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. 2003 Aug 1;31(15):4523–4530. doi: 10.1093/nar/gkg487

Low pH enhances Sp1 DNA binding activity and interaction with TBP

Takayuki Torigoe 1,2, Hiroto Izumi 1, Yoichiro Yoshida 1, Hiroshi Ishiguchi 1, Takashi Okamoto 3, Hideaki Itoh 2, Kimitoshi Kohno 1,*
PMCID: PMC169877  PMID: 12888513

Abstract

Sp1 is involved in the regulation of a wide variety of genes, including housekeeping genes and genes involved in tumor growth. Sp1 is a member of the C2-H2 zinc-finger family and is important for protection against cellular acidosis in cells that grow under hypoxic or acidic conditions, such as tumor cells. To obtain an insight into the molecular mechanisms underlying pH-dependent transcription by Sp1, both its DNA binding activity and its interaction with TATA binding protein (TBP) were investigated under various pH conditions. We show here that the DNA binding activity of Sp1 increased and Sp1 formed a stable interaction with TBP at low pH. These findings indicate that pH changes significantly modulate the activity of Sp1 and thus contribute to the cellular response under hypoxic or acidic conditions.

INTRODUCTION

Sp1 is a member of a large family of zinc finger proteins and was one of the first transcription factors identified in mammalian cells (1). The Sp1 gene is ubiquitously expressed in a wide variety of mammalian cells, suggesting that mammalian cells require Sp1 for a promoter of essential genes (2). In fact, GC boxes, which are Sp1 binding sites, are often located near a large number of genes involved in cell growth and development (3). Sp1 is required for the transcription of these genes, but its physiological functions are still being unraveled. For example, it has been shown that Sp1 is linked to the maintenance of methylation-free CpG islands (4). Inactivation of the mouse Sp1 gene results in retarded development and embryonic death at day 11 of gestation. Interestingly, the expression of the methyl-CpG binding protein, MeCP2, is significantly decreased in Sp1 knockout mouse embryos (5).

Transcription factors are generally composed of independent domains, for example a DNA binding domain and a transactivation domain, and interact with other proteins, as well as with DNA. Sp1 interacts with E2F and general transcription factors and these physical interactions provide plausible explanations of how proteins that interact with Sp1 could exert synergistic effects (6,7).

Hypoxia, acidosis and a transient decrease in intracellular pH are common characteristics of solid tumors (8). Tumor cells can survive under severely energy-deficient conditions. Therefore, in tumor cells it is favorable for Sp1 to function well at low pH, thus enabling the cells to proliferate rapidly under those conditions. In this work, we demonstrate that the DNA binding activity of Sp1 and its physical interaction with TATA binding protein (TBP) are increased at low pH. This suggests that low pH conditions synergistically induce Sp1-dependent transcription by promoting DNA–protein and protein–protein interactions that induce and stabilize the initiation complex at Sp1-dependent promoters.

MATERIALS AND METHODS

Plasmid constructs

The plasmids containing full-length cDNA fragments of human MeCP2 and TBP were generated by reverse transcription–PCR using total RNA from human epidermoid cancer KB cells. MeCP2 cDNA was cloned into the pGEX-4T vector (Pharmacia), and this is referred to as GST–MeCP2. To construct HA–TBP for expression in Escherichia coli, N-terminal HA-tagged TBP cDNA was cloned into the ThioHis vector (Invitrogen); thioredoxin was deleted from the vector by digestion with NdeI and Acc65I. The following oligonucleotides were used for construction of the cDNAs. MeCP2: 5′-ATGGTAGCTGGGATGTTAGGGC-3′; 5′-TCA GCTAACTCTCTCGGTCACG-3′. TBP: 5′-TGGATCAG AACAACAGCCTGC-3′; 5′-TTACGTCGTCTTCCTGAA TCC-3′. Sp1 cDNA fragment (encoding amino acids 30 to the C-terminus) was kindly provided by Dr Robert Tjian (University of California, Berkeley, CA). Sp3 and YB-1 cDNAs were constructed as described previously (9,10). These cDNA fragments were also cloned into the pGEX-4T vector, creating the constructs GST–Sp1, GST–Sp3 and GST–YB-1, respectively.

