The Basal Promoters for the Human Reduced Folate Carrier Gene Are Regulated by a GC-box and a cAMP-response Element/AP-1-like Element

BASIS FOR TISSUE-SPECIFIC GENE EXPRESSION*

Johnathan R. WhetstineDagger and Larry H. MatherlyDagger §

From the Dagger  Department of Pharmacology and the § Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201

Received for publication, September 4, 2000, and in revised form, October 30, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our laboratory previously identified two functional promoters (designated A and B) for the human reduced folate carrier (hRFC) gene that result in hRFC transcripts with differing 5'-untranslated regions. By transiently transfecting HT1080 and HepG2 cells with a series of 5' and 3' deletions in the hRFC-B and -A promoters, the minimal promoters were localized within 46 and 47 base pairs, respectively. Gel mobility shift assays with the hRFC-B basal promoter region revealed specific DNA-protein complexes involving a highly conserved GC-box and Sp1 or Sp3. In Drosophila SL2 cells, both Sp1 and the long Sp3 isoform potently transactivated the hRFC-B basal promoter; however, the short Sp3 isoforms were transcriptionally inert and resulted in a potent inhibition of Sp1 transactivation. For the hRFC-A basal promoter, a CRE/AP-1-like element was bound by the bZip superfamily of DNA-binding proteins. Cell-specific DNA-protein complexes were identified for hRFC-A (CREB-1 and c-Jun in HT1080 cells; CREB-1 and ATF-1 in HepG2 cells). When the GC-box and CRE/AP-1-like elements were mutated, a 60-90% decrease in promoter activity was observed in both cell lines. These results identify the critical regulatory regions for the hRFC basal promoters and stress the functional importance of the Sp and bZip families of transcription factors in regulating hRFC expression.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reduced folate cofactors are involved in one-carbon transfer reactions in anabolic pathways leading to the purines, thymidylate, serine, and methionine, essential precursors for the biosynthesis of DNA, RNA, and proteins (1). Mammalian cells lack the capability of synthesizing these essential cofactors. Furthermore, chemically, folates are hydrophilic anionic molecules with limited capacities to traverse biological membranes by diffusion alone. Accordingly, mammals have evolved highly sophisticated uptake processes for transporting folates into cells. The primary route for entry of reduced folates is the reduced folate carrier (RFC)1 (2-5). The biological role of RFC is to provide adequate rates of intracellular delivery of tetrahydrofolates to sustain DNA synthesis and cell proliferation.

In addition to the important role of RFC in tissue folate homeostasis, membrane transport via RFC is a critical determinant of the antitumor activity of antifolate therapeutics used in cancer chemotherapy (i.e. methotrexate (Mtx), Tomudex; 2-4) and alterations in RFC contribute to Mtx resistance (2-4). In cultured cells, high levels of Mtx transport are essential for complete inhibition of dihydrofolate reductase and for Mtx polyglutamate synthesis (2). Transport is frequently impaired in cultured cells after prolonged Mtx exposures in vitro (6-10) and in murine tumor cells in vivo after chemotherapy with Mtx (11). Similarly, in leukemic lymphoblasts from patients, a wide range of RFC was observed, and low levels of RFC transcripts were associated with small amounts of Mtx uptake (12, 13). Most recently, Guo et al. (14) report that osteosarcomas with decreased human RFC (hRFC) expression were associated with a poor response to chemotherapy, including Mtx. Taken together, these studies argue that the levels of RFC gene expression can potentially have a major impact on clinical outcome after chemotherapy with Mtx. RFC gene expression can also influence normal tissue development and could conceivably contribute to disease states associated with decreased accumulations of folate cofactors, exacerbating the effects of folate deficiencies on the development of cardiovascular disease (15) and fetal abnormalities (16) and on the carcinogenic process (17-19).

In recent years, cDNAs encoding RFC from various species have been isolated and found to restore many of the transport characteristics typically associated with this system (20-25). Furthermore, the structures of the RFC gene from hamster (26), mouse (27, 28), and human (29-31) cells have been established. Despite the close homologies between the rodent and human RFCs, the murine and hamster genes are notably smaller than the hRFC gene (23 and 15.3 kilobases, versus ~29 kilobases, respectively) (26, 27, 29). Although there is little similarity between the intron sizes for the human and rodent RFC genes, the intron-exon junctions are highly conserved (29-31).

Multiple cDNA isoforms were described for both the rodent (26-28, 32) and human (23, 30, 31, 33, 34) RFCs, differing in their 5'-untranslated regions (UTRs). For the mouse RFC gene, the multiple 5'-UTRs arise from alternate noncoding exons that are regulated by two distinct promoters (27, 28), each of which is regulated by multiple Sp1 binding sites (32). Our recent studies of the 5' upstream regulation of the hRFC gene confirmed the presence of 5' transcript heterogeneity resulting from multiple transcriptional start sites and variable splicing of alternate noncoding exons (designated exons 1 and 2 for the KS43 and KS32 5'-UTRs, respectively), each transcribed from separate GC-rich, TATA-less promoters (designated A and B or, previously, Pro32 and Pro43, respectively) (33). Similar results were described for hRFC by Tolner et al. (30) and Williams and Flintoff (31). In this report, we significantly extend this initial characterization of promoter structure and function by identifying the critical transcriptional elements involved in regulating basal levels of hRFC expression. Our results suggest a remarkable complexity of basal transcription that portends possibilities for tissue-specific hRFC transcriptional controls, in response to requirements for folate cofactors or other cell- or tissue-specific signals.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and Reagents-- [gamma -32P]ATP (3000Ci/mmol) was purchased from PerkinElmer Life Sciences. Synthetic oligonucleotides were purchased from Genosys Biotechnologies, Inc. (The Woodlands, TX). Lipofectin® was purchased from Life Technologies, Inc. Restriction and modifying enzymes, reporter gene vectors (pGL3-Basic, pGL3-Pro, pRLSV40), and other molecular biology reagents were obtained from Promega (Madison, WI). pPacSp1 and pPacO plasmid constructs were provided by Dr. Robert Tjian (University of California, Berkeley, CA), and the pPacSp3 and pPacUSp3 constructs were provided by Dr. Guntram Suske (Philipps-Universität, Marburg, Germany).

