Sp1 and Sp3 are involved in up-regulation of human deoxyribonuclease II transcription during differentiation of HL-60 cells San-Fang Chou 1 , Hui-Ling Chen 2 * and Shao-Chun Lu 1 * 1 Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan; 2 Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan Expression of DNase II in macrophages is potentially cru- cially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up-regulated at the transcriptional level during the phorbol 12-myristate- 13-acetate (PMA)-induced differentiation of HL-60 and THP-1 cells. In this study, we investigated the cis-regulatory elements and transcription factors involved in this process in HL-60 cells. cis-Regulatory elements in the DNase II pro- moter were located by 5¢ deletion and site-directed muta- genesis of promoter-luciferase constructs and transient transfection of HL-60 cells. Furthermore, the binding pro- teins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA-treated HL-60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 pro- teins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up-regulation of DNase II expression during the differentiation of HL-60 cells. Keywords: DNase II; Sp1; Sp3; HL-60; PMA. Deoxyribonuclease II (DNase II; EC.3.1.22.1) is a well known lysosomal acid endonuclease that hydrolyses DNA, producing 3¢-phosphoryl oligonucleotides [1,2]. DNase II activity and mRNA are detected in most human tissues, the highest levels being found in the adrenal gland, thyroid gland, lymph nodes, and pituitary gland [3]. DNase II activity is higher in macrophages than in various nonmacro- phage cell lines and is increased during the differentiation of HL-60 cells and peripheral blood monocytes to macro- phages [4]. Using a single radial enzyme diffusion method, Yasuda et al. [5,6] found that DNase II activity in the Japanese population can be classified into low-activity (DNASE2 L) and high-activity (DNASE2 H), resulting from a genetic polymorphism in the DNase II gene promoter region. However, no association has been found between DNase II activity and disease. Recently, research on DNase II has focused on its role in apoptosis. Barry & Eastman [7] demonstrated that DNase II mediates the digestion of internucleosomal DNA in apop- totic cells. Torriglia et al. [8] have shown that DNase II is involved in the degradation of fiber cell DNA during lens cell differentiation. Furthermore, McIlroy et al. [9] sugges- ted that DNase II is responsible for DNA fragmentation in apoptotic cells after they are engulfed by phagocytic cells. Mice with targeted disruption of the DNase II gene die at birth because of severe anemia [10] and/or asphyxiation [11]; after examination of the DNase II-null embryos, it was suggested that macrophage DNase II is required for degradation of nuclear DNA expelled during erythrocyte maturation [10] and for the digestion of DNA in apoptotic cells [11] during fetal development. These results suggest that macrophage DNase II plays a pivotal role in the removal of Ôunwanted DNAÕ. We previously reportedanincreaseinacid nuclease activity and DNase II mRNA levels during the myelomonocytic differentiation of HL-60 and THP-1 cells and demonstrated that the increase in DNase II mRNA levels was mainly due to transcriptional activation of the gene [4]. In the present study, our aim was to identify cis-regulatory element(s) and transcription factor(s) that mediate the transcriptional acti- vation of the human DNase II gene in HL-60 cells. Using transient transfection and electrophoretic mobility shift assay (EMSA), we demonstrated that binding of Sp1 and/ or Sp3 to three GC-boxes within the proximal region of the DNase II promoter is critical for DNase II transcription in phorbol 12-myristate-13-acetate (PMA)-treated HL-60 cells. Materials and methods Cell culture The human acute promyelocytic leukemia cell line, HL-60, obtained from the ATCC (Manassas, VA, USA), was Correspondence to S C. Lu, Department of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, no. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan 100. Fax: + 886 2 2391 5295, Tel.: + 886 2 2312 3456 ext 8224, E-mail: lsc@ccms.ntu.edu.tw Abbreviations: DNase II, deoxyribonuclease II; EMSA, electropho- retic mobility shift assay; PMA, phorbol 12-myristate-13-acetate. Enzyme: deoxyribonuclease II (DNase II; EC.3.1.22.1). *These authors contributed equally to this work. (Received 12 December 2002, revised 20 February 2003, accepted 3 March 2003) Eur. J. Biochem. 270, 1855–1862 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03551.x grown and induced to differentiate by PMA treatment as described previously [4]. Schneider’s Drosophila cell line 2 (SL2) cells (generously supplied by Y S. Chang, Graduate Institute of Basic Science, Chang-Gung University School of Medicine, Taiwan) were maintained in Schneider’s Insect Medium (Gibco, BRL) supplemented with 10% fetal bovine serum, 50 lgÆmL )1 of streptomycin, and 50 lgÆmL )1 of penicillin at 25 °C with atmospheric CO 2 . Plasmid construction A DNase II promoter-luciferase chimeric gene contain- ing nucleotides )1875 to +72 of the DNase II gene (pDNaseII()1875/+72)-Luc) was constructed as described previously [4]. To construct pDNaseII()934/+72)-Luc and pDNaseII()249/+72)-Luc, pDNaseII()1875/+72)-Luc was digested with SacIandXmaI to remove nucleotides )1875 to )935 or )1875 to ) 250, respectively, and the remaining DNA fragments were ligated using T4 DNA ligase. The fragments )149 to +72, )68 to +72, and )32 to +72 of the DNase II 5¢ flanking sequences were obtained by PCR from pDNaseII()1875/+72)-Luc using specific primers (Table 1), then the PCR products were cloned into the MluI/XhoI sites of the pGL3-basic vector (Promega) to produce pDNaseII()149/+72)-Luc, pDNaseII()68/+72)- Luc, and pDNaseII()32/+72)-Luc. In order to mutate the three GC boxes starting at nucleotides )135, )72 and )45, mutated oligonucleotides were synthesized (Table 1) and used to generate mutants of GC-I, GC-II, and/or GC-III on pDNaseII()249/+72)-Luc by an overlap extension method [11]. All clones were verified by restriction enzyme mapping and sequencing. The Sp1 (pPacSp1) and Sp3 (pPacUSp3) expression plasmids and their maternal plasmid, pPac0, were kindly provided by G. Suske (Philipps-Universitat, Marburg, Germany) [13]. Transfection of HL-60 and SL2 cells HL-60 cells were transfected using the DEAE-dextran procedure as previously described [4]. Briefly, cells (2 · 10 7 ) were collected by centrifugation, resuspended in 1 mL of 25 m M Tris/HCl buffer, pH 7.4, 5 m M KCl, 0.7 m M CaCl 2 ,137m M NaCl, 0.6 m M Na 2 HPO4, 0.5 m M MgCl 2 , containing 5 lgoftestplasmidDNA,5lg of phRL-TK DNA (Promega), and 50 lgÆmL )1 of DEAE-dextran (Sigma), and incubated at room temperature for 15 min. The cells were centrifuged and the pellet was washed, and resuspended in RPMI 1640 medium supplemented with 20% fetal bovine serum, then divided and cultured in the presence or absence of 30 n M PMA (Sigma) for another 48 h before being lysed by addition of 100 lL of Passive Lysis Buffer (Dual-Luciferase Reporter Assay System, Promega). Cell lysates from three dishes transfected with the same construct were pooled. Photinus and Renilla luciferase activities in the lysates were assayed using the Dual-Luciferase Reporter Assay System as described previously [4]. The light intensity produced by Photinus luciferase (test plasmid) was normalized to that produced by Renilla luciferase (control plasmid). Promoter activity was expressed relative to that of cells transfected with pGL3-b (relative value ¼ 1). At least three independent experiments in duplicate