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(Journal of Leukocyte Biology. 2000;68:772-777.)
© 2000 by Society for Leukocyte Biology

Transcription factor Sp3 activates the liver/bone/kidney-type alkaline phosphatase promoter in hematopoietic cells

Nozomi Yusa, Kunihito Watanabe, Satoru Yoshida, Naoki Shirafuji, Satoshi Shimomura, Kenzaburo Tani, Shigetaka Asano and Noriharu Sato

Department of Laboratory Medicine, The Institute of Medical Science, The University of Tokyo; The Institute of Bio-Medical Research, Teijin Ltd., Tokyo; and Department of Oncology/Hematology, The Institute of Medical Science, The University of Tokyo, Japan

Correspondence: Noriharu Sato, Department of Laboratory Medicine, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minatoku, Tokyo 108-8639, Japan. E-mail: nsato{at}ims.u-tokyo.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The promoter region of the liver/bone/kidney-type alkaline phosphatase gene was examined to define the cis-acting regulatory sequences and transcription factors responsible for its expression in hematopoietic cells. Transient transfection experiments revealed that regions deleted up to -154 base pairs upstream from the transcription initiation site had significant activities to induce bacterial chloramphenicol acetyltransferase gene. The shortest DNA fragment was found to contain three GC boxes in addition to a TATA box. Electrophoretic mobility shift assay and Southwestern analysis showed that Sp3 could bind to the fragment. Western blot analysis also detected Sp3 protein in eluate from the DNA probe mixed with the nuclear extracts. Through the use of Drosophila Schneider cells that lack the Sp1 family of transcription factors, Sp3 was shown to activate the basal promoter in a dose-dependent manner. When the amount of Sp3 was limited, the most proximal GC box was found to be critical for the basal promoter activity.

Key Words: gene regulation • transfection • blood cell • GC box


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of alkaline phosphatase in hematopoietic cells is restricted to neutrophilic granulocytes in the later stages of the maturation pathway. The enzyme, usually called neutrophil alkaline phosphatase (NAP) is the product of the liver/bone/kidney-type alkaline phosphatase gene [1 ]. Two promoters for the liver/bone/kidney-type alkaline phosphatase gene were discovered, and they were reported to be separated from each other by more than 21 kb [2 , 3 ]. The upstream promoter was used mainly in osteosarcoma [2 ] and the downstream one was found to be active in liver [3 ]. These results suggest tissue-specific expression of the gene under the respective promoters in various tissues [2 , 3 ]. Neutrophils were shown to use mainly the upstream promoter [4 ].

To examine further the molecular mechanisms underlying alkaline phosphatase induction in hematopoietic cells, we have cloned the DNA fragment that contains the upstream promoter region of the alkaline phosphatase gene. Using restriction enzyme sites, various 5’-deleted fragments of the promoter region were fused to bacterial chloramphenicol acetyltransferase (CAT) gene. When these constructs were transiently transfected into U937 and Jurkat cells, we detected measurable CAT activities in these cells, suggesting that nuclear factor(s) necessary for the activation of the promoter are ubiquitously present in these cells. The DNA fragment sufficient for the basal promoter activity was defined to -154 base pairs (bp) from the transcription initiation site that contains three GC boxes. When the fragment was used as the probe for electrophoretic mobility shift assay (EMSA), we could detect nuclear proteins that bound to the probe, one of which was supershifted by anti-Sp3 antibody. Using Western and Southwestern methods, Sp3 or a closely related protein was shown to bind to the probe as well. Furthermore, when the reporter plasmid containing the basal promoter region was cotransfected with Sp3 expression vector into Drosophila Schneider cells [5 ] significantly elevated levels of CAT activities were detected, suggesting that Sp3 is really active through GC boxes. The results also indicate that the most proximal GC box is indispensable in the presence of a suboptimal amount of Sp3, whereas each GC box is dispensable in the presence of a large amount of Sp3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of reporter plasmids
We cloned the 5’-flanking sequence of alkaline phosphatase gene with EcoRI ends from the human genomic library. The DNA fragment with approximately 2.5 kb was subcloned into pT7T3 19U vector (Pharmacia) and named p38C6. The DNA fragment (678 bp) produced by treatment of the p38C6 with Ava II as reported elsewhere contained the promoter region from -610 to +68 relative to the transcription start site [2 ]. Hind III linkers were ligated to both ends and the 678-bp fragment was subcloned into pT7T3 19U, now called pAlppF. pRSVCAT obtained from Dr. K. Ozawa, Jichi Medical School, Japan, was cut with both Nde I and Hind III to remove RSV LTR to make p0CAT. The 678-bp fragment with Hind III ends were ligated to p0CAT and was called p-610CAT. Using restriction enzyme sites found in the promoter region, including ApaL I, Hinc II, BssH II, Dde I, Cfr10 I, Apa I, we made 5’-deletions ligated to p0CAT. These reporter plasmids are called p-528CAT, p-446CAT, p-328CAT, p-296CAT, p-154CAT, p-43CAT, respectively (see Fig. 2A). The longest promoter sequence up to the 5’-EcoR I site (changed to Hind III site) with the same 3’-Ava II site (changed to Hind III site) was ligated to p0CAT and called p-1059CAT. p-1059{Delta}(3GC)CAT was made by deleting DNA fragment between -154 and -48 bp from p-1059CAT. The reporter plasmids containing mutated GC boxes (from GGGCGG to GGAAGG) were made by site-directed mutagenesis using p-154CAT as a template. The plasmids containing the most distal, middle, and most proximal GC box mutations were called p-154mGC1CAT, p-154mGC2CAT, and p-154mGC3CAT, respectively. Mutation of these GC boxes was confirmed by sequencing.

