREB-1 and c-Jun). We now report that three nearly identical tandem repeats (49–61 bp) in the hRFC-A upstream region are involved in regulating promoter activity. By in vitro binding assays, multiple transcription factors (e.g. AP2 and Sp1/Sp3) bound this region. When AP2 was cotransfected with the hRFC-A reporter construct into HT1080 cells, promoter activity increased 3-fold. In Drosophila SL2 cells, Sp1 transactivated promoter A and showed synergism with CREB-1. However, c-Jun was antagonistic to the effects of Sp1. A sequence variant in the hRFC-A repeated region was identified, involving an exact duplication of a 61-bp sequence. This variant had an allelic frequency of 78% in 72 genomic DNAs and resulted in a 63% increase in promoter activity. These results identify important regions in the hRFC-A promoter and critical roles for AP2 and Sp1, in combination with the bZip transcription factors. Moreover, they document a functionally novel polymorphism that increases promoter activity and may contribute to interpatient variations in hRFC expression and effects on tissue folate homeostasis and antitumor response to antifolates. Reduced folates are essential cofactors involved in one-carbon transfer reactions that lead to the synthesis of nucleotides and amino acids required for cell proliferation. Since mammals lack the capacity for de novo synthesis of reduced folates, these derivatives must be obtained from the diet (1Stokstad E.L.R. Picciano M.F. Stokstad E.L.R. Gregory J.F. Folic Acid Metabolism in Health and Disease. Wiley-Liss, New York1990: 1-21Google Scholar).The primary route for the transport of reduced folates into mammalian cells involves the ubiquitously expressed reduced folate carrier (RFC) 1The abbreviations used are: RFC, reduced folate carrier; Mtx, methotrexate; hRFC, human RFC; 5′-UTR, 5′-untranslated region; gDNA, genomic DNA; CRE, cAMP-response element; CREB, CRE-binding protein 1The abbreviations used are: RFC, reduced folate carrier; Mtx, methotrexate; hRFC, human RFC; 5′-UTR, 5′-untranslated region; gDNA, genomic DNA; CRE, cAMP-response element; CREB, CRE-binding protein(2Goldman I.D. Matherly L.H. Pharmacol. Ther. 1985; 28: 77-100Crossref PubMed Scopus (203) Google Scholar, 3Sirotnak F.M. Cancer Res. 1985; 45: 3992-4000PubMed Google Scholar, 4Jansen G. Jackman A.L. Anticancer Development Guide: Antifolate Drugs in Cancer Therapy. Humana Press Inc., Totowa, NJ1999: 293-321Google Scholar, 5Matherly L.H. Prog. Nucleic Acids Res. Mol. Biol. 2001; 67: 131-162Crossref PubMed Google Scholar). RFC is also the major cellular uptake system for antifolates used for cancer therapy, including methotrexate (Mtx) and Tomudex (2Goldman I.D. Matherly L.H. Pharmacol. Ther. 1985; 28: 77-100Crossref PubMed Scopus (203) Google Scholar, 3Sirotnak F.M. Cancer Res. 1985; 45: 3992-4000PubMed Google Scholar, 4Jansen G. Jackman A.L. Anticancer Development Guide: Antifolate Drugs in Cancer Therapy. Humana Press Inc., Totowa, NJ1999: 293-321Google Scholar, 5Matherly L.H. Prog. Nucleic Acids Res. Mol. Biol. 2001; 67: 131-162Crossref PubMed Google Scholar). High levels of transport by RFC are critical to the antitumor effects of antifolate inhibitors, and impaired transport results in decreased antitumor activity and a drug-resistant phenotype (6Schuetz J.D. Matherly L.H. Westin E.H. Goldman I.D. J. Biol. Chem. 1988; 263: 9840-9847Abstract Full Text PDF PubMed Google Scholar, 7Wong S.C. McQuade R. Proefke S.A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar, 8Jansen G. Mauritz R. Drori S. Sprecher H. Kathman I. Bunni M. Priest D.G. Noordhuis P. Schornagel J.H. Pinedo H.M. Peters G.J. Assaraf Y.G. J. Biol. Chem. 1998; 273: 30189-30198Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 9Gong M. Yess J. Connolly T. Ivy S.P. Ohnuma T. Cowanm K.H. Moscow J.A. Blood. 