were performed using each construct. SL2 cells were transfected using FuGENE 6 (Roach, Indianapolis, IN, USA) according to the manufacturer’s instructions. Briefly, 10, 50, 100, or 150 ng of expression vector (pPacSp1 or pPacUSp3) was mixed with 50 ng of pDNaseII()249/+72)-Luc, and the total amount of DNA adjusted to 200 ng with pPac0. The DNA was mixed with 0.6 lLofFuGENE6in100lL of serum-free Schneider’s Insect Medium (Gibco, BRL) and incubated at room temperature for 5 min. The DNA/FuGENE 6 mixture was then added to 24-well plates, each well containing 5 · 10 5 SL2 cells. Forty-eight hours after transfection, the cells were washed twice with NaCl/P i , then the luciferase activity was measured using the Luciferase Assay System (Promega). Luciferase activity was normalized to total cellular protein. Transfections were performed in duplicate and repeated two to four times to ensure reproducibility and to monitor transfection efficiency. Table 1. Sequences of the oligonucleotides used. mt, mutated. Location Sequence Note Oligonucleotides used for reporter constructs a Forward primers: )149 to )129: 5¢-CGG ACGCGTCGTGGGCGTGGTCTGGGC-3¢ pDNaseII()149/+72) )68 to ) 44: 5¢-AGGA ACGCGTACCCTCGTGATGTCCCCG-3¢ pDNaseII()68/+72) )32 to ) 11: 5¢-CAG ACGCGTTTAGGGAAGTGAAAGGCGCCA-3¢ pDNaseII()32/+72) Reverse primers: +72 to +51 5¢- CTCGAGCTGCTATGGGGCTGAGATCC-3¢ Oligonucleotides used for mutagenesis and EMSA b )151 to )129 5¢-CCCGTCGTGGGCGTGGTCTGGGC-3¢ GC-I )151 to )129 5¢-CCCGTCGTGG TATTGGTCTGGGC-3¢ mtGC-I )89 to ) 64 5¢-CGCGTCTCGGGGGAGTAGTCTGTACC-3¢ GC-II )89 to ) 64 5¢-CGCGTCTCGG TTTAGTAGTCTGTACC-3¢ mtGC-II )61 to ) 36 5¢-CGTGATGTCCCCGCCCCGGTTCCCAG-3¢ GC-III )61 to ) 36 5¢-CGTGATGTCCC AAACCCGGTTCCCAG-3¢ mtGC-III a The underlined ACGCGT and CTCGAG are MluI and XhoI restriction sites, respectively, created to facilitate cloning. b Mutated bases are underlined. 1856 S F. Chou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Nuclear extract preparation Nuclear extracts were prepared as described by Garban et al. [14], with some modifications. Briefly, cells were treatedwith30n M PMA for 60 h and collected by centrifugation, washed twice with ice-cold phosphate-buf- fered saline, and resuspended in 20 volumes of hypotonic lysis buffer (10 m M Hepes/KOH, pH 7.9, 10 m M KCl, 1.5 m M MgCl 2 ,0.5m M dithiothreitol, 0.1% NP-40, and 0.2 m M phenylmethanesulfonyl fluoride). After incubation of the mixture on ice for 15 min, nuclei were pelleted by centrifugation at 500 g for 5 min at 4 °C, washed once with hypotonic lysis buffer, and pelleted again, then nuclear proteins were extracted by incubation of the nuclei for 15 min at 4 °C with intermittent vortexing in 20 m M Hepes/KOH, pH 7.9, 25% glycerol, 420 m M NaCl, 1.5 m M MgCl 2 ,0.2m M EDTA, 0.5 m M dithiothreitol, 0.2 m M phenylmethanesulfonyl fluoride, and 1 · protease inhibitor cocktail (Roche); cell debris was removed by centrifugation at 12 900 g for 10 min at 4 °C. The Bradford method (DC Protein Assay, Bio-Rad) was used to measure the protein concentration in the extract, which was then stored in aliquots at )80 °C. Electrophoretic mobility shift assays The oligonucleotides and complementary oligonucleotides used in the EMSA (Table 1) were custom synthesized. The complementary primers were annealed to each other to produce a double-stranded DNA fragment, which was then 32 P-labeled using Taq DNA polymerase (Invitrogen) and [a- 32 P]dCTP (NEN Life Science Products, Boston, MA, USA). Binding reactions were performed by incubating 5 lg of nuclear extract and 600 fmol of 32 P-labeled double- stranded oligonucleotide, with or without competitor, for 30 min at room temperature in a final volume of 20 lLof binding buffer (20 m M Hepes, pH 7.9, 60 m M KCl, 6 m M MgCl 2 ,0.5m M EDTA, 10% glycerol, 1 m M dithiothreitol, 0.1 lgÆlL )1 of poly