Cell Culture-- The HT1080 human fibrosarcoma and the HepG2 human hepatoma cell lines were obtained from the American Type Culture Collection (Manassas, VA). Schneider's Drosophila SL2 cells were provided by Dr. Bonnie Sloane (Wayne State University, Detroit, MI). HT1080 cells were grown in RPMI 1640 medium with 10% heat-inactivated iron-supplemented calf serum (Hyclone Labs), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in an atmosphere of 5% CO2 and 95% air at 37 °C. HepG2 cells were cultured in minimum essential medium unmodified with 10% fetal bovine serum and antibiotics at 37 °C. SL2 cells were grown at room temperature in Schneider's Drosophila medium (Sigma-Aldrich) containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and antibiotics.

hRFC-Luciferase Reporter Constructs-- The full-length hRFC-B/-2016 (positions -2016 to -959) and hRFC-A/-958 (positions -958 to -277) promoters were prepared from a hRFC genomic clone (RFCg1-31) as described by Zhang et al. (33). 5'-Deleted hRFC-B (hRFC-B/-1088 and hRFC-B/-1043) and -A (hRFC-A/-501 and hRFC-A/-455) constructs were prepared by successive exonuclease III and S1 nuclease digestions (34), whereas 3' deletions (designated hRFC-B/MluI and hRFC-B/BsrBI) were introduced by restriction endonuclease digestions with MluI and BsrBI, respectively. hRFC-B/-1088 M1 and hRFC-B/-1088 M3, containing a mutated GC-box, and hRFC-A/-501 M1, containing a mutated CRE/AP-1-like element, were prepared by polymerase chain reaction using the following primers: hRFC-B/-1088 M1, 5'-GGACGGACCCGAACACCCCGCAGCCGCG-3'; hRFC-B/-1088 M3, 5'-GGACCCGAACAAACCGCAGCCGCGCGCCCGCCGCGCCGCCTT-3'; hRFC-A/-501 M1, 5'-CGGCCCCCAGCCTGCCCTCCGCGTCCTCCTGGGGCGCCAAGTCCCA-3'. The polymerase chain reaction conditions were as follows: 95 °C/30 s, 68 °C/45 s, 72 °C/45 s for 35 cycles. The amplicons were isolated from 2% LE-agarose and then subcloned into pGemT Easy plasmid (Promega). The inserts were subsequently subcloned into pGL3-Basic in the sense orientation. All mutations were verified by automated DNA sequencing.

Transient Transfections and Reporter Gene Activity-- HT1080 and HepG2 cells were seeded at 1 × 105 cells/well in 6-well dishes 24 h before transfection. The plates used for HepG2 cells were coated with 4 µg/ml poly-L-lysine. RFC-promoter constructs or empty pGL3-basic (4 µg for HT1080, 475 ng for HepG2 cells) were cotransfected with 25 ng of pRLSV40 plasmid using Lipofectin® (10 µg for HT1080, 3 µg for HepG2 cells) according to the manufacturer's protocol. After 24 h, the cells were washed, and then the appropriate medium was added. After an additional 24-48 h, lysates were prepared, and firefly luciferase activity was assayed using the dual luciferase kit (Promega) in a Turner 20/20 luminometer. Firefly luciferase activity was normalized with Renilla luciferase. Drosophila SL2 cells (5 × 105 cells/T12.5 cm2) were transfected with hRFC promoter constructs (1 µg) and 100 ng of pPacSp1, pPacO, pPacSp3, or pPacUSp3 using Fugene 6 (Roche Molecular Biochemicals; 3 µl) in complete media. Transfections were allowed to continue for 48 h before the cells were lysed, and luciferase activity was assayed with the single luciferase reporter assay system (Promega). Relative luciferase activities were normalized to protein concentrations of all the cell lysates. For all transfections, three or more experiments were performed in duplicate.

Gel Mobility Shift Assays-- Nuclear extracts from HT1080 and HepG2 cells were prepared by standard methods (35). 5-10 µg of nuclear extract was incubated for 15 min at room temperature with [gamma -32P]ATP end-labeled hRFC-B (-1088 to -1043) and -A (-501 to -455) double-stranded oligonucleotides in a binding buffer consisting of 20 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 0.1% Triton X, 6 µg of bovine serum albumin, 2 µg of poly[dI-dC], 0.5 mM dithiothreitol, and 10% glycerol. Oligonucleotide competition analysis was conducted with a 100-200-fold molar excess of commercial competitor oligonucleotides (CREB, AP-1, Sp1, mutant Sp1, and mutant CREB; Promega) or a 50-fold molar excess of unlabeled wild-type or mutant hRFC-B and hRFC-A oligonucleotides. For the supershift assays, the DNA-protein complexes were pretreated with anti-Sp1 (Geneka) and Sp3, c-Jun, c-Jun/AP-1, JunB, JunD, c-Fos, Nrf1, CREB-1, ATF-1, and ATF-2 antibodies (Santa Cruz Biotechnology, Inc.). DNA-protein complexes were separated on 4 or 6% nondenaturing polyacrylamide gels in 0.5× Tris borate-EDTA (pH 8.4) at 4 °C and at 35 mA. The gels were dried, and the complexes were visualized by autoradiography.