1997; 89: 2494-2499Crossref PubMed Google Scholar, 10Wong S.C. Zhang L. Witt T.L. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1999; 274: 10388-10394Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Moreover, impaired Mtx transport results from sustained exposures of human and murine tumor cells to Mtx in vitro (6Schuetz J.D. Matherly L.H. Westin E.H. Goldman I.D. J. Biol. Chem. 1988; 263: 9840-9847Abstract Full Text PDF PubMed Google Scholar, 7Wong S.C. McQuade R. Proefke S.A. Matherly L.H. Biochem. Pharmacol. 1997; 53: 199-206Crossref PubMed Scopus (39) Google Scholar, 8Jansen G. Mauritz R. Drori S. Sprecher H. Kathman I. Bunni M. Priest D.G. Noordhuis P. Schornagel J.H. Pinedo H.M. Peters G.J. Assaraf Y.G. J. Biol. Chem. 1998; 273: 30189-30198Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 9Gong M. Yess J. Connolly T. Ivy S.P. Ohnuma T. Cowanm K.H. Moscow J.A. Blood. 1997; 89: 2494-2499Crossref PubMed Google Scholar, 10Wong S.C. Zhang L. Witt T.L. Proefke S.A. Bhushan A. Matherly L.H. J. Biol. Chem. 1999; 274: 10388-10394Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and has been reported to develop in murine tumor cells in vivo during Mtx chemotherapy (11Sirotnak F.M. Moccio D.M. Kelleher L.E. Goutas L. Cancer Res. 1981; 41: 4447-4452PubMed Google Scholar).In studies of childhood acute lymphoblastic leukemia and osteosarcoma, diseases for which Mtx remains a cornerstone of modern therapies (12Matherly L.H. Taub J.W. Leukemia Lymphoma. 1996; 21: 359-368Crossref PubMed Scopus (34) Google Scholar,13Guo W. Healey J.H. Meyeers P.A. Ladanyai M. Huvos A.G. Bertino J.R. Gorlick R. Clin. Can. Res. 1999; 5: 621-627PubMed Google Scholar), an important role for human RFC (hRFC) was, likewise, implied. For instance, we previously reported an 88-fold range of hRFC expression in B-precursor acute lymphoblastic leukemia lymphoblasts and a proportional loss of Mtx transport capacity with changes in hRFC (14Zhang L. Taub J.W. Williamson M. Wong S.C. Hukku B. Pullen J. Ravindranath Y. Matherly L.H. Clin. Can. Res. 1998; 4: 2169-2177PubMed Google Scholar). Similarly, Guo et al. (13Guo W. Healey J.H. Meyeers P.A. Ladanyai M. Huvos A.G. Bertino J.R. Gorlick R. Clin. Can. Res. 1999; 5: 621-627PubMed Google Scholar) showed that low levels of hRFC gene expression in osteosarcomas accompanied decreased Mtx transport and a poor prognosis. These studies suggest that relative levels of hRFC gene expression can potentially have a major impact on clinical outcome following chemotherapy with Mtx.The differential expression of hRFC in these patient populations could be a result of altered promoter usage or transcriptional activity but could also be associated with interindividual variations in the regulatory regions of the hRFC gene. A better understanding of the molecular mechanisms that regulate hRFC gene expression and function in malignant and normal cells is critical, since this could foster improvements in the clinical use of antifolates for cancer therapy. Moreover, this could shed light on the intra- and extracellular signals that influence patterns of hRFC expression in normal human tissues and contribute to various pathophysiologic conditions associated with folate deficiency (e.g. cardiovascular disease (15Refsum H. Ueland P. Nygard O. Vollset S.E. Annu. Rev. Med. 1998; 49: 31-62Crossref PubMed Scopus (1832) Google Scholar), fetal abnormalities (16Butterworth Jr., C.E. Bendich A. Annu. Rev. Nutr. 1996; 16: 73-97Crossref PubMed Scopus (87) Google Scholar), neurological disorders (17Serot J.M. Christmann D. Dubost T. Bene M.C. Faure G.C. J. Neural Transm. 2001; 108: 93-99Crossref PubMed Scopus (101) Google Scholar), and cancer (18Choi S-W. Mason J.B. J. Nutrition. 