dI-dC, 160 lgÆmL )1 of BSA, 0.008% NP-40, and protease inhibitor). Competitors [either a 10- or 50-fold excess of unlabeled wild-type or mutant probe or a 0.6- to threefold excess of Sp1 consensus oligonucleotides (Promega)] were added to the mixture immediately after the labeled probe. For the supershift assay, the nuclear extract was incubated for 1 h on ice with rabbit polyclonal anti-Sp1 or anti-Sp3 IgG (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal antibody to the sterol response element binding protein-1 (SREBP-1; ATCC). The probe was then added and the mixture was incubated for a further 30 min at room temperature and immediately loaded onto a 5% nondenaturing polyacryl- amide gel containing 0.5 · Tris/borate/EDTA (45 m M Tris, 45 m M boric acid, 1 m M EDTA, pH 8.3) buffer. Electro- phoresis was carried out at 4 °C at 250 V. Gels were vacuum heat-dried and analyzed on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). Western blot analysis Nuclear proteins (20 lg of protein per lane) were separ- ated by SDS/PAGE on 10% gels and transferred to a poly (vinylidene difluoride) membrane, which was blocked overnight at 4 °C with blocking buffer (10 m M Tris/HCl, pH 8.0, 0.15 M NaCl, 0.1% Tween 20, and 5% fat-free milk). The blots were then incubated for 1 h at room temperature with 0.5 lgÆmL )1 of rabbit polyclonal anti-Sp1 or anti-Sp3 IgG (both from Santa Cruz Biotechnology) and for 40 min at room temperature with peroxidase-conjugated anti-(rabbit IgG) IgG (Amersham-Pharmacia Biotech), and bound antibody was detected using an improved chemi- luminescence detection system (NEN). Statistical analysis Data were analyzed using STATISCA for WINDOWS v4.5 (StatSoft, Tulsa, OK). Differences between mean values were evaluated using the Duncan’s multiple range test and were considered significant at P <0.05. Results Dissection of the 5¢ flanking sequence of the human DNase II gene To define the regulatory sequences required for transcrip- tion of the DNase II gene, HL-60 cells were cotransfected with a series of 5¢-deleted DNase II-Luc constructs and phRL-TK, a control plasmid containing the gene coding for Renilla luciferase driven by the TK promoter. After transfection, the cells were divided and cultured for 48 h in RPMI 1640 supplemented with 20% fetal bovine serum inthepresenceorabsenceof30n M PMA. As shown in Fig. 1, in non-PMA-treated cells, deletion of nucleotides )1875 to )249 had no significant effect on luciferase activity (P > 0.05); however, deletion to nucleo- tide )149 resulted in a significant lower luciferase activity compared to that seen with pDNaseII()1875/+72)-Luc (P < 0.05). Further deletion to nucleotide )68ledtoa further 82% drop in luciferase activity compared to that seen with pDNaseII()149/+72)-Luc (P < 0.01). Deletion to nucleotide )32 resulted in complete loss of luciferase activity. Fig. 1. Promoter activity of human DNase II-luciferase hybrid genes in HL-60 cells. Schematic representations of the 5¢-deleted promoter- luciferase constructs. The hybrid genes were constructed as described in the Materials and methods. All of the constructs were cotransfected with phRL-TK (internal control) into HL-60 cells using the DEAE- dextran method. Luciferase activity was normalized to Renilla luci- ferase activity and is shown as a relative activity compared to that for pGL3-b. The values are the means ± SD of at least three independent experiments. Ó FEBS 2003 Transcriptional regulation of human DNase II by Sp1 and Sp3 (Eur. J. Biochem. 