Transfection and assay for CAT, luciferase, and ß-galactosidase activities
U937 cells [6 ], Jurkat cells [7 ], HL-60 cells [8 ], and KY821 cells [9 ] were cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS). They were adjusted to 5 x 107 cells/mL, 250 µL of which was used as target cells for electroporation as described [10 ]. We employed one of the following plasmids as control to compensate for the transfection efficiency: SR{alpha}-luci (kindly provided by Dr. S. Watanabe, IMSUT, Japan), and pCH110 (Pharmacia Biotech). NFS-60 cells [11 ] were cultured in RPMI 1640 medium containing 10% FCS and 1% WEHI-3B conditioned medium. NFS-60 cells were transfected by the DEAE-Dextran method [12 ]. Usually, cells were harvested after 48 h of transfection and were treated with the lysis buffer as specified by the manufacturer (Promega Japan). Assay for CAT activity was done as described [13 ] and [14C]chloramphenicol (Amersham Japan) and its acetylated products were analyzed with the AMBIS radioanalytic imaging system (AMBIS Systems, San Diego, CA) after their separation by thin-layer chromatography.

Transfection into Schneider cells
Drosophila melanogaster Schneider line 2 cells were obtained from ATCC and were cultured as described [5 ]. Reporter plasmids were co-transfected with pPacUSp3 (kindly provided by Dr. G. Suske, Philipps-Unv., Marburg, Germany) by the calcium phosphate precipitation method [10 ]. Because control ß-galactosidase activity was affected by Sp3, mean CAT activities produced by the same amount of protein were compared when a dose-response experiment was performed.

EMSA
A 112-bp DNA fragment produced by restriction enzyme treatments of pAlppF with Cfr10 I and Apa I was used as a probe (probe A) . Nuclear extracts (5–10 µg) prepared as described [14 ] were added to 20 µL (final volume) of reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 % glycerol, 1 mM dithiothreitol (DTT), 1 mM EDTA, 100 µg/mL poly (dI-dC) · poly(dI-dC) (Pharmacia) together with a labeled probe with or without competitors. The unrelated competitor used in this study was the 120-bp DNA fragment of 5S rRNA sequence of Pneumocystis carinii amplified as described elsewhere [15 ]. The reaction mixture was left at room temperature (RT) for 30 min and separated by 5% polyacrylamide gel electrophoresis (PAGE). In the case of supershift assay, 2 µL of rabbit antibody against Sp1 or Sp3 (Santa Cruz Biotechnology) or control antibodies (Santa Cruz Biotechnology) were added 1 h before the labeled probe. After electrophoresis, the gel was dried and autoradiographed at -70°C with intensifying screens.

Southwestern and Western blot analyses
Nuclear extract was separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were treated for Southwestern analysis as described [16 ]. The membranes were exposed to X-ray films at -70°C. Western blotting analysis was performed as described [17 ]. The membranes were processed for enhanced chemiluminescence (ECL) Western blotting analysis (Amersham). Densitometric analysis was performed on immunoreactive bands with Densitron CR-20 (Jokoh, Tokyo, Japan).