To construct the C-terminus truncated proteins GST–Sp1ΔC, GST–Sp1 was digested with PaeI and NotI and then self-ligated after end-filling with T4 DNA polymerase. To obtain GST–Sp3ΔC, GST–Sp3 was digested with Acc65I and SalI and self-ligated after end-filling. GST–Sp1ΔN and GST–Sp3ΔN were constructed by PCR. The following oligonucleotides were used for construction of the truncated proteins. Sp1ΔN: 5′-GGTACCGATCCTGGCAAAAAGAAACAG-3′; 5′-TCAGAAGCCATTGCCACTG-3′. Sp3ΔN: 5′-GCATGC ACCTGTCCCAACTGTAAAGAAG-3′; 5′-TTACTCCAT TGTCTCATTTCCAGAAAC-3′.

Purification of GST fusion proteins

The GST fusion proteins were purified using a glutathione column (Pharmacia) according to the manufacturer’s protocol. The purified GST fusion proteins (100 ng) were separated on a 10% SDS–PAGE gel and detected with Coomassie Brilliant Blue staining.

Electrophoretic mobility shift assay (EMSA)

The purified GST fusion proteins were used directly for EMSA as described previously (10), with a few modifications. Reaction mixtures containing binding buffer [20 mM HEPES–KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 7.5% glycerol], 20 µM ZnCl2, 4 × 104 c.p.m. (2 ng) 32P-labeled oligonucleotide probe and 50 ng GST fusion protein were incubated for 30 min at room temperature in a final volume of 20 µl. The products were analyzed on non-denaturing 4% polyacrylamide gels using a bioimaging analyzer (BAS 2000; Fuji Photo Film, Tokyo, Japan). The following oligonucleotides were used for EMSA (9). GC box (V-ATPase subunit c promoter nucleotides –78 to –49): 5′-AGGCCCCGCCCCGTATGCTAATGAAGCACA-3′; 5′-GTGTGCTTCATTAGCATACGGGGCGGGGCC-3′. Met-GC box (mC indicates methylated C): 5′-GGCCCmC GCCCCGTATGCTAATGAAGCACA-3′; 5′-GGTGTGCTT CATTAGCATACGGGGmCGGGG-3′.

DNA binding assay

To investigate the Sp1–GC box interaction, we performed an in vitro DNA binding assay as described previously (11,12), with a few modifications. GST fusion proteins (100 ng) immobilized on 10 µl glutathione–Sepharose beads were washed three times with binding buffer (described above) at pH values ranging from 6.0 to 8.0 and incubated with 1 × 106 c.p.m. (50 ng) 32P-labeled oligonucleotide probes for 30 min at 4°C. The bound complexes were washed three times with binding buffer (at the relevant pH) and once with binding buffer at pH 8.0. 32P-labeled oligonucleotides recovered from the bound complexes were purified by phenol:chloroform extraction followed by ethanol precipitation. These oligonucleotides were analyzed on non-denaturing 15% polyacrylamide gels using a bioimaging analyzer (BAS 2000; Fuji Photo Film) and quantification of the radioactivity was performed using Cerenkov counting. The GC box oligonucleotide used for the DNA binding assay were described above and the following Y-box oligonucleotide was used for the DNA binding assay. Y-box: 5′-ACTCTGATTGGTCTCACTCC-3′; 5′-GAGACTAACCAGAGTGAGGA-3′.

Cell culture and chromatin immunoprecipitation assay

Human epidermoid cancer KB cells (13) were cultured in Eagle’s minimal essential medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% fetal bovine serum, 0.292 mg/ml l-glutamine, 100 U/ml penicillin and 100 µg/ml kanamycin. To vary the medium pH, the pH was adjusted with 20 mM 2-(N-morpholino)ethane sulfonic acid (MES) and 20 mM Tris (hydroxymethyl)aminomethane (14).

