Tài liệu Báo cáo khoa học: Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation ppt

Characterization of human deoxyribonuclease I gene
(DNASE1) promoters reveals the utilization of two
transcription-starting exons and the involvement of Sp1
in its transcriptional regulation
Yoshihiko Kominato
1
, Misuzu Ueki
2
, Reiko Iida
3
, Yasuyuki Kawai
4
, Tamiko Nakajima
1
, Chikako
Makita
1
, Masako Itoi
1
, Yutaka Tajima
1
, Koichiro Kishi
1
and Toshihiro Yasuda
2
1 Department of Legal Medicine and Medical Genetics, Gunma University, Japan
2 Division of Medical Genetics and Biochemistry, University of Fukui, Japan
3 Division of Legal Medicine, University of Fukui, Japan
4 Third Division of Internal Medicine, University of Fukui, Japan
Deoxyribonuclease I (DNase I, EC 3.1.21.1) is an
enzyme that preferentially attacks double-stranded

DNA by Ca
2+
- and Mg
2+
⁄ Mn
2+
-dependent endo-
nucleolytic cleavage to produce oligonucleotides with
5¢-phosphoryl- and 3¢-hydroxy termini [1,2]. DNase I
is considered to play a major role in the digestion of
dietary DNA, because in vertebrates it is secreted by
exocrine ⁄ endocrine glands such as the pancreas and
parotid gland into the alimentary tract [3–5]. However,
the presence of the enzyme in mammalian tissues other
than the digestive organs [6–8] suggested that it might
have other function(s) in vivo; endogenous DNase I
has been regarded as a candidate endonuclease respon-
sible for internucleosomal DNA degradation during
apoptosis [9]. Furthermore, Napirei et al. have shown
that extracellular (serum) DNase I participates in the
chromatin breakdown of necrotic cells, achieved by
its diffusion from the extracellular fluid into the
Keywords
alternative splicing; deoxyribonuclease I;
genes; promoter; Sp1
Correspondence
T. Yasuda, Division of Medical Genetics and
Biochemistry, Faculty of Medical Sciences,
University of Fukui, Eiheiji, Fukui 910-1193,
Japan

Fax: +81 776 61 8149
Tel: +81 776 61 8287
E-mail:
Database
The nucleotide sequences reported here
have been submitted to the GenBank/
EMBL/DDBJ Data Bank with accession
numbers AB188151 and AB188152
(Received 22 March 2006, revised 8 May
2006, accepted 15 May 2006)
doi:10.1111/j.1742-4658.2006.05320.x
Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown
to be altered by physiological and ⁄ or pathological processes. However, no
information is available on the regulation of DNase I gene (DNASE1)
expression in vivo or in vitro. We first mapped the transcription start sites
of DNASE1 in human pancreas and in the DNase I-producing human pan-
creatic cancer cell line QGP-1, and revealed a novel site  12 kb upstream
of exon 1, which was previously believed to be the single transcription-
starting exon. This initiation site marks an alternative starting exon,
designated 1a. Exons 1 and 1a were used simultaneously as transcription-
starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and
chromatin immunoprecipitation analysis with QGP-1 cells showed the pro-
moter region of exon 1a in which the Sp1 transcription factor is specifically
involved in promoter activity. This is the first to be identified as a tran-
scription factor responsible for gene expression of vertebrate DNase I
genes. Furthermore, RT-PCR analysis indicated alternative splicing of
human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two tran-
scripts among eight alternative splicing products identified can be transla-
ted to produce intact DNase I protein. These results suggest that human
DNASE1 expression is regulated through the use of alternative promoter

and alternative splicing.
Abbreviations
AMI, acute myocardial infarction; ChIP, chromatin immunoprecipitation; DNase I, deoxyribonuclease I; DNASE1, DNase I gene; EMSA,
electrophoretic mobility shift assay; PCI, percutaneous coronary intervention; SRED, single radial enzyme diffusion.
3094 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
cytoplasm and nucleus of such cells [10]. In a similar
context, DNase I has been postulated to be responsible
for the removal of DNA from nuclear antigens at sites
of high cell turnover and necrosis, and thus for the
prevention of systemic lupus erythematosus [11,12].
Recently, we demonstrated that an abrupt elevation of
serum DNase I activity occurs within  3 h of the
onset of symptoms in patients with acute myocardial
infarction (AMI) and that DNase I activity in serum
then exhibits a marked time-dependent decline within
12 h, returning to basal levels within 24 h [13]. More-
over, percutaneous coronary intervention (PCI), which
is performed to treat patients with stable angina pec-
toris, offers an in vivo model of mild myocardial ische-
mia in humans. Irrespective of a lack of alteration in
levels of other conventional cardiac markers such as
creatine kinase isoenzyme MB and cardiac troponin T,
serum DNase I levels rose significantly from basal lev-
els by 3 h after completion of the PCI procedure,
returning to basal levels by 12–24 h, in a manner sim-
ilar to in AMI [14]. These findings permitted us to sug-
gest that myocardial ischemia rather than injury
induces such elevation in serum DNase I activity.
However, the mechanisms for the elevation of serum
DNase I activity induced by ischemia during AMI or