Digoxigenin-labeled-DNA pull-down experiments
To analyze nuclear proteins binding to DNA probes, we prepared digoxigenin (Dig)-end-labeled primers to amplify DNA fragments containing three GC boxes and mutated three GC boxes as described above. The DNA fragments (0.5 µg) were mixed with the nuclear extracts (50 µg) in 10 mM Tris-HCl, pH 7.5, containing 50 mM NaCl, 5% glycerol, 1 mM DTT, 1 mM EDTA, 100 µg/mL poly (dI-dC) · poly (dI-dC), for 1 h at RT. Then anti-Dig antibody (Boehringer) and protein G-plus/protein A-agarose (Oncogene Science, Cambridge, MA) was added, and the mixture was incubated for another 2 h at 4°C. After washing, proteins binding to the DNA fragments were boiled in sample-loading buffer and separated by SDS-PAGE. The proteins were analyzed by the Western blot method through the use of anti-Sp3 antibody.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The 5’-flanking region of the alkaline phosphatase gene
The 5’-flanking region of the alkaline phosphatase gene was almost the same as reported before except for minor differences (Fig. 1 ). These include one nucleotide deletion at position 488 and insertion of a G at position 504 when compared with the DNA fragment reported previously [2 ]. In addition to the reported sequence, we have sequenced the remaining DNA fragment up to the 5’-EcoR I site (the sequence is available from DDBJ/EMBL/GenBank (accession no AB035417). Minor differences found in the promoter regions did not affect the GC boxes.



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  		Figure 1. Nucleotide sequence of the cloned DNA fragment from upstream promoter region of alkaline phosphatase gene. GC boxes are underlined. The TATA element is indicated by a box.

 
Basal promoter activity of the 5’-flanking region of the alkaline phosphatase gene
Using pRSVCAT, we first examined whether neutrophils could be transfected to examine the cis-regulatory region of the alkaline phosphatase gene. Little CAT activity was detected when 2.5 x 107 cells were used as target cells. Then, we examined several hematopoietic cell lines to determine whether they could be used as target cells for transient transfection. KY821 cells, NFS-60 cells, or HL-60 cells were not good targets by our methods. On the other hand, U937 cells and Jurkat cells were efficiently transfected. According to these results, we decided to focus U937 cells and Jurkat cells as target cells for the experiments throughout. Because these cell lines express little alkaline phosphatase activity by themselves or in response to cytokines, we examined factors affecting the basal promoter activity of the alkaline phosphatase gene. Various 5’-deleted constructs were used to determine the DNA sequence sufficient for the basal promoter activity. As shown in Figure 2B and C , significant promoter activity was detected between -1059 and -154 bp relative to the transcription start site. p-43CAT that had 11 nucleotides in addition to TATA box showed 20% or less CAT activity than the p-610CAT. In contrast, all plasmids that contained longer sequences than p-43CAT produced 50% or more of the CAT activity produced by the p-610CAT. These results indicate that the shortest DNA fragment sufficient for the basal promoter activity is located between -154 and -43 bp upstream from transcription initiation site. To confirm the significance of the 112 nucleotides for the basal promoter activity, we also examined p-1059{Delta}(3GC)CAT that had the longest promoter sequence except for the 112 nucleotides. As shown in Figure 2B and 2C , p-1059{Delta}(3GC)CAT showed little promoter activity in either case, indicating that the 112 nucleotides are indispensable for the basal promoter activity. Because the CAT activities produced by p-1059 plasmid and p-610 plasmid were almost the same, DNA sequence located upstream from the position -610 seemed to be neutral to the basal promoter activity.



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  		Figure 2. Structures of 5’-deletion constructs and assays for the promoter activities. (A) Schematic representations of 5’-deletion constructs. Open oval, GC box; shadowed rectangle, TATA box. Restriction enzyme sites are shown on top of p-1059CAT. (B) Transcriptional assays of promoter constructs (35 µg) transfected into U937 cells. Each bar represents the mean ± SD of three independent experiments and expressed as the percentage of p-610CAT that yielded 9067 cpm (mean value). (C) CAT in Jurkat cells transfected with various 5’-deletion constructs (35 µg). Each bar represents the mean ± SD of three independent experiments and expressed as the percentage of p-610CAT that yielded 59011 cpm (mean).