The chromatin immunoprecipitation assay was performed as described previously (9), with a few modifications. KB cells were cultured in normal medium or in acidic medium (pH 6.0) for 1 h. Then cross-linked protein–DNA was incubated with anti-Sp1 or normal rabbit IgG. DNA included in immunocomplexes was purified and resuspended in 10 µl H2O. Aliquots of 1 µl serial dilution were analyzed by PCR with the appropriate primer pairs. The V-ATPase subunit c promoter primers were 5′-CTGCAGACGACGCGCAGCCGCAGA GGAGGC-3′ and 5′-GCGCGAGACCGGTCCAACGCTG CGGAGATC-3′ and the VEGF (vascular endothelial growth factor) promoter primers were 5′-TGCGGGCCAGGCTT CACTGG-3′ and 5′-CCAAGCCTCCGCGATCCTCC-3′. Amplification was performed for a pre-determined optimal number of cycles. PCR products were separated by electrophoresis on 2% agarose gels, which were stained with ethidium bromide. The PCR product using the V-ATPase c promoter primers and that using the VEGF promoter primers contain four GC boxes. The anti-Sp1 antibody (catalogue no. sc-59) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Measurement of zinc content

Soluble GST–Sp1ΔN fusion protein (100 ng) bound to 10 µl of glutathione–Sepharose beads was washed three times with buffer X pH 6.0 or 8.0 (50 mM Tris–HCl, 120 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM PMSF, 1 mM DTT, 10% glycerol) and incubated in buffer X (pH 6.0 or 8.0) containing 0, 50 or 100 µM ZnCl2 for 30 min at 4°C. These samples were then washed five times with buffer X (pH 6.0 or 8.0) without ZnCl2. The quantity of zinc atoms bound to the zinc-finger domain of the GST–Sp1ΔN fusion protein was determined by atomic absorption spectroscopy using a polarized Zeeman atomic absorption spectrophotometer Z-8200 (Hitachi).

Pull-down assay and antibody detection

A pull-down assay was carried out essentially as described previously (10). GST fusion proteins bound to 15 µl of glutathione–Sepharose beads in buffer X (pH 8.0) (described above) were washed three times with buffer X and incubated with soluble anti-hemagglutinin (HA)–TBP fusion protein at 4°C for 2 h. The bound complexes were washed three times with buffer X. Pull-down samples were separated on 10% SDS–PAGE gels, immunoblotted with anti-HA–peroxidase antibody and visualized by chemiluminescence. To analyze the pH dependence of the interaction with HA–TBP, GST fusion proteins immobilized on 15 µl of glutathione–Sepharose beads in buffer X (pH ranging from 6.0 to 8.0) were washed three times with buffer X (relevant pH) and incubated with HA–TBP for 2 h at 4°C. Pull-down samples were washed three times with buffer X (relevant pH) and once with buffer X at pH 8.0 before SDS–PAGE, and detection was carried out as above.

The HA–peroxidase antibody was purchased from Roche Molecular Biochemicals.

RESULTS

Low pH enhances the DNA binding activity of Sp1

Figure 1A illustrates the synthesis of Sp1, Sp3 and the truncated proteins used in this study. Figure 1C indicates that both wild-type and N-terminus truncated Sp1 and Sp3 showed DNA binding activity, as reported previously (15). Analysis by SDS–PAGE followed by Coomassie Blue staining showed that each purification yielded a full-length fusion protein, as well as faster migrating proteins, which are frequently observed due to proteolysis (Fig. 1B).

Figure 1.

Figure 1

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Characterization of GST fusion proteins. (A) Schematic illustrations of the GST–Sp1 and GST–Sp3 fusion proteins and the truncated proteins used in this study. ID and AD indicate the inhibitory and activation domains, respectively. (B) Coomassie Brilliant Blue stained SDS–PAGE gel of purified GST fusion proteins. Purified proteins (100 ng) were electrophoresed on a 10% SDS–PAGE gel. Asterisks indicate the full-length GST fusion proteins. (C) DNA binding activity of GST fusion proteins. The labeled GC box oligonucleotide probe was incubated with 25 or 50 ng of the fusion proteins GST, GST–Sp1, GST–Sp1ΔC, GST–Sp1ΔN, GST–Sp3, GST–Sp3ΔC and GST–Sp3ΔN. The products were analyzed on non-denaturing 4% polyacrylamide gels using a bioimaging analyzer. F indicates the free probe.