Western Blot Analysis-- Nuclear extract proteins from HT1080 and HepG2 cells were electrophoresed on 7.5% polyacrylamide gel in the presence of SDS and electroblotted onto polyvinylidene difluoride membranes (DuPont) (36). Detection was with ATF-1, CREB-1, and c-Jun antibodies and enhanced chemiluminescence (Pierce). Light emission was recorded on x-ray film with various exposure times.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of the Basal Promoter Regions for the hRFC-B and -A Promoters-- Previously, our laboratory identified two unique promoters (designated A and B or Pro32 and Pro43) involved in regulating hRFC gene expression (33). Although regions of possible regulatory significance were suggested by 5' deletion analysis, neither the minimal promoters nor the major transcription factors that regulate basal transcriptional activity were elucidated. Accordingly, we prepared a refined series of deletion reporter constructs to better localize the minimal hRFC-A and -B promoters. These constructs were transiently transfected in HT1080 human fibrosarcoma and HepG2 human hepatoma cells, which express moderate to high levels of hRFC transcripts, with a 2-fold higher level of hRFC expression in HepG2 cells (data not shown). By using 5'-rapid amplification of cDNA ends for both cell types, we found that each cell line uses both hRFC promoters,2 making them a good in vitro model for studying hRFC gene expression.

For both cell lines, up to 928 bp could be deleted from the full-length construct (hRFC-B/-2016) with only a slight decrease (30%) of luciferase activity (Fig. 1, panel A). However, upon deletion of an additional 46 bp (to position -1043), promoter activity was completely abolished. When 141 bp were removed from the 3'-end of hRFC-B/-2016 (to position -1099) with MluI (hRFC-B/MluI), activity was similarly abolished.



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Fig. 1.   Deletion analysis of hRFC-B and -A promoter activity of hRFC gene. 5' and 3' deletions were introduced into the hRFC-B (panel A) and -A (panel B) promoters as described under "Experimental Procedures." Promoter constructs in pGL3-Basic were transiently expressed in HT1080 (4 µg of plasmid) and HepG2 (475 ng of plasmid) cells for luciferase assays. Data are reported as relative firefly luciferase activity, normalized to Renilla luciferase activity. S.E. are shown by the error bars.

For promoter A, the levels of luciferase activity were reduced by ~65% for both cell lines upon removal of 457 bp (hRFC-A/-501) from the full-length hRFC-A/-958 construct (Fig. 1, panel B). Luciferase activity was completely abolished by further deletion to position -455 (hRFC-A/-455) (Fig. 1, panel B). Removal of 261 bp from the 3'-end of the full-length hRFC-A/-958 construct (to position -541) with BsrBI (hRFC-A/BsrBI) abolished luciferase activity for promoter A.

These data strongly suggest that the basal promoter for hRFC-B resides within the 46-bp region from -1088 to -1043, and the minimal promoter for hRFC-A can be localized to within the 47 bp between positions -501 and -455.

Identification of Transcription Factors Binding to the Basal hRFC-B Promoters-- The 46-bp basal promoter B is GC-rich (87%), lacks a TATA box, and contains a highly conserved GC-box (position -1081 to -1075), flanked by a potential GT-box (-1077 to -1071), both of which are recognized by the family of Sp transcription factors (Fig. 2A). To assess transcription factor binding to these conserved elements, a double-stranded oligonucleotide (-1088 to -1043), including these elements (hRFC-B/-1088 Wt) was used in gel mobility shift assays. The 32P-labeled oligonucleotide was incubated with 5-10 µg of HT1080 or HepG2 nuclear extracts and analyzed on a nondenaturing polyacrylamide gel.



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Fig. 2.   Schematic of the hRFC-B and hRFC-A basal promoter constructs. Schematics are shown depicting the full-length hRFC-B (panel A) and hRFC-A (panel B) basal reporter constructs from Fig. 1, including the minimal promoters (capital letters) and the 5'-ends of exons 1 and 2, respectively (italicized letters). The GC/GT-box in hRFC-B and CRE/AP-1-like element in hRFC-A are also noted. In panel C, the wild-type and the mutated GC/GT-box and the CRE/AP-1-like elements in the oligonucleotides used in the gel mobility shift experiments and in the promoter constructs in the reporter gene assays are shown.

When incubated with the 46-bp basal promoter B oligonucleotide, both nuclear extracts resulted in three complexes (labeled 1, 2, and 3; Fig. 3A, lanes 2 and 9). All three complexes were competed with a 50-fold molar excess of unlabeled hRFC-B oligonucleotide (Self; lanes 3 and 10), establishing binding specificity. Likewise, the complexes were competed with a 100-fold molar excess of Sp1 consensus oligonucleotide (Fig. 3A, lanes 4 and 11). However, there was no competition with a 200-fold molar excess mutated Sp1 consensus oligonucleotide (Fig. 3A; lanes 5 and 12) or with hRFC-B oligonucleotides in which the GC-box (hRFC-B/-1088 M1; Fig. 2C) or both the GC- and GT-boxes (hRFC-B/-1088 M3; Fig. 2C) were mutated (Fig. 3B, lanes 4, 6, 10, and 12, respectively). However, when the flanking GT-box alone was mutated (hRFC-B/-1088 M2; Fig. 2C), competition was unaffected (Fig. 3B, lanes 5 and 11). Thus, the highly conserved GC-box (CCCGCCC) at position -1081 to -1075 is the major binding element in the hRFC-B basal region.



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Fig. 3.   Binding of Sp1 and Sp3 transcription factors to the -1081 to -1075 bp GC-box in the hRFC-B minimal promoter. Gel shift assays were conducted with HT1080 and HepG2 nuclear extracts as described under "Materials and Methods." Panel A, The -1088 to -1043-bp hRFC-B oligonucleotide was used as a probe in the absence and presence of competitor oligonucleotides or in the presence of anti-Sp polyclonal antibodies. Complexes were resolved on a 4% nondenaturing gel. Lane 1, probe alone (without nuclear extract); lanes 2 and 9, probe plus 5 µg of total nuclear extract for each cell line; lanes 3 and 10, 50-fold molar excess of the unlabeled hRFC-B/-1088 Wt oligonucleotide (Self); lanes 4 and 11, 100-fold molar excess of unlabeled Sp1 consensus oligonucleotide; lanes 5 and 12, 200-fold molar excess of mutated Sp1 consensus oligonucleotide; lanes 6-8 and 13-15, antibodies were added to the binding reactions, including anti-SP1 (lanes 6, 8, 13, and 15), anti-SP3 (lanes 7, 8, 14, and 15). The DNA-protein complexes are numbered, and the supershifted complexes are noted with letters. Panel B, lane 1, probe alone; lanes 2 and 8, probe plus 5 µg of total nuclear extract for each cell line; lanes 3 and 9, 50-fold molar excess of unlabeled hRFC-B/-1088 wild-type oligonucleotide; lanes 4 and 10, 50-fold molar excess of unlabeled hRFC-B/-1088 M1; lanes 5 and 11, 50-fold molar excess of hRFC-B/-1088 M2; lanes 6 and 12, 50-fold molar excess of hRFC-B/-1088 M3; lanes 7 and 13, 100-fold molar excess of Sp1 consensus oligonucleotide. The DNA-protein complexes are numbered.