PCI remain to be elucidated. Delineation of the
molecular basis for our observations is essential to
evaluate the elevated DNase I activity in the sera of
patients with AMI and to validate the use of serum
DNase I activity as a new diagnostic marker for the
early detection of AMI. To elucidate the molecular
basis of this phenomenon, it is important to under-
stand the regulatory mechanism of the human DNase I
gene (DNASE1) expression.
Previous molecular–genetic studies have shown that
the human DNase I gene consists of at least nine
exons spanning > 3.2 kb of genomic DNA at chromo-
some 16p13.3; exon 2 includes the translation initiation
codon (ATG) and the first exon is believed to include
only the 5¢-UTR of the mRNA [15,16]. However, com-
parison of the sequence of the 5¢-UTR of human pan-
creatic DNASE1 mRNA reported previously [15]
with the human genomic sequence showed that the
5¢-terminal 15 nucleotides of the 5¢-UTR are not found
in the human genomic sequence [16], whereas the 3¢
segment of 143 nucleotides in the 5¢-UTR matches the
genomic sequence. Thus, the transcription initiation
site of exon 1 has not yet been definitively identified.
To our knowledge, no information is available on the
regulation of vertebrate DNASE1 expression in vivo or
in vitro, including characterization of the promoter
region of the gene and the associated transcriptional
factors. Therefore, delineation of the transcriptional
regulation of human DNASE1 may provide clues to
the mechanisms underlying ischemia-induced elevation

of DNase I activity in vivo.
Here, we describe the identification of a novel tran-
scription starting exon, designated as exon 1a, in
human DNASE1 and characterization of the promoter
regions of the gene; Sp1 transcription factor plays an
important role in promoter activity in the 5¢-upstream
region of DNASE1 exon 1a.
Results
Mapping of transcription initiation sites in the
human DNASE1 gene
To identify the transcription initiation sites of
DNASE1 in pancreas, 5¢-RACE based on RNA ligase-
mediated and oligo-capping RACE [17] was performed
with cDNA synthesized from pancreas. Agarose gel
electrophoresis of the 5¢-RACE products showed a
slow-migrating major band and several faster-migra-
ting faint bands. Alternative splicing occurs in many
genes, and so DNA fragments were purified from the
major band and cloned into a sequencing vector. DNA
sequences were determined for seven transformant
clones. Four clones contained a 476-bp 5¢-RACE
DNA product, of which the 3¢ 380 bp were identical to
exons 1–3 from the 5¢-end of the reverse primer
DN+231 to the 5¢-end of the region where the
sequence of exon 1 reported previously [15] matched
with the sequence deposited in GenBank (Accession
no. AC006111) (Fig. 1A). The sequence of the 5¢ por-
tion beyond that point was identical to a 96-bp region
of the genomic DNA, indicating that the entire
sequence of exon 1 comprises 243 bp.

Human DNase I activity is mainly demonstrable in
pancreas, alimentary tract and pituitary [3,18], and
most serum DNase I appears to be produced from
those tissues. Moreover, our survey of DNase I-produ-
cing cell lines showed a high level of DNase I activity
and its transcripts in QGP-1 cells, which were estab-
lished by Kaku et al. [19] from a human pancreatic
islet cell carcinoma (possibly D cells) as a carcinoem-
bryonic antigen-secreting cell line. A similar 5¢-RACE
was performed with cDNA synthesized from total
RNA of QGP-1 cells. The DNA sequences of the
5¢-RACE products were determined for five transform-
ant clones. Four clones contained a 417-bp 5¢-RACE
DNA product that appeared to be a hybrid between
exon 1 and the upstream genomic DNA of DNASE1:
the sequence of the 3¢ portion in those products was
identical to that of exons 1–3 from the 5¢-end of the
reverse primer DN+231 to a position +156 relative
Y. Kominato et al. Promoters of human deoxyribonuclease I gene
FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3095
A
B
Fig. 1. The nucleotide sequence of the
5¢-flanking region in the human DNase I
gene. (A) The sequence located between
positions )100 and +250 relative to the
5¢-end in DNASE1 exon 1. +1 above the
sequence indicates the 5¢-end of exon 1.
Open circles indicate locations of 5¢-ends
of the DNASE1 transcripts, determined by

5¢-RACE using cDNA obtained from pan-
creas. The star indicates the 5¢-end of the
DNASE1 mRNA reported previously [3].
Exon 1 (nucleotides +1 to +243) is indica-
ted by uppercase letters within rectangles.
The vertical line between positions +155
and +156 represents the splicing junction
where exon 1a is ligated to the part of
exon 1 between positions +156 and +243.
(B) The sequence located between posi-
tions )200 and +150 relative to the tran-
scription start site in human DNASE1
exon 1a. Upstream counting is done from
+1 of exon 1. )11871 above the sequence
indicates the 5¢-end of exon 1a. The num-
bers in parentheses demonstrate the posi-
tion of the corresponding nucleotides
relative to the transcription start site in
exon 1a. Open circles indicate locations of
5¢-ends of the DNASE1 transcripts, deter-
mined by 5¢-RACE using cDNA obtained
from QGP-1 cells. Exon 1a (nucleotides
)11871 to )11770) is indicated by upper-
case letters within rectangles. Several puta-
tive transcription factor binding sites were
found using
TRANSFAC software and are indi-
cated by overbars. The underlines repre-
sent the locations of the two
oligonucleotide probes used for further ana-

lysis. The position and identity of mutations
at )11944 to )11941 are indicated in oligo-
nucleotide m91,50 and reporter construct
)197HmSp.
Promoters of human deoxyribonuclease I gene Y. Kominato et al.
3096 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
to the beginning of exon 1, as shown in Fig. 1A.
Beyond that point, however, the sequence of the 5 ¢
portion showed 100% identity with that of genomic
DNA  12 kb upstream of the DNASE1 exon 1
(Fig. 1B). More interestingly, the products lacked the
sequence between positions +1 and +155 in exon 1.
This comparison with the upstream genomic sequence
of DNASE1 allowed us to demonstrate the presence of
an alternative exon, which we named exon 1a. The
donor splice site between exon 1a and the subsequent
intron had GT, whereas the acceptor site between the
subsequent intron and the 5¢-end at position +156 in
exon 1 had AG. Therefore, the splice sites seem to be
compatible with a splicing junction.
Confirmation of utilization of exon 1a as
a transcription initiation exon and occurrence
of alternative splicing
To examine whether exon 1a is used as a transcription
starting exon in QGP-1 cells and pancreas, and to con-
firm the splicing junction between exon 1 and the
upstream DNA, RT-PCR was carried out using a pri-
mer specific for exon 1a and a reverse primer comple-
mentary to the sequence in exon 8. DNA fragments of
different sizes were amplified from the RNA of QGP-1

cells and pancreas (Fig. 2A). Determination of the
nucleotide sequences of the RT-PCR products revealed
1a 1 2 3 5 6 7 8 94Exons
DN-110
DN-144
DN+89
RT-PCR amplifi
e
dfragments
895 bp
877 bp
929 bp
789 bp
764 bp
A
C
H
I
J
DN+232 DN+254 DN+785
841 bp
D
755 bp
F
806 bp
E
730 bp
G
881 bp
B