To investigate the effect of pH on the DNA binding activity of Sp1, a DNA binding assay was carried out over the pH range 6.0–8.0 in HEPES binding buffer, using a 32P-labeled oligonucleotide probe containing a typical GC box. The binding of Sp1 to the GC box significantly increased at lower pH, whereas in a control experiment, YB-1 binding to an oligonucleotide containing a Y-box was unaffected by the pH change (Fig. 2A). As shown in Figure 2B, the DNA binding activity of Sp1 linearly increased as the pH was lowered from pH 8.0 to 6.0. The binding of Sp1 to the GC box at pH 6.0 is about 7-fold higher than that at pH 8.0. To determine whether the pH dependence of DNA binding is a common characteristic to other members of the Sp1 family, the pH dependence of Sp3 DNA binding was similarly examined. Although the Sp3 DNA binding activity also increased at lower pH, being approximately 3-fold higher at pH 6.0 than at pH 8.0 (Fig. 2B), the extent of the pH dependence was not as great as that of Sp1. In Figure 2C, the results of a DNA binding assay performed using only the zinc-finger domain of Sp1 (Sp1ΔN) and Sp3 (Sp3ΔN) are shown. The C-terminal region containing the zinc-finger domain was sufficient to maintain the pH dependence of the Sp1 and Sp3 DNA binding activity. Cerenkov counting of labeled GC box probe bound to GST (background binding) was about one-tenth of that to GST–Sp1 at pH 8.0.

Figure 2.

Figure 2

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Effect of pH on the Sp1 and Sp3 binding affinity to GC box DNA. (A–C) DNA binding assay using GST fusion proteins. GST fusion proteins immobilized onto glutathione–Sepharose beads were washed with binding buffer (pH range 6.0–8.0) and incubated with 32P-labeled oligonucleotide probes for 30 min at 4°C. 32P-labeled oligonucleotides bound to the GST fusion proteins were purified and serially diluted to 1, 1/3, 1/32 and 1/33 (left to right). (A) Samples were analyzed on non-denaturing 15% polyacrylamide gels using a bioimaging analyzer. (B) Radioactivities were quantified using Cerenkov counting. (C) DNA binding assays using GST–Sp1ΔN and GST–Sp3ΔN. The relative binding activities were calculated by setting the activity under the pH 7.0 condition as 1. Error bars indicate the standard deviation (SD).

In order to show that Sp1 binds specifically to the GC box DNA under low pH conditions in vivo, we utilized the chromatin immunoprecipitation assay as shown in Figure 3. PCR amplifications of both the V-ATPase subunit c and the VEGF promoters were carried out with DNA extracted from the immunocomplexes, because both genes are active in KB cells and there are multiple GC boxes in the core promoter region. Figure 3A shows that a significant increase in the V-ATPase subunit c promoter sequence was detected as 120 bp PCR products in the immunocomplexes prepared under low pH conditions compared with that prepared under normal conditions. The VEGF promoter sequence was detected as a 185 bp product and was also concentrated in the immunocomplexes prepared under low pH conditions (Fig. 3B). Further, the V-ATPase subunit c and VEGF promoter sequences were not observed when normal rabbit IgG was used.

Figure 3.

Figure 3

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Chromatin immunoprecipitation assay with anti-Sp1 antibody. Formaldehyde cross-linked chromatin was isolated from KB cells cultured in normal medium or acidic medium (pH 6.0) for 1 h. Chromatin was immunoprecipitated with anti-Sp1 antibody and normal rabbit IgG. Immunoprecipitated DNA was purified and analyzed by PCR using primers specific for either V-ATPase subunit c promoter (A) or VEGF promoter (B). Immunoprecipitated DNA was serially diluted to 1, 3–1, 3–2 and 3–3 (right to left). Amplification products were electrophoresed in 2% agarose gels containing ethidium bromide. M, DNA ladder mix Marker (MBI Fermentas; Lithuania). Arrowheads show 120 bp DNA of the V-ATPase subunit c promoter sequence (A) and 185 bp DNA of the VEGF promoter sequence (B).