2000; 130: 129-132Crossref PubMed Scopus (781) Google Scholar)).Recent studies suggest a remarkably complex regulation of hRFC the gene expression in tissues and tumors (19Whetstine J.R. Flatley R.M. Matherly L.H. Biochem. J. 2002; 367: 629-640Crossref PubMed Scopus (113) Google Scholar). We found that hRFC gene is ubiquitously but differentially expressed in human tissues and is regulated by seven noncoding exons (designated A1, A2, A, B, C, D, and E) and at least three promoters (A, B, and C) (19Whetstine J.R. Flatley R.M. Matherly L.H. Biochem. J. 2002; 367: 629-640Crossref PubMed Scopus (113) Google Scholar). Several of the noncoding exons are capable of alternative splicing. Altogether, there are as many as 18 unique hRFC transcripts with distinct 5′-UTRs linked to a common open reading frame (19Whetstine J.R. Flatley R.M. Matherly L.H. Biochem. J. 2002; 367: 629-640Crossref PubMed Scopus (113) Google Scholar). The exact function of each 5′-UTR has yet to be determined, however, an effect on hRFC mRNA stabilities, intracellular targeting, and/or translation efficiencies can be envisaged (20Roberts S.J. Chung K-N. Nachmanoff K. Elwood P.C. Biochem. J. 1997; 326: 439-447Crossref PubMed Scopus (22) Google Scholar, 21Elwood P.C. Nachmanoff K. Saikawa Y. Page S.T. Pacheco P. Roberts S. Chung K-N. Biochemistry. 1997; 36: 1467-1478Crossref PubMed Scopus (50) Google Scholar, 22Chen L., Qi, H. Korneberg J. Garrow T.A. Choi Y-J. Shane B. J. Biol. Chem. 1996; 271: 13077-13087Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 23Roy K. Mitsugi K. Sirotnak F.M. J. Biol. Chem. 1996; 271: 23820-23827Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 24Turner F.B. Andreassi II J.L. Ferguson J. Titus S. Tse A. Taylor S.M. Moran R.G. Cancer Res. 1999; 59: 6074-6079PubMed Google Scholar, 25Turner F.B. Taylor S.M. Moran R.G. J. Biol. Chem. 2000; 275: 35960-35968Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 26Kocarek T.A. Zangar R.C. Novak R.F. Arch. Biochem. Biophys. 2000; 376: 180-190Crossref PubMed Scopus (45) Google Scholar, 27Fiaschi T. Chiarugi P. Veggi D. Raugei G. Ramponi G. FEBS Lett. 2000; 473: 42-46Crossref PubMed Scopus (13) Google Scholar, 28Fournier B. Trunong-Bolduc Q.C. Zhang X. Hooper D.C. J. Bacteriol. 2001; 183: 2367-2371Crossref PubMed Scopus (58) Google Scholar).We recently began to identify and characterize critical transcription factor families involved in regulating the hRFC-B and -A promoters. For the basal promoter B, transactivation involved binding of Sp1 and Sp3 to a GC-box element. For hRFC-A, regulation involved binding of different members of the bZip superfamily, including c-Jun, CREB-1, and ATF-1, to a CRE/AP1-like element in the minimal promoter (29Whetstine J.R. Matherly L.H. J. Biol. Chem. 2001; 276: 6350-6358Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Most recently, we reported that the frequent use of promoter B in malignant tissues was associated with its regulation by multiple transcription factors, including oncogenic proteins such as c-Myc and Ikaros, and by histone acetylation (30Whetstine J.R. Witt T. Matherly L.H. Proc. Am. Assoc. Cancer Res. 2002; 43: 101Google Scholar). 2J. R. Whetstine, R. M. Flatley, and L. H. Matherly, submitted for publication. 2J. R. Whetstine, R. M. Flatley, and L. H. Matherly, submitted for publication.Accordingly, cell- and tissue-specific usage of alternate hRFC promoters/noncoding exons could serve to regulate relative expression and function of hRFC in response to differences in folate levels within tissues, intracellular distributions of transcription factors, or epigenetic events. It is this complexity of controls that probably ensures adequate levels of hRFC transcripts (and protein) in response to metabolic requirements for folates, or other tissue- or cell-specific signals.In this report, we expand our initial analysis of