DN+385 DN+721
DN-105
DN-9
+1
A
B
Fig. 2. RT-PCR analysis to detect the transcription-starting exon in
DNASE1 of QGP-1 cells and pancreas. (A) RT-PCR analysis. Total
RNA prepared from QGP-1 cells or pancreas was reverse-tran-
scribed with random primer, and the resulting single-strand cDNA
was used as a template for PCR analysis. The DNASE1 amplifica-
tion was performed using either distinct starting exon-specific pri-
mer DN)110 (left) or DN)144 (right) and a common reverse primer
DN+785 complementary to exon 8 of the DNASE1. PCR products
were electrophoresed through a 1.5% agarose gel and stained with
ethidium bromide. The amplified fragments were named A–J. A
1 kb Plus DNA Ladder was used as a molecular size marker. (B)
Splicing patterns of the amplified fragments A–J. Nucleotide
sequences of these fragments were determined and then com-
pared. Schematically represented DNASE1 was aligned with the
RT-PCR products amplified, using a set of each starting exon-speci-
fic primer (DN)110 or DN)144) and the DN+785 primer, which are
represented by arrows. Open boxes represent the DNASE1 exons,
and a vertical broken line indicates the splicing junction between
exon 1a and a portion of exon 1. The thick straight lines represent
the intron sequence. +1 indicates the position of the transcription
start site of exon 1. Dashed v-shaped lines in RT-PCR amplified
fragments A–J indicate regions that are removed by splicing,
whereas a dashed line in exon 7 of fragment C represents a dele-
tion of 39 bp. The thick lines indicate intron 6 of 75 bp in fragments

B and C, 3¢ portion of 35 bp in intron 5 in fragments C and D, and
3¢ portion of 25 bp in intron 6 in fragments F and I. The number at
the right of each RT-PCR product represents the length of the prod-
uct. The scheme also shows the location of the reverse primer
DN+89 used in another starting exon-specific PCR and quantitative
real-time RT-PCR, the locations of the primers DN)105 and DN)9
used in ChIP assays, the locations of the primers DN+232 and
DN+254 utilized in 5¢-RACE, and the locations of the primers
DN+385 and DN+721 used in 3¢-RACE.
Y. Kominato et al. Promoters of human deoxyribonuclease I gene
FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3097
that the sequence between positions +156 and +243
in exon 1 was linked with that of exon 1a  12 kb
upstream of exon 1 in both QGP-1 cells and pancreas
(Fig. 2B). In addition, another RT-PCR with a primer
corresponding to the 5¢-terminus of exon 1 and the
same reverse primer, followed by sequence determin-
ation, showed that DNA fragments derived from
exon 1, including the DNASE1 full-length and splicing
variants, were amplified in QGP-1 cells and pancreas.
Therefore, these results allow us to conclude that both
exon 1a and exon 1 are used simultaneously as tran-
scription-starting exons in QGP-1 cells and pancreas.
The RT-PCR products of different sizes observed in
QGP-1 cells and pancreas seem to arise from alternat-
ive splicing. The complex patterns of alternatively
spliced products are represented schematically in
Fig. 2B. Although the splicing patterns of the
DNASE1 transcripts were complex, the transcripts
could be classified into two types: first, the full-length

transcripts A and H and second, transcripts B–G, I
and J that all lack exon 3. Only two DNASE1 tran-
scripts, those corresponding to the amplified products
A and H, can be translated to produce intact DNase I
protein. By contrast, 3¢-RACE using total RNA from
QGP-1 cells and pancreas as a template gave a single
band with a sequence identical to that reported previ-
ously [16], in addition to the observation of a specific
cleavage ⁄ polyadenylation site located in the 3¢-flanking
region of the gene at position 142 downstream of the
stop codon. Thus, DNASE1 splicing variants appear
to share a common 3¢-UTR.
Quantitative real-time RT-PCR was performed
using each distinct starting exon-specific primer and
a common reverse primer complementary to exon 2
to determine the relative abundance by comparing
the copy number of transcripts containing exon 1a
with the number starting from exon 1. The abun-
dance of transcripts starting from exon 1 was 10-fold
higher than that of the transcripts starting from
exon 1a in pancreas, whereas it was half in QGP-1
cells.
To examine whether transcription starting from
both exon 1a and exon 1 results in the production of
DNase I enzyme, we transfected the expression plasmids
ex-pDN1a and ex-pDN1, containing the sequences cor-
responding to the DNASE1 cDNAs starting from either
exon 1a or exon 1, respectively, into COS-7 cells and
then determined the levels of DNase I activity secreted
into the medium of the cells transfected with each plas-

mid. DNase I activity could be seen in cells transfected
with either expression vector, although they differed
in levels of expressed DNase I activity (Fig. 3). The
ex-pDN1
Relative DNase I activities
in the supernatant of COS-7 cells
0
ex-pDN
+1
ex-pDN1a
0.5 1.0
ATG
+847
TGA
coding region
coding region
(+1) (+156)
(-11868) (-11770)
−5
+1 +847
exon 1
+1 +847
exon 1a
coding region
(+243)
Fig. 3. Demonstration of the DNase I activities expressed by the DNASE1 expression constructs containing distinct 5¢-UTR regions of
DNASE1 mRNA in COS-7 cells. DNase I expression vectors, ex-pDN1 and ex-pDN1a, containing the entire 5¢-UTR region of each transcripts
derived from exons 1 and 1a, respectively, were constructed and transfected into COS-7 cells. Constructs are shown in the left-hand panel.
The numbers over the diagrams indicate the position of the corresponding nucleotides relative to the translation start site, and the numbers
in parentheses below the diagram show the position of the corresponding nucleotides relative to the transcription start site in exon 1. In the