 
EMSA for the detection of DNA-binding proteins
Results of the transient transfection assay indicated that sequences sufficient for the basal promoter activity are located between -154 and -43 bp from the transcription start site. No putative binding sites for regulatory factors but GC boxes were found between -154 and -43 bp. Tools used for the binding site analysis for transcription factors include TFSEARCH (ver 1.3) and tfsites, available in our institute. EMSA was performed using the 112-bp DNA fragment as a probe (probe A). As shown in Figure 3 , we could detect nuclear proteins binding to the probe in U937 and Jurkat cell extracts. In competition experiments using both nuclear extracts, the slowest bands were completely abolished by the probe A added at 10-fold excess or more, whereas the unrelated competitor weakly affected the bands even at the highest concentration (Fig. 3A and 3B) . Because the fragment contained three GC boxes, we examined the effects of antibodies against Sp1 and Sp3 that were known to bind to the GC box. Supershifted bands were detected in lanes containing anti-Sp3 antibody but not in lanes containing anti-Sp1 antibody (Fig. 3C and 3D) , suggesting that Sp3 is at least one of the nuclear proteins binding the probe. In EMSA using nuclear extracts from Jurkat cells, we sometimes observe one clear slowest band and smears of radioactivity moving faster than that shown in Figure 3D . This may be a result of the difference between nuclear extract preparations.



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  		Figure 3. The nuclear protein binding to the alkaline phosphatase promoter. (A) Nuclear extracts from U937 cells were examined for binding proteins. Lane 1, probe A only (no extract); lane 2, probe A with nuclear extract; lane 3, 10-fold excess of cold probe A; lane 4, 50-fold excess of cold probe A; lane 5, 200-fold excess of cold probe A; lane 6, 10-fold excess of unrelated DNA; lane 7, 50-fold excess of unrelated DNA; lane 8, 200-fold excess of unrelated DNA; lane 9, nuclear extract only. Arrow indicates the band that is specifically abolished by the cold probe A and supershifted by the anti-Sp3 antibody (see below). (B) Nuclear proteins from Jurkat cells were examined as in panel A. (C) Nuclear extracts from U937 cells were examined by specific antibody for Sp1 or Sp3. Lane 1, probe A only; lane 2, probe A with nuclear extract; lane 3, anti-Sp1 antibody; lane 4, anti-Sp3 antibody; lane 5, anti-lck antibody (used as control-1); lane 6, anti-blk antibody (used as control-2). Filled arrow indicates shifted bands. Open arrow indicates the supershifted band. (D) Nuclear extracts from Jurkat cells were examined as described for panel C.

 
Southwestern analysis of the binding protein
The SDS-PAGE gel run with the Jurkat nuclear extracts was transferred to the PVDF membrane and was cut into two; one half was analyzed by the Southwestern method using probe A (lane A), and the other half was analyzed by Western method using anti-Sp3 antibody (lane B). As shown in Figure 4 , two proteins that bound to the probe with molecular mass of approximately 110 and 62 kDa (p110 and p62) were clearly detected in Jurkat nuclear extract (Fig. 4 , lane A). In the parallel experiment, the lane run with the Jurkat nuclear extract was analyzed with anti-Sp3 antibody. We could detect at least three major bands and several minor bands. It is noteworthy that the upper band detected with the Southwestern method (p110) coincided with one of the major bands in lane B and the lower band (p62) did with one of the minor bands, suggesting that these proteins are isoforms of Sp3 or closely related proteins.



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  		Figure 4. Southwestern and Western analysis of the binding protein in Jurkat cell nuclear extracts. Lane A, Southwestern analysis of nuclear proteins from Jurkat cell nuclear extract detected by the probe A. Lane B, Western analysis of nuclear proteins detected by anti-Sp3 antibody. Arrowheads indicate the positions of nuclear proteins detected by the probe A. Positions of molecular mass markers are shown on the left of lane A (kDa).

 
Digoxigenin-labeled DNA pull-down experiment
In vitro pull-down experiments using Dig-labeled DNA fragments followed by Western analysis showed that the amount of p110 binding to probe A was 5 times and that of p62 was 10 times more than that of mutated probe, respectively (Fig. 5 ). Several bands in lanes 2 and 3 that were not found in the untreated nuclear extract (lane 1) might be proteolytic fragments of proteins during in vitro incubation. The prominent bands detected around 64–68 kDa were not detected by Southwestern method but were detected by pull-down assay. The discrepancy might be due to differences of assay conditions; in the former, the proteins were fixed to the membrane during binding assay, whereas in the latter, the proteins were in solution in the binding buffer. The other possibility may be that these bands cannot bind to the probe by itself but are proteolytic products of larger protein(s) that can bind to the probe or can bind to the probe via other proteins.