Zinc dependence of Sp1 DNA binding activity

Next, we examined the effect of zinc on the DNA binding activity of Sp1. The addition of ZnCl2 enhanced the DNA binding activity of GST–Sp1 in a dose-dependent manner using EMSA (Fig. 4A). We then carried out a DNA binding assay to investigate the effect of zinc on Sp1 binding activity under different pH conditions. The amount of DNA–Sp1 complex formation is not affected by the addition of ZnCl2 under low pH conditions (Fig. 4B, solid line). In contrast, the addition of ZnCl2 significantly increases DNA–Sp1 complex formation at high pH in a dose-dependent manner (Fig. 4B, broken line). Thus, the Sp1 zinc-finger domain is susceptible to the enhancement of DNA binding activity by zinc ions at pH 8.0, but not at pH 6.0. Next, the zinc content of Sp1 was measured under two different pH conditions. The zinc content of GST–Sp1ΔN remained almost constant at low pH (pH 6.0) when ZnCl2 was added to the binding reaction. However, the zinc content of GST–Sp1ΔN was significantly increased by the addition of ZnCl2 at high pH (pH 8.0) (Fig. 4C).

Figure 4.

Figure 4

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Effect of zinc ions on the Sp1 and Sp3 binding affinity to GC box DNA. (A) Effect of zinc ions on the DNA binding activity of GST fusion proteins in EMSA. Labeled GC box oligonucleotide probes were incubated with GST fusion proteins (50 ng) in binding buffer containing 0, 5, 10 or 20 µM ZnCl2. The products were analyzed on non-denaturing 4% polyacrylamide gels using a bioimaging analyzer. F indicates the free probe. (B) Analysis of the effect of pH and ZnCl2 on Sp1 binding to GC box DNA in the DNA binding assay. GST–Sp1 fusion proteins immobilized on glutathione–Sepharose beads were washed with binding buffer (pH 6.0 or 8.0) containing 0, 10, 20, 40 or 80 µM ZnCl2 and incubated with a 32P-labeled GC box oligonucleotide probe for 30 min at 4°C. The bound complexes were washed with binding buffer (pH 6.0 or 8.0) containing the appropriate concentration of ZnCl2. Radioactivities were quantified using Cerenkov counting. The relative binding activities of Sp1 to GC box DNA, as a function of ZnCl2 concentration, at pH 6.0 and 8.0 are indicated by solid and broken lines, respectively. Error bars indicate the SD. (C) Measurement of the zinc content of GST–Sp1. GST–Sp1ΔN fusion protein bound to glutathione–Sepharose beads was washed three times with buffer X (pH 6.0 or 8.0) and incubated in buffer X (pH 6.0 or 8.0) containing 0, 50 or 100 µM ZnCl2 for 30 min at 4°C. These samples were then washed five times with buffer X (pH 6.0 or 8.0) without ZnCl2. The quantity of zinc atoms bound to the GST–Sp1ΔN fusion protein was analyzed by atomic absorption spectroscopy. The relative zinc contents were calculated by setting the content under the pH 8.0, 0 µM ZnCl2 condition as 1. Error bars indicate the SD.

Low pH also enhances binding activity of Sp1 to methylated DNA

Sp1 is critical for the maintenance of the methylation-free CpG islands and Sp1 can bind to an unmethylated GC box. The ability of Sp1 to bind methylated DNA was also analyzed. Using EMSA, Sp1 was found to bind to both GC box and methylated GC box DNA, but with a slightly lower affinity for methylated GC box DNA (Fig. 5A), as described previously (16,17). MeCP2 was the first member of the MBD (methyl-CpG binding domain) family found to recognize methylated CpG sites (18). Previous work has demonstrated that MeCP2 can recognize methylated GC box DNA and repress Sp1-dependent transcription (19). In order to compare the binding activity of Sp1 and MeCP2 to methylated GC box DNA, EMSA was performed using GST–MeCP2, purified as shown in Figure 1B. MeCP2 was able to bind to the methylated GC box DNA with an affinity over 4-fold higher than that for GC box DNA (Fig. 5A). We then investigated the effect of pH on Sp1 binding activity to methylated GC box DNA using a DNA binding assay. Sp1 bound more strongly to the methylated GC box at low pH (Fig. 5B), while MeCP2 binding to methylated GC box DNA was unaffected by pH (Fig. 5C).

Figure 5.