270) 1857 In contrast, in PMA-treated cells, luciferase activities were significantly higher than in untreated control cells and gradual deletion of 5¢ sequences from nucleotides )1875 to )249 resulted in a gradual increase in luciferase activity (Fig. 1). Maximal activity, seen with pDNaseII()249/+72)- Luc, was 152% that seen with pDNaseII()1875/+72)-Luc (P < 0.05). On further deletion to nucleotide )149, luci- ferase activity fell to 46% of the maximal activity (P < 0.01), and deletion to nucleotide )68 resulted in a substantial reduction to only 2% of the maximal activity. Deletion to nucleotide )32 again resulted in complete loss of luciferase activity. These results show that the region from nucleotide )249 to nucleotide )32 is required for maximal expression of DNase II in HL-60 cells, both in the presence and absence of PMA. Sequence analysis of nucleotides )249 to +72 using the MATINSPECTOR program [15] revealed three GC boxes, referred to as GC-I, GC-II, and GC-III (Fig. 2), starting at nucleotides )135, )72, and )45 relative to the start of transcription. Examination of GC boxes by in vitro mutagenesis and transfection To define the contribution of these three GC boxes to DNase II expression in HL-60 cells, they were mutated, individually or in combination, by an overlap extension method using pDNaseII()249/+72)-Luc as template, then the GC mutant constructs were transiently transfected into HL-60 cells, which were then cultured in the absence or presence of 30 n M PMA and their luciferase activity compared to that of cells transfected with the wild-type construct, pDNaseII()249/+72)-Luc (relative luciferase activity ¼ 100). In non-PMA-treated cells (Fig. 3, upper panel), single mutation of GC-I, GC-II, or GC-III resulted, respectively, in a significant reduction of 48, 70, or 36% in luciferase activity (P < 0.05), while mutation of all three GC boxes ledtoafallof96%(P < 0.01). In PMA-treated cells (Fig. 3, lower panel), mutation of GC-I, GC-II, or GC-III resulted in respective decreases in luciferase activity of 83, 63, or 53% (P < 0.05), and mutation of all three GC boxes resulted in complete loss of promoter activity (P < 0.01). These results show that all three GC boxes are required for maximal activity of the DNase II promoter in both control and PMA-treated HL-60 cells. Electrophoretic mobility shift assays To explore protein binding to these GC boxes, protein- DNA complex formation was examined in vitro using the electrophoretic mobility shift assay (EMSA). When nuclear extracts from control HL-60 cells were incubated with 32 P-labeled probe I, three weak DNA–protein complexes (C1, C2, and C3) were detected (Fig. 4A, lane 2). Significant increases in these complexes and the presence of two additional complexes were detected when the same probe was incubated with nuclear extracts from PMA-treated cells (Fig. 4A, lane 3). The intensity of these complexes was markedly decreased in the presence of a 10- or 50-fold molar excess of unlabeled probe I (lanes 4 and 5), but not in the presence of unlabeled GC-I mutated probe I (lanes 6 and 7). The formation of complexes C1, C2, and C3 was partially blocked by a 0.6- or threefold excess of GC consensus oligonucleotide (Promega) (lanes 8 and 9). In order to verify the involvement of Sp proteins, the nuclear extracts were incubated with anti-Sp1 or anti-Sp3 Ig before addition of Fig. 2. DNA sequence of the human DNase II promoter region. The GC-rich sequences, referred to as GC-I, GC-II, and GC-III, are marked above the sequence. The dashed lines under the sequence indicate the probes used in the EMSA. The numbers show the distance from the transcription start site (+1) [5]. The initiation codon is boxed. Fig. 3. Transient expression analysis of the three GC boxes in the proximal region of the human DNase II promoter. HL-60 cells were transfected with wild-type or GC mutants of pDNaseII()249/+72)- Luc, then were either left untreated (–PMA) or treated with PMA (+PMA) as described in the Materials and methods. The different mutants are shown on the left, the GC box mutated being indicated by a cross. The luciferase activity of the mutant constructs is expressed relative to that of the wild-type construct (relative value ¼ 100). The values are the mean ± SD of at least three independent experiments. 