Báo cáo khoa học: Sp1 and Sp3 are involved in up-regulation of human deoxyribonuclease II transcription during differentiation of HL-60 cells pptx

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 t

Sp1 and Sp3 are involved in up-regulation of human

deoxyribonuclease II transcription during differentiation of HL-60 cells

San-Fang Chou1, Hui-Ling Chen2* and Shao-Chun Lu1*

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
unwanted DNA

We previously reported an increase in acid 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)

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)1of penicillin at 25C with atmospheric CO2

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 SacI and XmaI 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· 107)

were collected by centrifugation, resuspended in 1 mL of

25 mM Tris/HCl buffer, pH 7.4, 5 mM KCl, 0.7 mM CaCl2, 137 mMNaCl, 0.6 mMNa2HPO4, 0.5 mMMgCl2, containing 5 lg of test plasmid DNA, 5 lg 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 nM 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 value¼ 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 lL of FuGENE 6 in 100 lL 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· 105 SL2 cells Forty-eight hours after transfection, the cells were washed twice with NaCl/Pi, 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.

Oligonucleotides used for reporter constructs a

Forward primers:

)149 to )129: 5¢-CGGACGCGTCGTGGGCGTGGTCTGGGC-3¢ pDNaseII( )149/+72) )68 to )44: 5¢-AGGAACGCGTACCCTCGTGATGTCCCCG-3¢ pDNaseII( )68/+72) )32 to )11: 5¢-CAGACGCGTTTAGGGAAGTGAAAGGCGCCA-3¢ pDNaseII( )32/+72) Reverse primers:

+72 to +51 5¢-CTCGAGCTGCTATGGGGCTGAGATCC-3¢

Oligonucleotides used for mutagenesis and EMSAb

)151 to )129 5¢-CCCGTCGTGGTATTGGTCTGGGC-3¢ mtGC-I

)89 to )64 5¢-CGCGTCTCGGGGGAGTAGTCTGTACC-3¢ GC-II

)89 to )64 5¢-CGCGTCTCGGTTTAGTAGTCTGTACC-3¢ mtGC-II

)61 to )36 5¢-CGTGATGTCCCCGCCCCGGTTCCCAG-3¢ GC-III

)61 to )36 5¢-CGTGATGTCCCAAACCCGGTTCCCAG-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.

Nuclear extract preparation

Nuclear extracts were prepared as described by Garban

et al [14], with some modifications Briefly, cells were

treated with 30 nM 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 mM Hepes/KOH, pH 7.9, 10 mM KCl,

1.5 mM MgCl2, 0.5 mM dithiothreitol, 0.1% NP-40, and

0.2 mMphenylmethanesulfonyl fluoride) After incubation

of the mixture on ice for 15 min, nuclei were pelleted by

centrifugation at 500 g for 5 min at 4C, washed once with

hypotonic lysis buffer, and pelleted again, then nuclear

proteins were extracted by incubation of the nuclei for

15 min at 4C with intermittent vortexing in 20 mM

Hepes/KOH, pH 7.9, 25% glycerol, 420 mM NaCl,

1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol,

0.2 mMphenylmethanesulfonyl fluoride, and 1· protease

inhibitor cocktail (Roche); cell debris was removed by

centrifugation at 12 900 g for 10 min at 4C 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

32P-labeled using Taq DNA polymerase (Invitrogen) and

[a-32P]dCTP (NEN Life Science Products, Boston, MA,

USA) Binding reactions were performed by incubating

5 lg of nuclear extract and 600 fmol of32P-labeled

double-stranded oligonucleotide, with or without competitor, for

30 min at room temperature in a final volume of 20 lL of

binding buffer (20 mMHepes, pH 7.9, 60 mMKCl, 6 mM

MgCl2, 0.5 mMEDTA, 10% glycerol, 1 mMdithiothreitol,

0.1 lgÆlL)1of poly dI-dC, 160 lgÆmL)1of 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 mMTris,

45 mM boric acid, 1 mM EDTA, pH 8.3) buffer

Electro-phoresis was carried out at 4C 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 4C with blocking buffer (10 mMTris/HCl,

pH 8.0, 0.15M 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)1of 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

in the presence or ab sence of 30 nMPMA

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 )68 led to a 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.

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 MATINSPECTORprogram [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 byin 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 nM PMA and their luciferase activity

compared to that of cells transfected with the wild-type

construct, pDNaseII()249/+72)-Luc (relative luciferase

activity¼ 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

led to a fall of 96% (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

32P-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.

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 SC3bappeared (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·) excess of a GC

con-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 comcom-plexes (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.

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.

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

DrosophilaSL2 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 11bpromoters 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 These findings indicate that Sp1 and Sp3 play a

pivotal role in the transcriptional activation of DNase II in

HL-60 cells during PMA-induced differentiation

Acknowledgements

We are greatly indebted to Dr Guntram Suske (Philipps-Universitat,

Marburg, Germany) for providing the pPacSp3, pPacUsp3, pPacSp1

and pPac0 plasmids We also thank Dr Yu-Sun Chang (Graduate

Institute of Basic Science, Chang-Gung University School of Medicine,

Taiwan) for providing Drosophila SL2 cells This work was supported

by research grants 89M012 from the National Taiwan University

Hospital, and NSC90-2320-B-002-117 from the National Science

Council of Taiwan.

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