expression vector ex-pDN, the Kozak sequence just upstream from the coding region of DNASE1 is contained and indicated by a closed
box. In the expression vector ex-pDN1, the gray box represents the whole sequence of exon 1. In the expression vector ex-pDN1a, the
sequence between +4 and +102 relative to the transcription start site of exon 1a, indicated by the open box, is ligated with the part of
exon 1 between positions +156 and +243. The resulting DNase I activity in the medium secreted from each transfected cells was normal-
ized by coexpressed b-galactosidase activity, and is shown in the right panel. The mean values and standard deviations were calculated from
five independent experiments. The activity of the expression plasmid ex-pDN was assigned an arbitrary value of 1.0. The DNase I activities
of the cells transfected with ex-pDN1 were statistically significantly lower than those of cells transfected with ex-pDN or ex-pDN1a
(P<0.05).
Promoters of human deoxyribonuclease I gene Y. Kominato et al.
3098 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
findings indicate that transcripts starting from either
exon 1a or exon 1 are translated to produce intact
DNase I protein.
Characterization of the promoter region of
exons 1a and 1 in the human DNase I gene
Because 5¢-RACE analysis identified two transcription-
starting exons used in DNASE1, we characterized the
promoters that regulate transcription of the DNASE1
messages containing exons 1 or 1a. To examine pro-
moter activity in the 5¢-flanking region of exon 1 in
DNASE1, we first obtained the )1386M construct by
introducing the )1386 to +268 sequence of DNASE1
into the promoterless pGL3–basic vector upstream of
the luciferase coding sequence. The reporter plasmid
was transfected into QGP-1 cells, followed by assay of
luciferase activities (Fig. 4A). pGL3–promoter vector
containing the SV40 promoter and pGL3–basic vector
without the promoter sequence were used as positive
and negative controls, respectively. The relative lucif-
erase activity of the )1386M construct was at least

eightfold higher than that of pGL3–basic vector and
was half that of pGL3–promoter vector. These findings
demonstrate the promoter activity of the 5¢-flanking
region of exon 1 in DNASE1. Deletion of the
upstream end of the 5¢-flanking region of exon 1 from
position )1386 to )231, )116, or )78 did not result in
any significant change. However, deletion of the
sequence from position )78 to )54 resulted in the loss
of 50% of the luciferase activity. These results imply
that the )78 to )55 region is required for DNASE1
proximal promoter activity in the 5¢-flanking region of
exon 1.
Similarly, we obtained the )2081H construct by
introducing the )13952 to )11781 sequence of
DNASE1 exon 1a relative to the transcription start site
of exon 1 into the promoterless pGL3–basic vector
upstream of the luciferase coding sequence, followed
by transient transfection into QGP-1 cells. The relative
luciferase activity of the )2081H construct was at least
14-fold higher than that of the pGL3–basic vector and
was not inferior to that of the pGL3–promoter vector
(Fig. 4B), indicating promoter activity of the 5¢-flank-
ing region of exon 1a in DNASE1. These results are
consistent with the finding that DNASE1 utilizes two
transcription starting exons in QGP-1 cells. Deletion
of the upstream end of the 5¢-flanking region of
exon 1a from position )13952 to )11965 did not result
in any significant change. However, deletion of the
sequence from position )11965 to )11944 elicited an
approximate twofold increase in luciferase activity,

indicating that negative regulatory elements are present
in the )11965 to )11944 region. Furthermore, deletion
of the upstream end from position )11944 to )11931
resulted in a fivefold decrease in luciferase activity,
suggesting that elements important for distal promoter
function are contained within the deleted region.
Inspection of the sequence between )73 and )60
upstream of the transcription start site of exon 1a
revealed a putative binding site for Sp1 transcription
factor and related proteins, as shown in Fig. 1B. To
evaluate whether the Sp1-binding site in the DNASE1
distal promoter is crucial for expression, a mutated
binding site was introduced into the )197H construct,
resulting in the loss of 80% of luciferase activity
(Fig. 4B). The data show that the Sp1 site is import-
ant for the DNASE1 distal promoter function
involved in transcription from exon 1a. To demon-
strate whether the sequence between )11944 and
)11931 bound Sp1 transcription factor, EMSA was
carried out using nuclear extracts prepared from
QGP-1 cells (Fig. 5). The oligonucleotide 90,51 probe
produced a major up-shifted band when the probe
was incubated with the nuclear extracts (lanes 1 and
7). Formation of the up-shifted complex, indicated by
the arrow, was decreased by the addition of compet-
ing unlabeled self oligonucleotide or Sp1 oligonucleo-
tide (lanes 2 and 4), but not by addition of
oligonucleotide m90,51 containing the same mutation
of the Sp1 site in )197HmSp construct as well as
oligonucleotide mSp1 with a mutated Sp1-binding site

(lanes 3 and 5). Consistently, formation of the DNA–
protein complex was significantly reduced when the
oligonucleotide m90,51 probe was incubated with the
nuclear extract (lane 6). These observations suggest
that an Sp1-like protein binds to the putative Sp1-
binding site between )73 and )64 relative to the tran-
scription start site of exon 1a. To investigate whether
Sp1 itself binds to the oligonucleotide 90,51 probe, a
supershift assay was performed. Although the tran-
scription factor pancreatic duodenal homeobox-1 pro-
tein (PDX-1) binds to the sequence C(C ⁄ T) and can
heterodimerize with PBX [20], an anti-PDX-1 IgG
failed to supershift the DNA–protein complex (lane
8). However, an anti-Sp1 IgG supershifted the DNA–
protein complex in association with reduction in the
amount of the complex (lane 9), suggesting that Sp1
binds to the putative site in the region from )90 to
)51 relative to the transcription start site of exon 1a.
Because the Sp1-binding site might be recognized by
other members of the Sp1 transcription factor family,
including Sp2, Sp3, and Sp4 [21], a chromatin immu-
noprecipitation (ChIP) assay was carried out with
antibodies against Sp1 and Sp1-related proteins. The
precipitated DNA was subjected to PCR with specific
Y. Kominato et al. Promoters of human deoxyribonuclease I gene
FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3099
primers for the endogenous DNASE1 distal promoter
region. Analysis revealed that Sp1 binds strongly to
the DNASE1 promoter, whereas neither Sp2 nor Sp4
bind to the promoter (Fig. 6). However, Sp3 seemed