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  		Figure 5. Dig-labeled-DNA pull-down assay. Jurkat cell nuclear extract (input) was run in lane 1 and detected by anti-Sp3 antibody. Lane 2, proteins binding to the probe A detected by anti-Sp3 antibody. Lane 3, proteins binding to the mutated probe A that has GC to AA substitutions in three GC boxes. Arrowheads indicate positions of the bands detected by Southwestern assay. Asterisks indicate proteolytic fragments of Sp3-related proteins.

 
Cotransfection of Schneider cells with minimal basal promoter carrying either intact or mutated GC boxes and Sp3 expression vector
To obtain the unequivocal evidence supporting the important role for Sp3 transcription factor in the basal promoter activity, we used Schneider cells that are known to have no endogenous Sp1 family members (18, 19). As shown in Figure 6 , the CAT activities were strongly stimulated by the cotransfection with Sp3 in a dose-dependent fashion. Which one of the three GC boxes is important for Sp3 function? To address this issue, we prepared reporter plasmids carrying mutated GC boxes. As shown in Figure 7A , when 1 µg of Sp3 expression vector was co-transfected, no difference in CAT activity was noted among these reporter plasmids, suggesting that each one of the three GC boxes might be dispensable, provided that the other two were intact. On the other hand, when the amount of Sp3 was limited, mutation of the most proximal GC box greatly affected the CAT activity (Fig. 7B and 7C) . Thus, in the presence of 0.5 µg of Sp3 expression vector (Fig. 7B) , p-154mGC3CAT yielded 40% of control. Furthermore, when Sp3 expression vector was reduced to 0.1 µg (Fig. 7C) , p-154mGC3CAT produced 15% of control, whereas p-154mGC1CAT and p-154mGC2CAT showed 130 and 98% of control, respectively. The CAT activities obtained by p-154mGC3CAT and p-154mGC1CAT are statistically significant. This indicates that the most proximal GC box is indispensable for the promoter function under the circumstances when Sp3 expression is very limited.



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  		Figure 6. Effects of various doses of Sp 3 on basal promoter activity when transfected into Schneider cells. A total of 15 µg containing the reporter plasmid (10 µg), Sp3 expression vector, and the empty vector was used. Each represents the percentage of acetylated forms and are expressed as the mean ± SD from triplicate experiments. The amounts Sp3 expression vectors added are shown in abscissa.

 


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  		Figure 7. Effects of mutation in one of three GC boxes on the promoter activity stimulated by the various amounts of Sp3 expression vector. The total dose of 15 µg DNA containing effector and reporter plasmids adjusted with empty vector was transfected into Schneider cells. (A) CAT activities obtained by each reporter plasmid (14 µg) when stimulated by 1 µg of Sp3 expression vector. Activities are shown as the percentage of p-154CAT that yielded 51,769 cpm (mean of triplicates). (B) CAT activities obtained by each reporter plasmid (14 µg) with 0.5 µg of Sp3 expression vector. Values are shown as the percentage of p-154CAT that yielded 22,988 cpm (mean of triplicates). (C) CAT activities obtained by each reporter plasmid (14 µg) with 0.1 µg of Sp3 expression vector. CAT activities are shown as the percentage of p-154CAT that produced 10,853 cpm (mean of triplicates). *P < 0.05; ***P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The reporter construct deleted to -154 bp upstream from the transcription start site had significant promoter activity, whereas that containing -43 to +68 bp DNA fragment had little activity. These results suggest that the basal promoter activity is provided mainly by the fragment between -154 and -43 bp. This DNA fragment contains three GC boxes that are located upstream of the TATA box. The GC box is found in many promoters [20 , 21 ] and Sp1 family of transcription factors including Sp1, Sp2, Sp3, and Sp4 are shown to bind to the consensus GC box or GT box sequences [22 , 23 ]. Sp4 was reported to be detectable mainly in brain [23 ]. Sp2 was found to have little affinity to GC box, and it binds to GT box [24 ]. Accordingly, there remained two Sp1 family of transcription factors for the possible candidate(s) working on the promoter. We could not show, however, a significant participation of Sp1 protein in binding to the basal promoter region by EMSA. Although a truncated form of Sp1 protein was reported in undifferentiated HL-60 cells [25 ], we could detect a significant amount of full-length Sp1 protein in Jurkat cell and U937 cell nuclear extracts by Western blot analysis (data not shown), excluding the possibility that proteolytic activity in these nuclear extracts might mask Sp1 activity. Because the Schneider cell assay revealed the activity of Sp1 protein on the basal promoter (data not shown), the assay conditions employed for the EMSA or Southwestern method might favor the detection of Sp3 protein. On the contrary, we detected Sp3 in Jurkat cell and U937 cell nuclear extracts that bound to the basal promoter region. Sp3 was first reported as the factor that inhibits the function of Sp1 [26 ]. Recently, accumulating evidence indicates that Sp3 also has the ability to stimulate transcription of various promoters [27 28 29 ], suggesting that Sp3 is really a bifunctional transcriptional regulator [30 ].