Based on our competition studies with the promoter B oligonucleotide, supershift analyses were performed. Antibodies to Sp1 and Sp3 were added to the nuclear extracts before the binding reactions. The Sp1 antibody resulted in a supershift in both cell lines (complex A; Fig. 3A, lane 6 and 13). In the presence of the Sp3 antibody, both lower complexes (2 and 3) were supershifted (complex B; Fig. 3A, lanes 7 and 14). When both antibodies were added simultaneously to each nuclear extract, all three complexes were supershifted (complex C; Fig. 3A, lanes 8 and 15). In separate experiments (not shown), we found that purified Sp1 also bound the basal promoter B oligonucleotide. Collectively, these results demonstrate that both Sp1 and Sp3 can bind to the 46-bp minimal promoter B through the highly conserved GC-box at positions -1081 to -1075.

Identification of Transcription Factors Binding to the Basal hRFC-A Promoter-- The 47-bp hRFC-A basal promoter is 76% GC-rich, also TATA-less, and has a CRE/AP-1-like element at positions -485 to -471 (Fig. 2B). In gel mobility shift assays with the 47-bp hRFC-A oligonucleotide (-501 to -455), a single DNA-protein complex was detected on a nondenaturing 6% polyacrylamide gel for both HepG2 and HT1080 cells (Fig. 4, lanes 1 and 7). The complex was competed with a 50-fold molar excess of unlabeled basal promoter A oligonucleotide (Self; Fig. 4, lanes 2 and 8) and with excess (100-fold) commercial AP-1 and CREB consensus oligonucleotides (Fig. 4, lanes 4 and 10, 5 and 11, respectively). However, competition was significantly diminished with a commercial mutant CREB oligonucleotide (Fig. 4, lanes 6 and 12). When a single point mutation was introduced into the CRE/AP-1-like binding site in the hRFC-A oligonucleotide (hRFC-A/-501 M1, Fig. 2C), competition was almost completely inhibited (Fig. 4, lanes 3 and 9). In data not shown, the bZip DNA binding domain from CREB-1 (Santa Cruz Biotechnology) also formed a DNA-protein complex with the 47-bp hRFC-A oligonucleotide.



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Fig. 4.   Binding of CREB/ATF- and AP-1-like proteins to the CRE/AP-1-like element in the hRFC-A minimal promoter. Gel shift assays were conducted with HT1080 and HepG2 nuclear extracts as described under "Materials and Methods." Panel A, The -501 to -455-bp oligonucleotide was used as a probe in the absence and presence of competitor sequences. Complexes were resolved on a 6% nondenaturing gel. Lanes 1 and 7, probe plus 10 µg of total nuclear extract for each cell line; lanes 2 and 8, 50-fold molar excess of unlabeled hRFC-A/-501 wild-type oligonucleotide (Self); lanes 3 and 9, 50-fold molar excess of unlabeled hRFC-A/-501 M1 oligonucleotide; lanes 4 and 10, 100-fold molar excess of AP-1 consensus oligonucleotide; lanes 5 and 11, 100-fold molar excess of CREB consensus oligonucleotide; lanes 6 and 12, 200-fold molar excess of mutated CREB consensus oligonucleotide. The specific DNA-protein complex is designated with an arrow.

Based on our competition results with hRFC-A consensus sequences, supershifts were conducted with antibodies to various members of the CREB/ATF and AP-1 families of transcription factors. On a 6% nondenaturing polyacrylamide gel, CREB-1 antibody supershifted complexes in both HT1080 and HepG2 cells (complex A; Fig. 5A, lanes 3 and 11, respectively). A supershifted complex was detected in HepG2 cells with an ATF-1 antibody (complex B; Fig. 5A, lane 12); however this shift was not detected in HT1080 cells (Fig. 5A, lane 4). Antibodies to c-Jun or c-Jun/AP-1 (recognizes all Jun family members), JunD, JunB, c-Fos (recognizes all Fos family members), or Nrf1 did not result in detectable supershifts for either HT1080 or HepG2 cell lines on a 6% gel (Fig. 5A, lanes 5-9, 13, and 14; not shown for JunD, JunB, or Nrf1 for HepG2 cells). However, a supershift with c-Jun and c-Jun/AP-1 antibodies could be detected on a 4% gel in HT1080 cells (complex C; Fig. 5B, lane 1; data not shown for c-Jun/AP-1). Neither c-Jun nor c-Jun/AP-1 antibodies resulted in a supershift with the HepG2 nuclear extract (Fig. 5B, lane 4, shows data for c-Jun antibody). For the other antibodies included on the 6% gel, shown in Fig. 5A, identical results were obtained on a 4% gel (data not shown).