to bind weakly to the promoter, although EMSA
failed to show a supershifted band with anti-Sp3 IgG
(data not shown). These results provide direct evi-
dence for Sp1 binding to the DNASE1 distal promo-
ter. The data demonstrate that Sp1 is involved in the
DNASE1 distal promoter function in transcription
from exon 1a.
Discussion
In previous studies, we were the first to determine the
genomic structure of the mammalian DNase I gene;
the human gene is located on chromosome 16p13.3, is
 3 kb long and contains nine exons interrupted by
eight introns [16]. Subsequently, the mouse [22], rat
[23] and bovine [24] genes have been shown to be sim-
ilar to the human gene. In this study, we investigated
the upstream region of the human gene. 5¢-RACE ana-
lysis of QGP-1 cells and pancreas revealed the presence
of a novel exon, named exon 1a, 12 kb upstream of
the original exon 1 in human DNASE1. Furthermore,
because the full-length 5¢-UTR of the transcript from
exon 1 was determined, the 5¢ boundary of exon 1
could be confirmed and was not in agreement with ear-
lier studies [15]. RT-PCR analysis and promoter assay
of the 5¢-upstream regions of both exons 1 and 1a clar-
ified that human DNASE1 utilizes two transcription-
starting exons simultaneously for expression of the
gene. Accordingly, the gene organization of human
DNASE1 should be corrected to 10 exons interrupted
by nine introns spanning  15 kb of genomic DNA.
Although the DNA region corresponding to exon 1a

in human DNASE1 could not be found on inspection
of the rat and mouse databases, further investigation
might identify an alternative transcription-starting
exon in DNASE1 of mammals other than humans.
Multiple promoters and transcription initiation sites
are frequently used to create diversity and flexibility in
−78
+1 +268
−1053
−231
luc
−1.0−2.0kb
Relative luciferase activities
in QGP-1 cells
0 0.5
luc
luc
luc
luc
luc
luc
luc
luc
luc
1
pGL3-Basic Vector
−1053M
−231M
−137M
−78M

−54M
−33M
−28M
−17M
+1M
luc
−116M
luc
−1386M
−138
A
6
B
−13952
−13866
−12068
−13202
−12395
luc
−1.0−2.0kb
Relative luciferase activities
in QGPI cells
0 1
luc
luc
luc
luc
luc
luc
luc

luc
luc
luc
2
pGL3-Basic Vector
−1995H
−2081H
−1331H
−524H
−197HmS
−94H
−73H
−60H
−45H
−14H
luc
−197H
−11871
−11781
Fig. 4. Summary of relative luciferase activities of the reporter constructs containing the different length of 5¢ upstream sequence of the
human DNASE1 exon 1 (A) or exon 1a (B). The different length of 5¢ upstream sequence of the human DNASE1 exon 1 or exon 1a (horizon-
tal bars) were inserted into the upstream of the firefly luciferase coding sequence of pGL3–basic vector. The numerals over the diagrams
are the nucleotide positions relative to the transcription starting site of exon 1. Constructs are shown in the left-hand panel; construct names
are given at the left of the bar and the locations of the inserted fragment are shown. The circle represents the CCCC fi AGAG substitution
at )11944 and )11941 introduced in reporter construct )197HmSp. Each construct as depicted on the left was transiently transfected into
QGP-1 cells. One microgram of firefly luciferase reporter construct and 0.01 lg of SV40 ⁄ Renilla luciferase were used for each analysis. The
cells were harvested for firefly and Renilla luciferase after culture for 38 h. The obtained firefly luciferase activity was normalized, which is
shown in the right-hand panel. Mean values and standard deviations were calculated from more than three independent experiments. The
activity of pGL3–promoter vector containing the SV40 promoter was arbitrary, given the value of 1.0.
Promoters of human deoxyribonuclease I gene Y. Kominato et al.

3100 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
the regulation of gene expression [25]. The level of
transcription initiation can vary between alternative
promoters, the turnover or translation efficiency of
mRNA isoforms with different leader exons can differ,
and alternative promoter usage can lead to the genera-
tion of protein isoforms differing in amino acid
sequence. Human DNASE1 pre-mRNA is transcribed
from different transcription start sites, exons 1 and 1a,
resulting in generation of two kinds of gene transcript.
However, only the sequences of the 5¢-UTR are differ-
ent between them, because these transcripts share all
the other exons, exons 2–9, which contain the entire
coding region. Thus, use of alternative promoters in
DNASE1 results in no generation of protein isoforms.
The 5¢-UTR of eukaryotic mRNA influences the initi-
ation step of protein synthesis and thereby in part
determines the translational efficiency of the transcript
[26]. In fact, as shown in Fig. 3, the translational effi-
ciency of the transcript from exon 1 was about half
of that from exon 1a. We used genetyx software
(GENETYX Corp., Tokyo, Japan) to search for poss-
ible secondary structure in the nucleotide sequence of
the 5¢-UTR in the transcripts starting from exons 1
and 1a, and found that the 5¢-UTR of the transcript
from exon 1 has a higher content of stem-loop struc-
ture than does that from exon 1a. Because stable
stem–loop structures are known to cause significant
suppression of translation, the distinctive secondary
structures of the 5¢-UTRs in these transcripts could