Figure 5

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Effect of pH on Sp1 and MeCP2 binding to methylated GC box DNA. (A) DNA binding activity of Sp1 and MeCP2 to methylated GC box DNA in EMSA. Labeled GC box or methylated-GC box oligonucleotide probes were incubated with 50 ng GST fusion proteins in binding buffer containing 20 µM ZnCl2. The products were analyzed on non-denaturing 4% polyacrylamide gels using a bioimaging analyzer. F indicates the free probe. (B and C) Effect of pH on Sp1 and MeCP2 binding affinity to methylated GC box DNA in the DNA binding assay. GST–Sp1 (B) and GST–MeCP2 (C) fusion proteins immobilized on glutathione–Sepharose beads were washed with binding buffer (pH 6.0, 7.0 or 8.0) and incubated with 32P-labeled GC box or methylated-GC box oligonucleotide probes for 30 min at 4°C. The bound complexes were washed with binding buffer (pH 6.0, 7.0 or 8.0). Radioactivities were quantified using Cerenkov counting. The relative binding activities were calculated by setting the activity to GC box DNA under the pH 7.0 condition as 1. Error bars indicate SD.

TBP interacts preferentially with Sp1 under low pH conditions

It has been shown that the glutamine-rich activation domains of Sp1 interact with TBP (20). Therefore, it is of interest to examine whether the physical interaction of Sp1 with TBP is also pH dependent. As shown in Figure 6A, both Sp1 and Sp3 interacted directly with TBP in vitro under standard conditions. Next, we examined the effect of pH on the interaction of Sp1 or Sp3 with TBP under different pH conditions (pH 6.0, 7.0 or 8.0). As demonstrated in Figure 6B, TBP interacted strongly with both Sp1 and Sp3 at low pH (pH 6.0). In contrast, at pH 7.0, the interaction of Sp1 with TBP was markedly reduced to about one-tenth of that at pH 6.0, while the interaction of Sp3 with TBP was not affected. At higher pH (pH 8.0), the interaction of both Sp1 and Sp3 with TBP was considerably reduced.

Figure 6.

Figure 6

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pH-dependent interaction of Sp1 and Sp3 with TBP. (A) Interaction of Sp1/3 with TBP in vitro. GST, GST–Sp1 and GST–Sp3 immobilized on glutathione–Sepharose beads were washed three times with buffer X and incubated with soluble HA–TBP fusion protein for 2 h at 4°C. The bound complexes were washed three times with buffer X. Immobilized protein samples were electrophoresed on 10% SDS–PAGE gels, analyzed by immunoblotting with anti-HA–peroxidase antibody and detected by chemiluminescence. (B) Effect of pH on the interaction of Sp1 and Sp3 with TBP. GST, GST–Sp1 and GST–Sp3 immobilized on glutathione–Sepharose beads in buffer X (pH ranging from 6.0 to 8.0) were washed three times with the same buffer and incubated with soluble HA–TBP fusion protein for 2 h at 4°C. The bound complexes were washed three times with buffer X (pH 6.0 to 8.0) and once with buffer X at pH 8.0. Pull-down samples were analyzed on 10% SDS–PAGE gels, immunoblotted with anti-HA–peroxidase antibody and detected by chemiluminescence.

DISCUSSION

A number of biological phenomena are affected by changes in microenvironmental conditions such as temperature, ionic strength (osmolarity) and pH. The cellular pH is intricately involved in the cellular functions of tumor cells, such as invasion/metastasis (21), drug resistance (22), genomic instability (23) and malignant progression (24). The extracellular pH of a solid tumor is generally more acidic than that of normal tissue, since solid tumors adopt a highly glycolytic status for energy production. The ability to up-regulate proton extrusion may be important for cell survival in a tumor (25). CpG island regions span the promoters of housekeeping, growth-associated and tumor suppressor genes, and the Sp1 family proteins are responsible for the transcription of these genes. Tumor cells may utilize the pH-dependent transcription system of growth-associated genes that enables growth under conditions of cellular acidosis.

In this report, we have demonstrated that both the DNA binding activity of Sp1 and its interaction with TBP are enhanced at low pH (Figs 2 and 6). The data presented in Figure 4 also indicate that the DNA binding affinity of Sp1 to GC box DNA under pH 8.0 conditions is significantly increased in a zinc-dependent manner. On the other hand, GC box DNA binding activity of Sp1 at low pH is not. These findings are consistent with the local pH of a solid tumor usually being low and the observation that highly proliferative tumor cells can grow at low zinc concentrations (26,27).