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Fig. 5.   Supershift analysis of CREB/ATF and AP-1 transcription factor binding to the hRFC-A minimal promoter and Western analysis of cell-specific factors. Gel shift assays were conducted with HT1080 and HepG2 nuclear extracts, as described under "Materials and Methods." Panel A, The -501 to -455-bp fragment was used as a probe in the absence and presence of anti-CREB/ATF and anti-AP-1 antibodies and resolved on a 4% nondenaturing polyacrylamide gel. Lane 1, probe alone; lanes 2 and 10, probe plus 10 µg of total nuclear extract for each cell line; lanes 3 and 11, anti-CREB-1 antibody; lanes 4 and 12, anti-ATF-1 antibody; lanes 5-7 and 13, various anti-Jun antibodies (c-Jun, JunD, JunB, and c-Jun/AP-1, respectively); lanes 8 and 14, anti-c-Fos antibody; lane 9, anti-Nrf-1 antibody. Panel B, supershift complexes were resolved on a 6% nondenaturing polyacrylamide gel. Lanes 1 and 3, probe plus 10 µg of total nuclear extract for each cell line; lanes 2 and 4, anti-c-Jun antibody. Panel C, nuclear extracts (5, 10, 15 µg of protein) were electrophoresed and transferred to polyvinylidene difluoride as described under "Materials and Methods." The membranes were probed with antibodies specific to CREB-1, ATF-1, and c-Jun proteins. Detection was by enhanced chemiluminescence.

Collectively, these results identify a multiplicity of transfactor binding associations with the hRFC-A basal promoter involving members of the superfamily of bZip DNA-binding proteins and the conserved CRE/AP-1-like motif at positions -485 to -471. The identification of complexes involving different members of the bZip superfamily with the hRFC-A minimal promoter suggests that the nature of the DNA-protein complexes formed may be cell-specific. This may reflect differences in the levels of the individual transcription factors between the cell lines.

To assess this possibility, Western analysis was used. As shown in Fig. 5C, CREB-1 was detected at high levels in both cell lines, thus explaining the presence of CREB-1/hRFC-A complexes on our supershifts. However, c-Jun was detected only in HT1080 cells, and ATF-1 was identified only in HepG2 cells (Fig. 5C). These striking differences between c-Jun and ATF-1 are in further agreement with our supershift data. Taken together, they suggest that different cellular levels of c-Jun, CREB-1, and ATF-1 determine the types of DNA-protein complexes that are observed.

Confirmation of Transcription Elements and Promoter Transactivation by Mutagenesis and Transient Transfections-- From our gel shift experiments (Figs. 3-5), the GC-box (position -1081 to -1075) in hRFC-B and the CRE/AP-1-like sequence (-485 to -471) in hRFC-A were implicated in transactivation of the minimal promoters since the DNA-protein complexes were selectively abolished by competitive oligonucleotides (Sp1, and CREB and AP-1, respectively) and were supershifted with specific antibodies to the individual transcription factors (Sp1, Sp3, CREB-1, ATF-1, and c-Jun). However, commercial mutant oligonucleotides (Sp1 and CREB) or hRFC oligonucleotides with mutations in the highly conserved GC-box (hRFC-B/-1088 M1 and hRFC-B/-1088 M3) and the CRE/AP-1-like (hRFC-A/-501 M1) elements were no longer effective competitors (Figs. 3 and 4).

To confirm important transactivating roles for these regions, reporter gene constructs for hRFC-B/-1088 and hRFC-A/-501 containing these point mutations were transiently expressed in HT1080 and HepG2 cells (Fig. 6, A and B, respectively). In HT1080 cells (Fig. 6A, upper panel), the hRFC-B/-1088 M1 and M3 constructs were only 10 and 7%, respectively, as active as wild-type, whereas only slightly greater levels (45 and 35%, respectively) were detected in HepG2 cells (Fig. 6A, lower panel). In both HT1080 and HepG2 cells (Fig. 6, panel B), the hRFC-A promoter construct containing a mutated CRE/AP-1-like element (hRFC-A/-501 M1) resulted in a 60% decrease in luciferase activity compared with the hRFC-A/-501 wild-type construct. These data clearly demonstrate the critical transactivating roles for the GC-box in the hRFC-B minimal promoter and for the CRE/AP-1-like element in the hRFC-A basal region.



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Fig. 6.   Mutational analysis of the GC-box and CRE/AP-1-like consensus sequences in the hRFC-B and the hRFC-A minimal promoters. HT1080 and HepG2 cells were transfected with wild-type and mutant hRFC reporter constructs (depicted in Fig. 2C) as described under "Material and Methods." Panel A, the relative luciferase activities for the wild-type and mutated hRFC-B reporter constructs were plotted for the HT1080 and HepG2 cells. Panel B, the relative luciferase activities for wild-type (Wt) and mutated hRFC-A reporter constructs were plotted for the HT1080 and HepG2 cells. The star indicates that the differences between wild-type and mutant activities were statistically significant (p < 0.05). The error bars represent the S.E.

Sp1 and Sp3 Transactivation of the hRFC-B Basal Promoter in Drosophila SL2 Cells-- Sp1 has been primarily shown to act as a positive transactivating factor (37, 38). Sp3 exists as three isoforms, generated by different translation starts (39), all of which can act (to varying degrees) as activators or repressors of transcription, depending on the cell and promoter context (37-43). To compare the effects of Sp1 and the long and short isoforms of Sp3 on the hRFC-B promoter activity, transient transfection experiments were performed with Drosophila SL2 cells, which provide a negative background for the Sp family of transcription factors (44-46). hRFC-B/-2016 and hRFC-B/-1088 reporter constructs were transfected with expression vectors for Sp1 (pPacSp1) (44) or the short (pPacSp3) or long (pPacUSp3) isoforms of Sp3 (41) under the control of a SL2- specific promoter. Relative luciferase activities for the hRFC reporter constructs were compared with that in cotransfections with an empty expression vector (pPacO). As shown in Fig. 7A, cotransfection with pPacSp1 induced an ~500-fold increase in relative luciferase activity for hRFC-B/-1088 over cotransfection with pPacO. An ~240-fold stimulation by Sp1 was observed for hRFC-B/-2016. Conversely, there was no stimulation of hRFC-A/-501 basal promoter by Sp1. Thus, both the full-length and basal hRFC-B constructs are exquisitely sensitive to transactivation by Sp1. The greater stimulation of the basal construct in this assay implies that negative Sp1 elements or other upstream regions in the full-length promoter are able to interfere with Sp1 transactivation of hRFC-B basal region.