hRFC-A, a major promoter in liver, bone marrow, and immortalized cell lines (19Whetstine J.R. Flatley R.M. Matherly L.H. Biochem. J. 2002; 367: 629-640Crossref PubMed Scopus (113) Google Scholar), as well as in primary malignant cells.2 Our results demonstrate that AP2 and Sp1 binding to a series of unique tandem nucleotide repeats in the hRFC-A upstream region is critical to promoter transactivation. Moreover, we document the presence of a high frequency polymorphism in the repeated region, involving the insertion of an additional 61 nucleotides that influence hRFC-A transcriptional activity. This is the first report of a functional polymorphism in the hRFC gene that may contribute to interpatient variations in hRFC gene expression and response to antifolate cancer chemotherapeutics.RESULTSIn Vitro Binding of USF-1, AP2, and Mzf-1 to the hRFC-A PromoterOur previous studies of the regulation of the hRFC gene confirmed that promoter activity was associated with a 452-bp GC-rich region immediately upstream from exon A that was devoid of a TATA-box but contained a number of putative transcription elements (31Zhang L. Wong S.C. Matherly L.H. Biochem. J. 1998; 332: 773-780Crossref PubMed Scopus (33) Google Scholar). The basal hRFC-A promoter was localized to within 47 bp (positions −501 to −455; see Fig. 1) and included a CRE/AP1-like consensus sequence capable of binding the bZIP family of transcription factors, including CREB-1, ATF-1, and c-Jun (29Whetstine J.R. Matherly L.H. J. Biol. Chem. 2001; 276: 6350-6358Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 31Zhang L. Wong S.C. Matherly L.H. Biochem. J. 1998; 332: 773-780Crossref PubMed Scopus (33) Google Scholar). However, the contributions of individual transcription factors capable of binding cis elements upstream of this minimal region were not established.A characteristic feature of the original promoter A sequence involves the presence of three nearly identical tandem repeats of 49–61 bp and spanning 170 bp of upstream sequence from position −775 to −606 (Fig.1). Potential binding sites identified in this region include E-box (−884 to −867), Mzf-1 (site 1, −897 to −889; site 2, −765 to −752), AP2 (site 1, −864 to −853; site 2, −771 to −760), and GC-box (site 1, −689 to −674; site 2, −629 to −613) elements (Fig.1). In transient transfections of hRFC-A deletion clones, striking variations in promoter activity were observed upon deleting this region (31Zhang L. Wong S.C. Matherly L.H. Biochem. J. 1998; 332: 773-780Crossref PubMed Scopus (33) Google Scholar). The most significant changes occurred between positions −903 to −809 and between −721 and −558 (31Zhang L. Wong S.C. Matherly L.H. Biochem. J. 1998; 332: 773-780Crossref PubMed Scopus (33) Google Scholar).In vitro binding assays were used to identify the key proteins that interact with the first 298 bp of the hRFC-A promoter between positions −903 and −606. This region was analyzed by DNase I footprinting with HT1080 nuclear extracts, and gel shift assays were then used to identify the probable transcription factors capable of binding to the hRFC-A upstream sequence (Fig.2).Figure 2Cis elements within promoter A bind multiple transcription factors in vitro. DNase I footprinting (positions −940 to −570) and gel shift assays were performed with HT1080 nuclear extracts as described under “Materials and Methods.” Panel A, the DNase I footprinted region from −883 to −852 is shown in the left panel. Lanes 1 and 3, probe alone; lane 2, probe and nuclear extract. The protected regions correspond to potential AP2 and E-box sites. In the center andright panels, the hRFC-A/−903 double-stranded oligonucleotide (containing Mzf-1(1), E-box, and AP2(1) elements) was used as a probe for gel shift assays with HT1080 nuclear extracts.Lane 4, probe alone (without nuclear extract); lanes 5 and 10, probe plus nuclear extract; lane 6, 150-fold molar excess of the unlabeled wild type hRFC-A/−903 oligonucleotide (self); lanes 7,8, and 11, 150-fold molar excess of the unlabeled E-box consensus sequence (lane 7), Mzf-1 consensus sequences (Mzf1 1–4; lane 8), or AP2 consensus sequence (lane 11); lanes 9 and 12, antiserum to USF-1 or AP2 β, respectively, was added to the binding reactions. Panel B, the left panelshows the DNase I footprinted region from position −777 to −735, corresponding to Repeat 1 (R1). Lanes 1 and3, probe alone; lane 2, probe and nuclear extract. The protected regions correspond to potential Mzf-1 (Mzf-1(2)) and AP2 (AP2(2)) sites. The center and right panels show gel shift results with a32P-end-labeled hRFC-A/R1 oligonucleotide incubated with nuclear extract. Lane 4, probe alone; Lanes 5 and 11, hRFC-A/R1 plus nuclear extract; Lane 6, 150-fold molar excess of unlabeled wild type hRFC-A/R1 oligonucleotide (self); lanes 7 and8, unlabeled Mzf-1 consensus sequences (Mzf1 1–4 and Mzf1 5–13, respectively); lane 9, AP2 consensus sequence; lane 10, AP2 β antiserum was added to the binding reactions; lanes 11–13, 32P-end-labeled mutant R1 mAP2(2) (lane 11), R1mMzf-1(2) (lane 12), or R1 mAP2(2)/mMzf-1(2) (lane 13) oligonucleotides were incubated with nuclear extract. C, the DNase I footprinted region from −676 to −711 is shown in the left panel, corresponding to Repeat 2 (R2).Lanes 1 and 3, probe alone; lane 2, probe and nuclear extract. The protected region corresponds to a potential GC/GT-box (GC-box(1)). In the right panel, the gel shifts show double-stranded oligonucleotide hRFC-A/R2 incubated without (lane 4) or with (lane 5) nuclear extract. The complexes were competed with the unlabeled hRFC-A/R2 oligonucleotide (lane 6) and a GC-box (lane 7) consensus sequence. In lanes 8 and9, antibodies were added to the nuclear extract including that for Sp1 (lane 8) and Sp3 (lane 9). ForA–C, the complexes for each gel shift probe are labeled with letters and arrowheads, and the supershifted complexes are marked with arrows. The nonspecific band is noted with a cross. Gel-shifted complexes were resolved on a 5% nondenaturing gel, and DNase I footprints were resolved on a denaturing 8% gel.View Large Image Figure ViewerDownload (PPT)−903 to −840DNase I footprint analysis identified two protected regions from position −883 to −867 (a putative E-box) and from −865 to −852 (a putative AP2 site; AP2(1)) (Fig.2 A, compare lanes 1 and 3with lane 2). Protein binding to a putative Mzf-1 element (positions −897 to −889) was also localized to the most 5′-end of the footprint probe (data not shown).The proteins that bound the −903 to −840 region in HT1080 nuclear extracts were identified with the hRFC-A/−903 double-stranded oligonucleotide probe in gel shift assays. Three DNA-protein complexes were detected (labeled A–C in Fig.2 A, lanes 5 and 10) and effectively competed by excess unlabeled hRFC-A/−903 probe (lane 6). Complex A was competed with an E-box consensus oligonucleotide (lane 7) and was supershifted with an antibody to an E-box-binding protein (USF-1; lane 9) (35Gregor P.D. Sawadogo M. Roeder R.G. Genes Dev. 1990; 4: 1730-1740Crossref PubMed Scopus (433) Google Scholar,36Pognonec P. Roeder R.G. Mol. Cell. Biol. 1991; 11: 5125-5136Crossref PubMed Scopus (75) Google Scholar). An AP2 consensus oligonucleotide competed with complex B (lane 11), and an AP2 β antibody resulted in a decreased DNA/protein signal and a supershifted complex (lane 12, arrow). Although complex C was effectively competed with a Mzf-1 consensus sequence (Mzf-1 1–4) (lane 8), the identity of the protein in this complex could not be verified due to the lack of a commercially available Mzf-1 antibody. When each of these elements was individually mutated in the hRFC-A/−903 oligonucleotide and the32P-mutant probes used directly in gel shift assays, protein binding was abolished (data not shown). Collectively, these data confirm binding of USF1, AP2, and a Mzf-1-like protein to the −903 to −840 region of the hRFC-A promoter.