lead to differences in translation efficiency between
them.
Recently, rat DNase I pre-mRNA was reported to be
alternatively spliced in the kidney, leading to the gen-
eration of two types of transcript of 1.3 and 1.5 kb [27];
the former showed an internal deletion of a 132-bp
segment present in exon 1, and these transcripts were
produced from the same transcription starting exon.
Similarly, the human DNASE1 pre-mRNA undergoes
extensive alternative splicing in pancreas and QGP-1
cells, resulting in the generation of normal transcripts
and many alternative splicing variants initiated from
both exons 1a and 1, as shown in Fig. 2B. Alternative
splicing transcripts without exon 3 shift the reading
frame and generate a stop codon in exon 4, resulting in
no production of active DNase I enzyme. Furthermore,
differences in splicing patterns between QGP-1 cells and
pancreas (Fig. 2A) suggest that splicing patterns of
DNASE1 pre-mRNA could change in a cell- and ⁄ or
tissue-specific manner. Therefore, alternative splicing in
DNASE1 may potentially serve as a regulatory device
to modulate levels of the gene products.
No information on the regulation of gene expression
in vertebrate DNase I gene, such as characterization of
the promoter region and transcription factors respon-
sible for gene expression, has been obtained so far. In
this study, characterization of the promoter region in
Fig. 5. Sp1 specifically binds to the DNASE1 distal promoter at a
site between )91 and )51 relative to the transcription start site of
exon 1a. EMSA was performed with nuclear extract from QGP-1

cells. DNA–protein interaction was investigated using radiolabeled
probe 90, 51 (lanes 1–5, 7–8) or m 90, 51 (lane 6) in the presence
or absence of a 200-fold molar excess of competing unlabeled
oligonucleotides or antibodies as indicated. The major shifted com-
plex is indicated by the arrow. Oligonucleotides Sp1 and mSp1 con-
tained the wild and mutant types of Sp1 site, respectively (lanes 4
and 5). The nuclear extract was preincubated with anti-PDX-1 (lane
8) or anti-Sp1 IgG (lane 9). A supershifted band with antibody to
Sp1 is indicated by the arrowhead.
Fig. 6. ChIP assays of the Sp1-binding status at the endogenous
DNASE1 distal promoter in QGP-1 cells with anti-Sp1, -Sp2, -Sp3,
and -Sp4 IgG. The amplified DNASE1 distal promoter sequences in
the input and bound fractions are shown. The PCR products of
97 bp were electrophoresed through a 2% agarose gel and stained
with ethidium bromide.
Y. Kominato et al. Promoters of human deoxyribonuclease I gene
FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3101
the human DNASE1 gene was performed with QGP-1
cells; using promoter deletion analysis we could survey
the upstream regions responsible for transcription from
exons 1 and 1a (Fig. 4), in the latter of which a poten-
tial Sp1 site in the promoter contributes to its basal
activity. EMSA experiments (Fig. 5), ChIP assay
(Fig. 6) and the introduction of mutations in the
reporter gene construct revealed that binding of Sp1 to
the upstream region of DNASE1 exon 1a controlled
the basal distal promoter activity. This is the first to
be identified as a transcription factor responsible for
gene expression of vertebrate DNase I genes. Sp1 is
the founding member of a growing family of transcrip-

tion factors that bind and act through GC boxes,
being important in the transcription of many cellular
genes. Sp1 is abundantly distributed in most cell types,
irrespective of differences in levels of its expression
among different cell types [28]. Shiokawa and Tanuma
used polyA
+
RNA dot blot hybridization to show that
DNASE1 is expressed in a wide variety of human tis-
sues [6], being compatible with direct involvement of
Sp1 in expression of the gene. Furthermore, we dem-
onstrated in preliminary studies that upregulation of
distal DNASE1 promoter activity in QGP-1 cells by
hypoxia might depend on the Sp1-binding site
(Kominato et al., unpublished data). Because levels of
DNase I activity are known to differ greatly among
human tissues [3,7], it is likely that additional control
mechanisms such as alternative splicing and ⁄ or other
post-transcriptional regulation are responsible for tis-
sue-specific distribution of DNase I enzyme activity.
Experimental procedures
Cells
Human pancreatic cancer cell line QGP-1 (JCRB0183) was
obtained from Health Science Research Resources Bank
(Osaka, Japan). The cells were grown in RPMI-1640 con-
taining 10% fetal bovine serum (Invitrogen Corp., Carls-
bad, CA), 50 UÆmL
)1
penicillin and 50 lgÆmL
)1

streptomycin.
5¢- and 3¢-RACE analysis
5¢-RACE was performed using the 5¢-GeneRacer kit (Invi-
trogen Corp.) according to the manufacturer’s instruction.
Total RNAs isolated from QGP-1 cells using the acid
guanidine thiocyanate ⁄ acid phenol method [29] and human
pancreas (BD Biosciences Clontech, Palo Alto, CA) were
employed for the RACEs. Five micrograms of each total
RNA was treated with bovine intestinal phosphatase, fol-
lowed by incubation with tobacco acid pyrophosphatase
and ligation with the GeneRacer RNA oligo. cDNA
was synthesized by using Superscript III and the DNASE1-
specific primer DN+254, the sequence of which was 5¢-
TAGGTGTCTGGTGCATCCTG-3¢.5¢-Ends were PCR
amplified from these cDNA templates with a primer to the
GeneRacer RNA oligo and the DNASE1-specific primer
DN+232, the sequence of which was 5¢-TGAGGTTGTC
CAGCAGCTTC-3¢.3¢-RACE was performed using the
3¢-RACE system (Invitrogen Corp.) according to the manu-
facturer’s instruction. Five micrograms of each total RNA
was subjected to 3¢-RACE under the same conditions
described previously [30]. The sequences of the DNASE1-
specific forward primers used were 5¢-GACACCTTCAA
CCGAGAGCC-3¢ (DN+385) and 5¢-ATGCTGCTCCG
AGGCGCCGT-3¢ (DN+721). Conditions for these amplif-
ications were 95 °C for 9 min, 40 cycles of 94 °C for 1 min,
55 °C for 1 min, 72 °C for 2 min, followed by incubation
at 72 °C for 10 min. PCR amplifications were performed in
a50lL reaction mixture containing 10 pmol of each pri-
mer, 1.25 units of AmpliTaq Gold (Applied Biosystems,