1858 S F. Chou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 the labeled probe. When anti-Sp1 IgG was used, complex C1 disappeared and a new complex, SC1, with a higher molecular mass was formed (lane 11), and, when anti-Sp3 IgG was used, bands C2 and C3 disappeared and bands SC3a and SC3b appeared (lane 12). Coaddition of the two antibodies resulted in the loss of bands C1, C2, and C3 (lane 13). In contrast, the use of a control monoclonal antibody against SREBP did not affect the formation of any of the complexes (lane 14). EMSA experiments using labeled probe II (Fig. 4B) or probe III (Fig. 4C) gave results similar to those shown in Fig. 4A, the main differences being that only three complexes were identified in both control using probe II or probe III and that the GC consensus oligonucleotide eliminated the formation of most of the complexes identified using probe II or probe III (Fig. 4B,C, lanes 8 and 9), but only partially competed for the DNA–protein complexes formed with probe I (Fig. 4A, lanes 8 and 9). These results suggest that both Sp1 and Sp3 are able to bind to the GC boxes and that binding of Sp1 forms complex C1 and binding of Sp3 forms complexes C2 and C3. PMA treatment increases Sp1 and Sp3 protein expression in HL-60 cells Western blotting was used to estimate levels of Sp1 and Sp3 in nuclear extracts from control and PMA-treated HL-60 cells. Using anti-Sp1 IgG, two protein bands with approxi- mate molecular masses of 105 and 95 kDa were detected. The intensity of the 105 kDa band was significantly increased in the PMA treated cells than in the control cells, whereas that of the 95 kDa band was not changed (Fig. 5A). Using anti-Sp3 IgG, three proteins with approxi- mate molecular masses of 110, 70, and 60 kDa were seen, the levels of which were again greatly increased by PMA treatment (Fig. 5B). Overexpression of Sp1 results in increased DNase II promoter activity in SL2 cells Although the EMSA showed more binding of Sp1 and Sp3 to GC boxes in PMA-treated cells compared to untreated cells (Fig. 4), it was not known whether binding of Sp1 and/or Sp3 functionally transactivated the DNase II promoter. To determine whether this was the case, Drosophila SL2 cells were cotransfected with Sp1 or Sp3 expression plasmid (pPacSp1 or pPacUSp3, respect- ively) and either the wild-type pDNaseII()249/+72)-Luc construct or the same construct mutated in all three GC boxes. As shown in Fig. 6A, using wild-type pDNaseII()249/+72)-Luc, a dose-dependent increase in luciferase activity was seen in the presence of increasing amounts of the pPacSp1 plasmid, and a similar, but much smaller, effect was seen using pPacUSp3. In contrast, when pDNaseII()249/+72)-Luc mutated in all three GC boxes was used (Fig. 6B), pPacSp1 or pPacUSp3 had very little effect on luciferase activity. These results show that Fig. 4. Electrophoretic mobility shift assays using probes containing the GC boxes. EMSAs were carried out on nuclear extracts from control (lane 2) or PMA-treated (lanes 3–14) HL-60 cells as described in the Materials and methods using probe I (A), II (B), or III (C) (shown in Fig. 2). Competitions were performed using a 10-fold (10·) or 50-fold (50·) molar excess of unlabeled wild-type or mutant oligonucleotide competitors or a 0.6-fold (0.6·) or threefold (3·)excessofaGCcon- sensus oligonucleotide. Supershift assays were performed using anti- Sp1 and/or anti-Sp3 IgG (lanes 11–13). Anti-(SREBP-1) IgG (lane 14) was used as a negative control. The positions of DNA–protein com- plexes (C) and DNA–protein–antibody complexes (SC) are indicated. Fig. 5. Western blot analysis of Sp1 and Sp3 in nuclear extracts of HL-60 cells. Twenty micrograms of nuclear extracts from untreated (–) or PMA-treated (+) HL-60 cells was separated on a 10% SDS- polyacrylamide gel and immunoblotted using polyclonal anti-Sp1 (A) or anti-Sp3 (B) IgG as described in the Materials and methods. Ó FEBS 2003 Transcriptional regulation of human DNase II by Sp1 and Sp3 (Eur. J. Biochem. 