−775 to −715Footprint analysis of the −775 to −715 region identified protected regions spanning positions −775 to −761 and −758 to −742 (Fig. 2 B, compare lanes 1 and 3 with lane 2). These sites correspond to candidate AP2 (−771 to −760; AP2(2)) and Mzf-1 (−765 to −752; Mzf-1(2)) elements (37Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2421) Google Scholar) that overlap in the first repeated region (designated R1 in Fig. 1). When an end-labeled double-stranded hRFC-A/R1 oligonucleotide probe (positions −775 to −715) was incubated with a HT1080 nuclear extract, a series of DNA-protein complexes was detected (complexes A–D in Fig.2 B, lanes 5 and 11) and were competed by a molar excess of unlabeled probe (lane 6, self). Mzf-1 competitor oligonucleotides (Mzf-1 1–4 and 5–13) added to the binding reactions in molar excess resulted in a slightly diminished signal for all complexes (lanes 7 and 8, respectively). An AP2 consensus oligonucleotide potently competed for complexes A and C while perturbing complex D (lane 9). Similarly, an AP2 antibody (AP2 β antiserum) perturbed complexes A, C, and D and resulted in a supershifted complex (lane 10; noted with an arrow).The identities of the DNA-protein complexes were further assessed on gel shifts with end-labeled hRFC-A/R1 oligonucleotides with mutations in the potential AP2(2) and/or Mzf-1(2) sites (lanes 12–14). Thus, mutation of the AP2(2) element (hRFC-A/R1 mAP2(2)) resulted in decreased levels of all four complexes (lane 12). When the Mzf-1 site was mutated (hRFC-A/R1 mMzf-1(2)), the intensities of complexes A, B, and D were all decreased, whereas complex C increased in intensity (lane 13). When both the Mzf-1 and AP2 elements were mutated (hRFC-A/R1 mAP2(2)/mMzf-1(2)), there was a complete abolition of protein binding (lane 14). These results demonstrate the interactive binding of AP2 and Mzf-1-like proteins to the AP2(2) and Mzf-1(2) elements in the hRFC-A Repeat 1 region.−714 to −655 and −654 to −606A single protected region was identified by DNase I footprinting of the hRFC-A/Repeat 2 antisense sequence (R2 in Fig. 1; positions −714 to −655). This protected sequence (positions −677 to −693; Fig. 2C) corresponds to a putative GC/GT-box (GC-box(1), positions −689 to −674) (38Philipsen S. Suske G. Nucleic Acids Res. 1999; 27: 2991-3000Crossref PubMed Scopus (531) Google Scholar, 39Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (978) Google Scholar) and is identical to the protected GC-box(2), (positions -629 to -613) in the hRFC-A/Repeat 3 sequence (R3 in Fig. 1; positions −654 to −606) (data not shown). The hRFC-A/R2 oligonucleotide probe was used in gel shift assays to identify the DNA-protein complexes between positions −714 and −665. The hRFC-A/R3 oligonucleotide, including the identical 48 bp in Repeat 3, was also synthesized and used in gel shift assays to identify the complexes between positions −654 and −606.For both probes, three specific complexes (designated A–C) were detected (Fig. 2 C, lane 5, shows data for the hRFC-A/R2 probe; the hRFC-A/R3 probe results in identical complexes when compared with hRFC-A/R2, so the data are only shown for the hRFC-A/R3 probe). Since the DNA-protein complexes were completely competed by a GC-box consensus oligonucleotide and were supershifted with Sp1 and Sp3 antibodies (lanes 7–9, respectively; supershifts are noted with arrows), all complexes clearly involved members of the Sp family of transcription factors. When the GC-box elements were mutated, the