Foster City, CA), 1.5 mm MgCl
2
, 150 lm dNTP, and 1·
buffer. After cloning of the PCR-amplified products into a
pCR2.1 plasmid vector (Invitrogen Corp.), the nucleotide
sequences of the amplified fragments were determined,
using the BigDye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems) with both M13 forward and reverse
primers. The sequencing run was performed on a Genetic
Analyzer (model 310, Applied Biosystems) and all the
DNA sequences were confirmed by reading both strands.
The nucleotide sequences reported here have been submit-
ted to the GenBank ⁄ EMBL ⁄ DDBJ Data Bank with acces-
sion numbers AB188151 and AB188152.
RT-PCR
cDNA was synthesized from total RNA (2 lg) of QGP-1
cells using random hexamers and Superscript III (Invitro-
gen Corp.). One and two of 20 lL of the resulting single
strand cDNA reaction were used as templates for RT-PCR
and quantitative real-time RT-PCR, respectively. Similarly,
2 lg of total RNA prepared from human pancreas was also
reverse-transcribed with random primer, and the synthes-
ized cDNA was utilized as template in RT-PCR. Starting
exon-specific amplification of the DNASE1 mRNA was
performed using each distinct starting exon-specific primer
and the reverse primer DN+785, of which the sequence
was 5¢-GCCATAGGCAGCCTGGAAGT-3¢, complement-
ary to exon 8 of DNASE1. The sequences of the starting
exon-specific primers were 5¢-GCCTTGAAGTGCTTCTTC
AGAGAC-3¢ (DN)144) and 5¢-GCACAACACAGGGAA

GCTTGG-3¢ (DN)110), corresponding to the sequence in
exons 1 and 1a of the DNASE1, respectively. Another start-
ing exon-specific amplification of the DNASE1 message was
performed using each distinct starting exon-specific primer
Promoters of human deoxyribonuclease I gene Y. Kominato et al.
3102 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
and the reverse primer DN+89, of which the sequence was
5¢-ATGTTGAAGGCTGCGATCTTCAG-3¢, complement-
ary to exon 2 of the DNASE1. Conditions for these amplifi-
cations were 95 °C for 9 min, 30 cycles of 94 °C for 1 min,
60 °C for 1 min, and 72 °C for 2 min, followed by incuba-
tion at 72 °C for 10 min. Determination of the nucleotide
sequences of the amplified fragments were described above.
Plasmids
The different length of 5¢-upstream sequence of exons 1 and
1a in the DNASE1 were PCR-amplified using sequence-spe-
cific primers corresponding to the sequence deposited in the
GenBank with Accession no. AC006111 and subcloned into
a firefly luciferase reporter vector, the pGL3–basic vector
(Promega, Madison, WI). Nomenclature used for the var-
ious reporter constructs is based on the nature of the inser-
ted fragments. Letter symbol M reflects the restriction
enzyme cleavage site of MlnI at +268 relative to the tran-
scription start site of exon 1 in the DNSAE1, letter symbol
H reflects the restriction enzyme cleavage site of HindIII at
+90 relative to the transcription start site of exon 1a,
whereas numerals indicate the endpoints of the primers used
for PCR. For example, )197H construct contains the frag-
ment bordered with PCR primer sequence starting at )197
relative to the transcription start site at one end and HindIII

site at the other.
The DNASE1 expression plasmid ex-pDN was constructed
by preparing a PCR-amplified fragment containing the entire
coding sequence of the human DNASE1 message by using an
Expanded High Fidelity PCR system (Roche) with the total
RNA from QGP-1 cells and a primer set of the forward pri-
mer DN-5R and the reverse primer DN+854X (sequences
5¢-CG
GAATTCTCAGGATGAGGGGCATGAAG-3¢ and
5¢-CG
CTCGAGGCTGCTCACTTCAGCATCAC-3¢, res-
pectively) and directionally ligating the fragment into the
EcoRI ⁄ XhoI site of pcDNA3.1(+) vector (Invitrogen Corp.).
The nucleotides underlined in the primer sequences above
represent EcoRI or XhoI sites. The 5¢-UTRs of the DNASE1
messages were separately amplified by RT-PCR of total
QGP-1 RNA with a primer set of the common reverse primer
DN+89 and either of the starting exon-specific forward
primers DN)243R or DN)183R, the sequences of which
were 5¢-CGGAATTCACATTTGCCCCAGGGAAGGTC-
3¢ and 5¢-CGGAATTCTCCTGCCCAGGACCCGAGG-3¢,
respectively. The DNASE1 expression plasmids ex-pDN1
and ex-pDN1a, containing the sequences corresponding to
the 5¢-UTRs of the transcripts from exons 1 and 1a, respect-
ively, were constructed by use of the overlap extension
method [31] with those PCR-amplified fragments and the
plasmid ex-pDN. The plasmids pD(1) and pD(1a) were con-
structed by cloning the PCR-amplified fragments obtained
with either of the starting exon-specific forward primers
DN)144 or DN)110 and the common reverse primer

DN+89, respectively, into a pCR2.1 plasmid vector.
For all the constructs, sequencing was performed over
the entire region of the inserted sequences. Plasmid DNA
was purified by using HiSpeed Plasmid Kit (QIAGEN
GmbH, Hilden, Germany).
Transfection and luciferase assay
Transient transfection experiments into QGP-1 cells were
performed with Lipofectamine Plus reagent (Invitrogen
Corp.); 1 lg of firefly luciferase reporter and 0.01 lgof
pRL-SV40 Renilla luciferase reporter (Promega) were used
for each analysis. QGP-1 cells were split, 18–24 h prior to
transfection, into a six-well tissue culture plate (Becton
Dickinson Labware, Franklin Lakes, NJ) at 1 · 10
5
ÆmL
)1
.
At the time of transfection, cells were washed once with
Opti-MEM I-reduced serum medium (Invitrogen Corp.)
containing neither fetal bovine serum nor l-glutamine.
Plasmid DNA was suspended in 100 lL of Opti-
MEM I-reduced serum medium, followed by mixture with
6 lL of Lipofectamine Plus reagent at room temperature
for 10 min. Four microliters of Lipofectamine Plus reagent
were diluted in 100 lL of Opti-MEM I-reduced serum med-
ium. The two solutions were combined at room tempera-
ture for 15 min, followed by the addition of 0.8 mL of
Opti-MEM I-reduced serum medium. The mixture was then
overlaid onto the cells. The cells were incubated for 3 h
prior to addition of 1 mL of Opti-MEM I-reduced serum