Southwestern analysis detected two protein bands that specifically bound to the probe (p110 and p62). Because various isoforms of Sp3 gene products have been reported [31 ], other bands that did not bind to the probe might also have derived from the messages for Sp3 gene. Densitometric analysis of Western blot indicated that p110 was approximately five times more than p62, suggesting that Sp3 of 110 kDa is the major protein active in the Jurkat nuclei. In pull-down assays using Dig-labeled intact probe we could detect the same bands. With the mutated probe, faint bands of the same size were detectable after long exposure, although the amount of reactivity was less than 20% of the intact probe. The results suggest that mutated GC boxes may still have a little affinity for Sp3 protein, albeit much reduced when compared with intact GC boxes. In addition, we could detect several other bands that were not found by Western analysis. These may have been proteolytic products of intact proteins. Finally, we showed the potent activity of Sp3 protein on the basal promoter by using Schneider cells that lack endogenous Sp1 family of transcription factors.

Site-directed mutagenesis was undertaken to elucidate which GC box is important for the Sp3 function. When Sp3 was overexpressed, we could detect little difference among three mutated reporter plasmids. Perhaps in this situation most of the two intact GC boxes may be occupied by Sp3 protein. Furthermore, because the mutated GC box seems to have weak affinity for Sp3 protein, overexpression might have forced Sp3 to bind to the mutated GC box, resulting in the enhanced promoter activity in every case. On the other hand, when Sp3 expression was limited, mutation at the most proximal GC box suppressed CAT activity to 15% of control. In this case, only one of the two intact GC boxes may be occupied by Sp3 protein. Furthermore, p-154mGC1CAT showed 130% of control, suggesting that the most distal GC box may be least important for the basal promoter and that the middle GC box may be second-most important. In human glucagon-like peptide-1 receptor, the most distal Sp1 binding site was reported to act as a repressor [19 ]. Our result may be compatible with this report.

NAP is a GPI-anchored membrane protein and NAP was shown to be induced by granulocyte colony-stimulating factor (32). Granulocyte colony-stimulating factor is known to affect various neutrophil functions (33, 34). Although NAP function in neutrophil is largely unknown, we speculate that NAP may play some role in neutrophil functions such as phagocytosis. In fact, alkaline phosphatase was shown to enhance phagocytosis in rat fibroblasts (35). We hope that studies on the regulation of NAP gene may lead to the regulation of neutrophil function at the molecular level.


   ACKNOWLEDGEMENTS
 
The authors are grateful to Carl W. Miller, Ph.D., UCLA, for helpful comments. We also thank Dr. G. Suske for pPacUSp3, Dr. K. Ozawa for pRSVCAT, and Dr. S. Watanabe for SR{alpha}-luci. We are also indebted to Dr. I. Saito (IMSUT, Japan) for providing us with oligonucleotides.

Received April 6, 2000; revised June 2, 2000; accepted June 2, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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T. Aoyama, T. Okamoto, S. Nagayama, K. Nishijo, T. Ishibe, K. Yasura, T. Nakayama, T. Nakamura, and J. Toguchida
Methylation in the Core-promoter Region of the Chondromodulin-I Gene Determines the Cell-specific Expression by Regulating the Binding of Transcriptional Activator Sp3
J. Biol. Chem., July 2, 2004; 279(27): 28789 - 28797.
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