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Fig. 7.   Effects of co-transfected Sp1 and Sp3 isoforms on hRFC-B promoter activity in Drosophila SL2 cells. SL2 cells were co-transfected with 1 µg of the hRFC promoter constructs in pGL3-Basic and pPacSp1, pPacSp3, and pPacUSp3 or empty pPacO vector. Luciferase activity was assayed after 48 h as described under "Materials and Methods." Panel A, luciferase activity was measured in cotransfections of pPacSp1 with hRFC-pGL3-Basic constructs including hRFC-B/-2016, hRFC-B/-1088, and hRFC-A/-501. Data are also shown for transfections in which empty pGL3-Basic was substituted for the promoter-pGL3 constructs. Panel B, dose-dependent transactivation of hRFC-B/-1088 with pPacSp1, pPacSp3, and pPacUSp3. Transfections were supplemented with pPacO vector so that the total of pPacSp plus pPacO vectors was 110 ng. Panel C, cotransfections were performed with hRFC-B/-1088 and a total of 110 ng of expression vector, as shown. The error bars represent S.E.

Fig. 7B shows the effects of transfecting SL2 cells with increasing amounts of pPacSp1, pPacSp3, and pPacUSp3 on hRFC-B/-1088 luciferase activity. Both pPacSp1 and pPacUSp3 exhibited a dose-dependent transactivation of hRFC-B/-1088, differing in potency by up to 4-fold. However, the short Sp3 isoforms were completely inert (Fig. 7B). In combination experiments (panel C), the stimulatory effects of Sp1 and the long Sp3 isoform on hRFC-B transcription were completely additive. Conversely, the short Sp3 isoforms caused a striking decrease (70%) in the ability of Sp1 to transactivate basal promoter B. Thus, Sp1, in combination with the short and long isoforms of Sp3, can exert positive or negative effects on the transactivation of the hRFC-B minimal promoter, apparently by competing for the same DNA consensus sequence. This could represent another level of cell- or tissue-specific regulation for the hRFC gene.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of this study was to identify the basal promoters for the hRFC gene and the transcriptional elements that are responsible for regulating hRFC gene expression. By 5' and 3' deletion analysis, the minimal A and B promoters were localized to positions -501 to -455 and -1088 to -1043, respectively. Gel mobility shift assays including competition and supershift analysis were used to identify the critical transcriptional elements in these core promoter regions. Thus, specific binding by members of the Sp family of transcription factors (Sp1 and Sp3 isoforms) to the highly conserved GC-box in promoter B and members of the superfamily of bZip DNA-binding proteins (CREB-1, ATF-1, and c-Jun) to an CRE/AP-1-like element in promoter A could be demonstrated. This promoter duality should provide a potentially powerful means of ensuring adequate levels of hRFC transcripts (and protein) in response to requirements for folate cofactors or in response to cell- or tissue-specific signals.

Since promoter B is GC-rich, TATA-less, Sp1-regulated, and constitutively expressed, it resembles those for other housekeeping genes, such as dihydrofolate reductase (47) and thymidylate synthase (48), involved in one-carbon transfer pathways that result in purines, pyrimidines, and amino acids. The transcobalamin II receptor (vitamin B12 receptor) is also regulated by Sp1 binding to a GC-box element with an identical consensus sequence to that in basal promoter B (49). The common basal regulatory regions of these assorted genes could potentially explain their ubiquitous expression and may reflect the absolute requirements for these biosynthetically interrelated gene products for cell proliferation.

Sp1 is typically an activator of transcription; however, Sp3 has been found to exert both agonistic and antagonistic effects on gene expression (37-43). Both factors are ubiquitously expressed (37, 38), perhaps explaining why transcripts transcribed from promoter B have predominated in virtually every cell line examined to date.3 When the effects of Sp1 and Sp3 on basal promoter B were examined in Drosophila SL2 cells, both Sp1 and the long Sp3 isoform were able to stimulate transcription; however, the short isoforms of Sp3 were transcriptionally inactive. In cotransfection experiments, the effects of Sp1 and the long Sp3 isoform were completely additive, yet transactivation by Sp1 was potently repressed in the presence of the short Sp3 isoforms. These results strongly suggest that Sp1 and the Sp3 isoforms bind to the same GC-box in basal promoter B and may have different effects on hRFC expression if the stoichiometric ratios between these factors are varied. Changes in cellular levels of Sp1 and Sp3 isoforms could conceivably exert profound effects on the overall expression of hRFC-B in different cell and tissue types. This adds another level of complexity and cell specificity to the already intricate regulation of hRFC gene expression resulting from the use of dual promoters with different transcription elements.

Likewise, the activity of promoter A could be regulated in a tissue-specific manner by different members of the bZip superfamily of DNA binding factors, which bind to the hRFC-A CRE/AP-1-like element. On our gel shifts, CREB-1 was able to form a DNA-protein complex with basal promoter A in both HepG2 and HT1080 cells. Conversely, an ATF-1 promoter complex was only identified in HepG2 nuclear extracts, and a c-Jun complex was only detected in HT1080 cells. Interestingly, these cell-specific differences were closely paralleled by differences in the levels of ATF-1 (detectable in HepG2 cells, virtually undetectable in HT1080 cells) and c-Jun (extremely low levels in HepG2 cells and high levels in HT1080 cells) in nuclear extracts prepared from both cell lines. High levels of CREB-1 were detected in both cell lines. Of course, these findings could possibly be extended to other cell or tissue types for which other members of the AP-1 and CREB/ATF families may be present and result in additional homo-/heterodimeric complexes that bind at the CRE/AP-1-like element in promoter A. It is the complexity of the bZip superfamily and the intricate interrelationships between these factors (50) that underscore the regulation of the hRFC-A minimal promoter and may explain previous reports of the effects of antagonists and agonists of second messenger pathways on hRFC function (51, 52). Clearly the mechanisms that determine differential promoter activities and alternate promoter usage from one cell or tissue type to another is an area that needs to be further explored.

