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

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

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

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.

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

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,

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-

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

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

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

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

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

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