competitions seen with the wild type oligonucleotides were abolished (data not shown).Mutagenesis of Transcription Factor Binding Elements in the hRFC-A Upstream RegionTo further assess the importance of the major binding sites identified in the hRFC-A upstream region by in vitro binding assays, the individual elements were mutated. The mutant hRFC-A promoter constructs in pGL3 Basic vector were transiently transfected into HT1080 cells, and luciferase activities were compared with that of the wild type hRFC-A/−903 construct. When the Mzf-1(1), E-box, Mzf-1(2), and GC-box(1) sites (Fig. 1) were individually mutated, insignificant changes in promoter A activity were observed (<10%;p > 0.05). However, the effects of other individual mutations were significant (p < 0.05) and ranged from a 23 ± 5% (for AP2(1)) or 25 ± 3% (for AP2(2)) increase in promoter activity to a 37 ± 6% decrease in activity (GC-Box(2)). Interestingly, when both the AP2(2) and Mzf-1(2) elements were mutated, a striking 37 ± 5% decrease in promoter activity was observed, consistent with our gel shift findings (Fig.2 B) that imply a functional interaction between AP2 and Mzf-1-like factors bound to these sites. Collectively, our mutation results suggest that the AP2(1) and AP2(2), Mzf-1(2), and GC-box(2) binding sites are important for promoter A activity.Cotransfections of Promoter A with AP2 and Mzf-1 Expression ConstructsAP2 and Mzf-1 expression constructs were cotransfected into HT1080 cells with the hRFC-A/−903 promoter construct to assess their effects on promoter activity. Whereas Mzf-1, alone, was transcriptionally inert in this assay, AP2 (α, β, and γ) markedly stimulated (∼3-fold) luciferase activities (Fig. 3). When Mzf-1 was expressed in combination with the AP2 isoforms, there was no further increase in luciferase activity from that with AP2 alone (data not shown). These data further support the notion of a critical role for the family of AP2 proteins in the regulation of hRFC-A transcription.Figure 3Effects of co-expressed AP2 and Mzf-1 on hRFC-A promoter activity. HT1080 cells were cotransfected with the hRFC-A/−903 promoter construct and 200 ng of Mzf-1 or 100 ng of the AP2 expression vectors. Luciferase activities were assayed with the Single Luciferase Kit and normalized to β-galactosidase activities. Relative luciferase activities for cotransfections of HSV-AP2α, HSV-AP2β, HSV-AP2γ, and pCDNA3-Mzf-1 constructs with the hRFC-A/−903 reporter construct were compared with that for a cotransfection of hRFC-A/−903 and empty cytomegalovirus vector. Theerror bars represent S.E.View Large Image Figure ViewerDownload (PPT)Sp1 Regulates Promoter A Activity in Drosophila SL2 CellsSp1 primarily acts as a transactivating factor (38Philipsen S. Suske G. Nucleic Acids Res. 1999; 27: 2991-3000Crossref PubMed Scopus (531) Google Scholar, 39Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (978) Google Scholar). Sp3 exists as three isoforms, generated by different translation starts (40Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar), all of which can act (to varying degrees) as activators or repressors of transcription, depending on the cell and promoter context (38Philipsen S. Suske G. Nucleic Acids Res. 1999; 27: 2991-3000Crossref PubMed Scopus (531) Google Scholar, 39Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (978) Google Scholar, 40Kennett S.B. Udvadia A.J. Horowitz J.M. Nucleic Acids Res. 1997; 25: 3110-3117Crossref PubMed Scopus (233) Google Scholar, 41Hagen G. Müller S. Beato M. Suske G. Nucleic Acis Res. 1992; 20: 5519-5525Crossref PubMed Scopus (522) Google Scholar, 42Hagen G. Müller S. Beato M. Suske G. EMBO J. 1994; 13: 3843-3851Crossref PubMed Scopus (650) Google Scholar, 43Ihn H. Trojanowska M. 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