In our studies, the involvement of the GC-box and the CRE/AP-1-like elements on transactivation of the hRFC basal promoters was confirmed by mutating the consensus sequences and transfecting the reporter constructs into HT1080 and HepG2 cells. The mutated GC-box resulted in ~90% or more reduction in promoter activity in HT1080 cells but only 55% or more reduction in HepG2 cells. Similarly, when the CRE/AP-1-like site in hRFC-A promoter was mutated by altering one nucleotide, relative luciferase activity decreased by 60% in both cell lines. Our inability to uniformly abolish promoter activity in these experiments may reflect partial factor binding to the mutated regions (for CRE/AP-1) or conformational interactions between the basal promoters and further downstream regions in the hRFC-A or -B promoter constructs. Alternately, this could reflect important roles of accessory proteins that coordinate transcription factor binding or, perhaps, reflect contributions from elements in the downstream regions of the hRFC promoter constructs. Clearly, the level of transcription ultimately achieved depends on a large number of factors, including the levels of the individual transcription factors and the presence of upstream and downstream inhibitory and activating elements, and the overall promoter architecture.

In conclusion, our results demonstrate that the hRFC-A and -B minimal promoters can be localized to within 47 and 46 bp, respectively. We demonstrate that the GC-box and the CRE/AP-1-like consensus sequences are the major sites of regulation for the basal promoters; however, our findings do not eliminate the possibility that other accessory proteins or downstream transcription elements may also contribute to basal transcriptional activity. Our results suggest a remarkable and surprising complexity of basal hRFC transcription, which portends many possibilities for tissue-specific control of hRFC transcription, in response to cellular requirements for folate cofactors, nucleotides, or other exogenous signals. Future studies will focus on the interrelationships between the basal transcription for each promoter and upstream and downstream regulatory regions. The results of these studies should provide insights into the roles of changes in hRFC gene expression in nutritional folate deficiency and human disease, including possible therapeutic strategies for increasing hRFC gene expression, and also in relation to response and resistance of cancer in patients undergoing chemotherapy with antifolates.


    ACKNOWLEDGEMENTS

We thank Dr. Guntram Suske (University of Marburg, Germany) and Dr. Robert Tjian (University of California, Berkeley, CA) for providing the pPacSp3, pPacUSp3, pPacSp1, and pPacO plasmids for our studies.


    FOOTNOTES

* This study was supported by National Institutes of Health Grant CA53535 (NCI).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Experimental and Clinical Therapeutics Program, Karmanos Cancer Institute, 110 E. Warren Ave., Detroit, MI 48201. Tel.: 313-833-0715 (ext. 2407); Fax: 313-832-7294; E-mail: matherly@kci.wayne.edu.

Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M008074200

2 J. R. Whetstine and L. H. Matherly, manuscript in preparation.

3 J. R. Whetstine and L. H. Matherly, unpublished data.


    ABBREVIATIONS

The abbreviations used are: RFC, reduced folate carrier; hRFC, human RFC; Mtx, methotrexate; 5'-UTRs, 5'-untranslated regions; bp, base pair(s); CRE, cAMP-response element; CREB, CRE-binding protein.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Stokstad, E. L. R. (1990) in Folic Acid Metabolism in Health and Disease (Picciano, M. F. , Stokstad, E. L. R. , and Gregory, J. F., eds) , pp. 1-21, Wiley-Liss, Inc., New York
2. Goldman, I. D., and Matherly, L. H. (1985) Pharmacol. Ther. 28, 77-100[CrossRef][Medline] [Order article via Infotrieve]
3. Jansen, G. (1999) in Anticancer Development Guide: Antifolate Drugs in Cancer Therapy (Jackman, A. L., ed) , pp. 293-321, Humana Press Inc., Totowa, NJ
4. Sirotnak, F. M. (1985) Cancer Res. 45, 3992-4000[Medline] [Order article via Infotrieve]
5. Sirotnak, F. M., and Tolner, B. (1999) Annu. Rev. Nutr. 19, 91-122[CrossRef][Medline] [Order article via Infotrieve]
6. Schuetz, J. D., Matherly, L. H., Westin, E. H., and Goldman, I. D. (1988) J. Biol. Chem. 263, 9840-9847[Abstract/Free Full Text]
7. Wong, S. C., McQuade, R., Proefke, S. A., and Matherly, L. H. (1997) Biochem. Pharmacol. 53, 199-206[CrossRef][Medline] [Order article via Infotrieve]
8. Jansen, 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., and Assaraf, Y. G. (1998) J. Biol. Chem. 273, 30189-30198[Abstract/Free Full Text]
9. Gong, M., Yess, J., Connolly, T., Ivy, S. P., Ohnuma, T., Cowanm, K. H., and Moscow, J. A. (1997) Blood 89, 2494-2499[Abstract/Free Full Text]
10. Sadlish, H., Murray, R. C., Williams, F. M. R., and Flintoff, W. F. (2000) Biochem. J. 346, 509-516[CrossRef][Medline] [Order article via Infotrieve]
11. Sirotnak, F. M., Moccio, D. M., Kelleher, L. E., and Goutas, L. J. (1981) Cancer Res. 41, 4442-4452
12. Zhang, L., Taub, J. W., Williamson, M., Wong, S. C., Hukku, B., Pullen, J., Ravindranath, Y., and Matherly, L. H. (1998) Clin. Can. Res. 4, 2169-2177[Abstract]
13. Gorlick, R., Goker, E., Trippett, T., Steinherz, P., Elisseyeff, Y., Mazumdar, M., Flintoff, W. F., and Bertino, J. R. (1997) Blood 89, 1013-1018[Abstract/Free Full Text]
14. Guo, W., Healey, J. H., Meyeers, P. A., Ladanyai, M., Huvos, A. G., Bertino, J. R., and Gorlick, R. (1999) Clin. Can. Res. 5, 621-627[Abstract/Free Full Text]
15. Refsun, H., Ueland, P., Nygard, O., and Vollset, S. E. (1998) Annu. Rev. Med. 49, 31-62[CrossRef][Medline] [Order article via Infotrieve]
16. Butterworth, C. E., Jr., and Bendich, A. (1996) Annu. Rev. Nutr. 16, 73-97[CrossRef][Medline] [Order article via Infotrieve]
17. Albanes, D., and Simone, A. (1994) Nutr. Cancer 22, 101-119
18. Mason, J. B., and Levesque, T. (1996) Oncology 10, 1727-1743[Medline] [Order article via Infotrieve]
19. Kim, Y. I. J. (1999) Nutr. Biochem. 10, 66-88[CrossRef]
20. Dixon, K. H., Lanpher, B. C., Chiu, J., Kelley, K., and Cowan, K. H. (1994) J. Biol. Chem. 269, 17-20[Abstract/Free Full Text]
21. Williams, F. M. R., Murray, R. C., Underhill, T. M., and Flintoff, W. F. (1994) J. Biol. Chem. 269, 5810-5816[Abstract/Free Full Text]
22. Moscow, J. A., Gong, M., He, R., Sgagias, M. K., Dixon, K. H., Anzick, S. L., Meltzer, P. S., and Cowan, K. H. (1995) Cancer Res. 55, 3790-3794[Abstract]
23. Wong, S. C., Proefke, S. A., Bhushan, A., and Matherly, L. H. (1995) J. Biol. Chem. 270, 17468-17475[Abstract/Free Full Text]
24. Williams, F. M. R., and Flintoff, W. F. (1995) J. Biol. Chem. 270, 2987-2992[Abstract/Free Full Text]
25. Nguyen, T. T., Dyer, D. L., Dunning, D. D., Rubin, S. A., Grant, K. E., and Said, H. M. (1997) Gastroenterology 112, 783-791[Medline] [Order article via Infotrieve]
26. Murray, R. C., Williams, F. M. R., and Flintoff, W. F. (1996) J. Biol. Chem. 271, 19174-19179[Abstract/Free Full Text]
27. Brigle, K. E., Spinella, M. J., Sierra, E. E., and Goldman, I. D. (1997) Biochim. Biophys. Acta 1353, 191-198[Medline] [Order article via Infotrieve]
28. Tolner, B., Roy, K., and Sirotnak, F. M. (1997) Gene 189, 1-7[CrossRef][Medline] [Order article via Infotrieve]
29. Zhang, L., Wong, S. C., and Matherly, L. H. (1998) Biochim. Biophys. Acta 1442, 389-393[Medline] [Order article via Infotrieve]
30. Tolner, B., Roy, K., and Sirotnak, F. M. (1998) Gene 211, 331-341[CrossRef][Medline] [Order article via Infotrieve]
31. Williams, F. M. R., and Flintoff, W. F. (1998) Somatic Cell Mol. Genet. 24, 143-156[Medline] [Order article via Infotrieve]
32. Tolner, B., Singh, A., Esaki, T., Roy, K., and Sirotnak, F. M. (1999) Gene 231, 163-172[CrossRef][Medline] [Order article via Infotrieve]
33. Zhang, L., Wong, S. C., and Matherly, L. H. (1998) Biochem. J. 332, 773-780[Medline] [Order article via Infotrieve]
34. Gong, M., Cowan, K. H., Gudas, J., and Moscow, J. A. (1999) Gene 233, 21-31[CrossRef][Medline] [Order article via Infotrieve]
35. Ausuble, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1998) Current Protocols in Molecular Biology , Vol. 12 , pp. 1.1-12.1.9, John Wiley & Sons, Inc., New York
36. Matsudaira, P. (1987) J. Biol. Chem. 262, 10035-10038[Abstract/Free Full Text]
37. Philipsen, S., and Suske, G. (1999) Nucleic Acids Res. 27, 2991-3000[Abstract/Free Full Text]
38. Suske, G. (1999) Gene 238, 291-300[CrossRef][Medline] [Order article via Infotrieve]
39. Kennett, S. B., Udvadia, A. J., and Horowitz, J. M. (1997) Nucleic Acids Res. 25, 3110-3117[Abstract/Free Full Text]
40. Hagen, G., Müller, S., Beato, M., and Suske, G. (1992) Nucleic Acids Res. 20, 5519-5525[Abstract]
41. Hagen, G., Müller, S., Beato, M., and Suske, G. (1994) EMBO J. 13, 3843-3851[Abstract]
42. Ihn, H., and Trojanowska, M. (1997) Nucleic Acids Res. 25, 3712-3717[Abstract/Free Full Text]
43. Chen, S. J., Artlett, C. M., Jimenez, S. A., and Varga, J. (1998) Gene 215, 101-110[CrossRef][Medline] [Order article via Infotrieve]
44. Courey, A. J., and Tjian, R. (1988) Cell 55, 887-898[Medline] [Order article via Infotrieve]
45. Courey, A. J., Holtzman, D. A., Jackson, S. P., and Tjian, R. (1989) Cell 59, 827-836[Medline] [Order article via Infotrieve]
46. Pascal, E., and Tjian, R. (1991) Genes Dev. 5, 1646-1656[Abstract]
47. Azizkhan, J. C., Jensen, D. E., Pierce, A. J., and Wade, M. (1993) Crit. Rev. Eukaryotic Gene Expression 3, 229-254[Medline] [Order article via Infotrieve]
48. Horie, N., and Takeishi, K. (1997) J. Biol. Chem. 272, 18375-18381[Abstract/Free Full Text]
49. Li, N., Seetharam, S., and Seetharam, B. (1998) J. Biol. Chem. 273, 16104-16111[Abstract/Free Full Text]
50. Shaywitz, A. J., and Greenberg, M. E. (1999) Annu. Rev. Biochem. 68, 821-861[CrossRef][Medline] [Order article via Infotrieve]
51. Said, H. M., Ma, T. Y., Ortiz, A., Tapia, A., and Valerio, C. K. (1997) Am. J. Physiol. 272, C729-C736[Abstract/Free Full Text]
52. Kumar, C. K., Moyer, M. P., Dudeja, P. K., and Said, H. M. (1997) J. Biol. Chem. 272, 6226-6231[Abstract/Free Full Text]


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