270) 1859 Sp1 and/or Sp3 transactivated the DNase II promoter through the GC boxes. Discussion We have previously shown that DNase II promoter activity increases following chronic exposure of HL-60 cells to PMA, accounting for the observed increase in DNase II mRNA and protein levels and activity [4]. In this study, we showed that 249 bp upstream of the transcription start site were essential for maximal promoter activity in both untreated HL-60 cells and HL-60 cells treated with PMA for 48 h (Fig. 1). Within this region, three GC boxes were located starting at nucleotides )135, )72, and )45. Muta- tion of any one of these GC boxes resulted in decreased promoter activity in both untreated and PMA-treated HL-60 cells, while mutation of all three led to complete loss of promoter activity (Fig. 3), suggesting a critical transcrip- tional role of these GC boxes in HL-60 cells. When analyzing genetic polymorphism of a high (DNASE2*H) and a low (DNASE2*L) DNase II activity allele in man, Yasuda et al. [6] found that DNASE2*H has a G residue at nucleotide )75 of the DNase II promoter, whereas DNASE2*L has an A residue, and a transient transfection assay showed that the DNASE2*H promoter has fivefold higher transcriptional activity than the DNA- SE2*L promoter in HepG2 cells. Nucleotide )75 is located within GC-II, which we found to be critical for DNase II promoter activity in HL-60 cells. Yasuda et al. [6] also showed that deletion of nucleotides )151 to )137, contain- ing GC-I, results in a drastic decrease in promoter activity in HepG2 and TCO-1 cells. In experiments in which we transfected HepG2 cells with wild-type and GC-mutated pDNaseII()934/+72)-Luc, the promoter activity of the GC-I or GC-II mutated form was 42 or 24%, respectively, that of the wild-type construct (data not shown). These results suggest that GC-I is also essential for basal promoter activity of the DNase II gene in HepG2 cells. Figure 4 shows that the binding of Sp1 and Sp3 to the GC boxes was increased in PMA-treated cells. This result could be attributed, at least partly, to significantly increased levels of Sp1 and Sp3 proteins in PMA-treated cells (Fig. 5). Up-regulation of Sp1 protein levels by PMA has been demonstrated in THP-1 cells [16], but Sp3 protein levels were not evaluated. In Drosophila SL2 cells, cotransfection of an Sp1 or Sp3 expression plasmid with wild-type pDNaseII()249/+72)-Luc resulted in an Sp1/Sp3 dose- dependent increase in DNase II promoter, this effect being lost when all three GC boxes were mutated (Fig. 6). Taken together, these results suggest that the PMA-induced expression of Sp1 and Sp3 is involved in the PMA-mediated up-regulation of DNase II expression. In addition to an increase in protein levels, Sp1 may regulate gene expression by changing DNA binding affinity or transcriptional activity. Several reports have shown that phosphorylation or glycosylation of Sp1 regulates its binding and transcrip- tional activities [17–19]. Using anti-Sp1 IgG, two protein bands, with approximate molecular masses of 95 and 105 kDa, were detected on Western blots of nuclear extracts (Fig. 5). The intensity of the 105 kDa band, presumably the phosphorylated form of Sp1 [20], was significantly increased in PMA-treated cells, whereas that of the 95 kDa band was not altered. It is possible that increased levels of the 105 kDa Sp1 contribute to the increased Sp1 binding to GC boxes and DNase II promoter