medium containing 20% serum to the DNA-containing
medium, followed by incubation for 14 h. Subsequently,
those cells were cultured at 37 °C under either normoxic or
hypoxic conditions prior to the measurement of the activit-
ies of firefly and Renilla luciferase. Cell lysis and luciferase
assays were performed using the Dual Luciferase Reporter
Assay System (Promega). Light emission was measured by
Wallac 1420 ARVOMX (Perkin–Elmer). The values were
obtained in relative light units. Variations in transfection
efficiency were normalized to the activities of Renilla lucif-
erase expressed from cotransfected pRL-SV40 Renilla
luciferase reporter vector. COS-7 cells were transiently
cotransfected by using Lipofectamine Plus reagent (Invitro-
gen Corp.) with 2 lg of the DNASE1 expression vectors
and 0.6 lg of the pSV–b-galactosidase vector (Promega),
followed by assay of DNase I and b-galactosidase activities,
according to a previously described method [32].
Preparation of nuclear extracts and EMSA
The nuclear extract and probe were prepared as reported
previously [33], and the sequence of oligonucleotide 90,51
probe corresponds to nucleotides )90 to )51 relative to
the transcription start site of exon 1a in the DNASE1.
EMSA was carried out according to the method des-
cribed previously [33]. The different double-stranded oligo-
nucleotides were obtained by annealing two chemically
synthesized strands. The double-stranded Sp1 and mSp1
Y. Kominato et al. Promoters of human deoxyribonuclease I gene
FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3103
oligonucleotides containing the wild-type and the mutant
version of Sp1 site, respectively, were purchased from

Santa Cruz Biotechology (Santa Cruz, CA). A 200-fold
molar excess of unlabeled competitors over the radiolabe-
led probe was used for competition analyses. For super-
shift experiments, 2 lL of polyclonal rabbit anti-PDX-1
IgG or anti-Sp1 IgG (Santa Cruz Biotechnology) was
added to the nuclear extract, and preincubated on ice for
15 min prior to the addition of radiolabeled probe.
ChIP analysis
ChIP assay was performed using chromatin immunoprecipi-
tation assay kit (Upstate, Lake Placid, NY) according to the
manufacturer’s instruction. Anti-Sp1 IgG was purchased
from Upstate, whereas anti-Sp2, anti-Sp3 and anti-Sp4 IgGs
were obtained from Santa Cruz Biotechnology. PCR was
performed to amplify the region between ) 105 and )9 relat-
ive to the transcription start site of exon 1a in DNASE1
using primers DN)105 and DN)9, the sequences of which
were 5¢-CCAGCCTGGCTGGTTATCAGTCC-3¢ and 5¢-
GAGCTCTTCCACACCAGACGCA-3¢, respectively. Con-
ditions for these amplifications were 95 °C for 9 min, 37
cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for
2 min, followed by incubation at 72 °C for 10 min. The PCR
products were electrophoresed through a 2% agarose gel and
were stained with ethidium bromide. The sequences of the
amplified fragments were determined as described above.
Enzyme assay
Enzyme activity of DNase I was determined by the single
radial enzyme diffusion (SRED) method as described previ-
ously [7]. The human specific DNase I activity in the med-
ium secreted from QGP-1 cells was calculated by
subtraction of bovine DNase I activity in the fresh medium

from the whole DNase I activity in the supernatant of
QGP-1 cells. DNase I activity was assayed for cell extract
prepared from QGP-1 cells by sonication. Protein assay
was carried out using a protein assay kit (Bio-Rad, Rich-
mond, CA) with BSA as a standard, and 10 lg of the cell
extract was applied to the gel plate for SRED method. One
unit of enzyme activity assayed corresponds to 0.6 ng of
purified human DNase I [34].
Quantitative real-time RT-PCR
Quantitative real-time RT-PCR was performed using Light-
Cycler
TM
(Roche Diagnostics GmbH, Mannheim, Ger-
many) and SYBR
Ò
Premix Ex Taq (TaKaRa, Shiga,
Japan). Specific amplifications of individual starting
exon-specific variants of the DNASE1 were performed
using starting exon-specific forward primer, DN-110 or
DN-144, and the common reverse primer DN+89.
Conditions for both amplifications were 95 °C for 10 s, 40
cycles of 95 °C for 5 s, 60 °C for 20 s. Quantitative PCR
was performed in a 20-lL reaction mixture containing
4 pmol of each primer, 10 lLof2· SYBR Premix Ex Taq,
and 2 of 20 lL single-strand cDNA reactions. To determine
the absolute copy number of the target transcripts in indi-
vidual cDNA reaction mixtures, the plasmids pD(1) and
pD(1a) were used to generate a calibration curve. The plas-
mid templates were measured using a spectrophotometer,
and copy numbers were calculated from the absorbance at

260-nm. For each assay, a standard curve was prepared
using serial dilutions of template plasmid DNA with known
copy numbers in log steps from 2 · 10
7
copies to 2 · 10
2
copies in a 2-lL volume. All samples to be compared were
run in the same assay. After completion of the PCR ampli-
fication, the data were analysed with the lightcycler
ver.3.5 (Roche). The threshold cycle was calculated using
the sequence detection software as the cycle number at
which the fluorescence of the reporter dye crossed the
threshold in log-linear range of PCR. The copy numbers of
the respective DNASE1 cDNA were quantified by interpo-
lating the results from the threshold cycles.
Acknowledgements
This work was supported in part by Grant-in-Aid for
Scientific Research from the Ministry of Education, Sci-
ence, Sports and Culture, Japan (15209023, 17659196 to
TY, 17659161 to MU and 16209023 to KK).
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