The G-rich oligonucleotides used in this study are prone to adopt alternative conformations. We observed a similar autoradiogram of eluted probes which bound to GST–Sp1 in the DNA binding assay (Fig. 2A), indicating that double-stranded probe can bind to GST–Sp1 under both low and high pH conditions. However, this is not direct evidence that the DNA binding assays were carried out with double-stranded probes. It may be possible that an effect of pH on DNA conformation existed in the DNA binding assays.

Intracellular pH often varies in response to cell growth, transformation and apoptosis (25). Tumor cells have acquired an ability to maintain a transmembrane proton gradient, even under hypoxic conditions. However, intracellular pH decreases in solid tumors and subsequently the cells undergo apoptosis. Cells have developed several membrane transport mechanisms for regulating the intracellular pH. These transport systems are up-regulated by treatment with anticancer agents and by hypoxia (9,28). The promoter regions of these pH regulators contain GC boxes (29,30), suggesting that Sp1 can transactivate the anti-apoptotic genes in response to pH changes. Acidic pH also induces VEGF expression, which contributes to cell survival by increasing the blood supply to solid tumors (31). Several GC boxes are also located in the VEGF promoter. The chromatin immunoprecipitation assay demonstrated that the V-ATPase subunit c and the VEGF promoter sequences containing four GC boxes were efficiently recovered in complexes prepared under low pH culture conditions (Fig. 3). These data indicate that enhancement of Sp1 DNA binding activity and of the Sp1–TBP interaction may contribute to the up-regulation of VEGF or V-ATPase under conditions of cellular acidosis.

The concentrations of Sp1 and Sp3 vary between different cell types and the relative abundance of Sp1 and Sp3 could regulate the activities of target genes. We found that the pH-dependent enhancement of DNA binding activity for Sp1 is higher than that for Sp3. Since the zinc-finger domains of Sp1 and Sp3 are strongly homologous, this difference is probably due to differences in the amino acid sequence of the C-terminal region. Furthermore, we also found that there are some differences in the interaction of TBP with Sp1 and Sp3 under neutral to alkaline conditions (Fig. 6B). This indicates that the cellular pH, as well as the ratio of Sp1/Sp3 expression, is critical for target gene expression. Sp1 has also been shown to interact with other cellular proteins, such as transcription factor E2F and co-activators (6,7,32). It would be interesting to determine whether the interaction of Sp1 with these proteins is pH dependent in tumor cells.

Sp1 is required to prevent methylation of CpG islands (4). It is also essential for normal mouse embryogenesis. Sp1 null mice express many putative Sp1 target genes and, in addition, their CpG islands remain unmethylated because of MeCP2 expressed at a lower level (5). As shown in Figure 5, the binding of Sp1 to methylated GC boxes is enhanced at low pH (Fig. 5B), whereas the binding of MeCP2 to the same sequence was unaffected by pH (Fig. 5C). These results indicate that Sp1 has a higher affinity for a methylated GC box than does MeCP2 under low pH conditions such as in a solid tumor, thus enabling it to transactivate growth-associated genes during tumor progression, even when the GC boxes in the core promoter lesions are methylated.

Transcription factor binding to specific target DNA sequences to activate or repress the expression of associated genes is a basic mechanism of gene expression. In this paper, we have provided evidence that the specificity and stability of both protein–DNA and protein–protein complexes are influenced by the physical environment and, more specifically, by pH. Zinc plays a role in a wide variety of physiological systems, including growth and differentiation, and zinc deficiency accelerates esophageal carcinogenesis in p53 knockout mice (33). In general, cancer patients show a marked decrease in plasma zinc levels, probably due to malnutrition. Under zinc-deficient conditions, tumor cells can activate growth-related genes through Sp1. Our results indicate that Sp1 can function even at low zinc concentrations when the cellular pH is low (Fig. 4), and this may provide a significant advantage for tumor cell growth. Hence, the current study provides additional clues to the understanding of the novel characteristics of Sp1 and the role of Sp1 in solid tumors. However, direct proof for an in vivo function of Sp1 will be necessary to substantiate the conclusions based on in vitro data.

Acknowledgments

ACKNOWLEDGEMENTS

This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a research grant from the Princess Takamatsu Cancer Research Fund (99-23106), by Astra Zeneca research grant 2002 and by the Japan Medical Association.

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