activity. Other mechanisms, such as interactions with other factors, may also be involved in increasing the DNA binding and transcriptional activities of Sp1. As shown in Fig. 4A, two DNA–protein complexes other than C1, C2, and C3 were detected in PMA-treated cells, the formation of which was not affected by addition of anti-Sp1 or anti-Sp3 IgG, indicating they contain proteins other than Sp1 or Sp3. It is not clear whether these unknown factors interact with Sp1 or Sp3, facilitating their binding to GC-I and enhancing their transcriptional acti- vity. On the basis of these results, we cannot rule out the possibility that other factors binding to probe I may interact with Sp1 or Sp3, and promote their DNA binding and transcription activity. Fig. 6. Cotransfection of Drosophila SL2 cells with the human DNase II promoter-Luc chimeric gene and an Sp1 or Sp3 expression plasmid. (A) Drosophila SL2 cells were transfected with 50 ng of wild-type pDNaseII()249/+72)-Luc and increasing amounts (10–150 ng) of Sp1 (pPacSp1) or Sp3 (pPacUSp3) expression plasmid. (B) SL2 cells were cotransfected with 50 ng wild-type or GC-mutated pDN- aseII()249/+72)-Luc and 10 ng of pPacSp1 or pPacUSp3. Luciferase activity was normalized to the protein concentration of the cell lysate and expressed relative to that of cells transfected with wild type or GC-mutated pDNaseII()249/+72)-Luc and pPac0. The values pre- sented are the mean ± SD of at least three independent experiments performed in duplicate. 1860 S F. Chou et al. (Eur. J. Biochem. 270) Ó FEBS 2003 Early studies indicated that Sp1 is responsible for recruiting TATA-binding protein [21] and guiding tran- scriptional initiation [22] at promoters without a TATA box. Recent studies showed that Sp1 is implicated in the transcriptional activation that occurs following a number of different stimuli. Biggs et al. [23] showed that it is involved in the PMA-induced expression of the WAF/CIP1 gene in U937 cells, while Sakamoto and Taniguchi [24] demonstra- ted that Sp1 binding to the PMA-response element mediates the PMA-induced up-regulation of the interferon-c receptor gene in THP-1 cells. Schmitz et al. [16,25] showed that Sp1 acts in concert with AP2 to mediate the PMA-induced transcription of lysosomal acid lipase and acid sphingo- myelinase in THP-1 cells. In this study, we show that Sp1 is involved in PMA-induced expression of DNase II in HL-60 cells. Although, Sp3 has been reported to repress the promoters of the genes coding for uteroglobin [26], the thrombin receptor [27], and HTLV-III [28] by competitively binding to Sp1 binding sites. In this study, transfection of Drosophila SL2 cells with an Sp1 or Sp3 expression plasmid showed that Sp1 is a strong activator, and Sp3 a weak activator, of the DNase II promoter (Fig. 6). In HL-60 cells, PMA treatment also resulted in increased levels of Sp3 protein, the greatest increase being seen in the levels of the 110 kDa protein (Fig. 5). These Sp3 proteins with different molecular masses are presumably derived from 5¢ and internal initiation sites [29]. Noti [30], using an antisense strategy to knock out endogenous Sp3 in HL-60 cells, demonstrated that it is involved in the activation of the CD 11c and CD 11b promoters. The contribution of Sp1 and Sp3 to DNase II promoter activation during HL-60 cell differentiation requires further investigation. In summary, we have demonstrated that DNase II transcription increases during the PMA-initiated differenti- ation of HL-60 cells. Three GC boxes, found within the 249 bp upstream of the DNase II promoter, are essential for both basal and PMA-mediated induction of DNase II transcription. These sites bind Sp1 and Sp3, and protein levels and binding of Sp1 and Sp3 are increased in PMA- treated cells. 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