Ets-1/ Elk-1 is a critical mediator of dipeptidyl-peptidase III
transcription in human glioblastoma cells
Abhay A. Shukla, Misti Jain and Shyam S. Chauhan
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
Introduction
Dipeptidyl-peptidase III (DPP-III), a cytosolic amino-
peptidase has been purified and characterized from dif-
ferent tissues of various animal species such as rat and
human skin [1,2], bovine and human cataractous lens
[3], rabbit and human erythrocytes [4], rat brain [5]
and pancreas [6], monkey brain [7], human placenta
[8], neutrophils [9], Saccharomyces cerevisiae [10] and
Drosophila melanogaster [11]. All mammalian DPP-IIIs
require zinc ions for their maximum activity and have
therefore been termed metalloaminopeptidases. The
crystal structure of yeast DPP-III has been described
by Baral et al. [12], providing an insight into its cata-
lytic mechanism and mode of substrate binding. No
endogenous substrate for this enzyme has yet been
identified. However, it has broad specificity for a num-
ber of polypeptides, suggesting its involvement in the
terminal stage of intracellular protein catabolism.
Interestingly, DPP-III activity has been reported to
increase in retroplacental serum [8] suggesting that it is
synthesized in placental cells and released into the
maternal circulation. In view of its high affinity for
angiotensin II and III [13], the potential role of this
Keywords
5¢-RACE; electrophoretic mobility shift
assays; promoter; site-directed
mutagenesis; transcription factors
Correspondence
S. S. Chauhan, Room No. -3009,
Department of Biochemistry, All India
Institute of Medical Sciences, New Delhi
110029, India
Fax: +91 11 2658 8663
Tel: +91 11 2659 3272
E-mail: s_s_chauhan@hotmail.com
Database
The nucleotide sequence of the human
DPP-III promoter has been submitted to the
GenBank database under the accession
number FJ793449
(Received 9 November 2009, revised 25
December 2009, accepted 1 February 2010)
doi:10.1111/j.1742-4658.2010.07603.x
Dipetidyl-petidase III is a metallopeptidase involved in a number of physi-
ological processes and its expression has been reported to increase with the
histological aggressiveness of human ovarian primary carcinomas. Because
no information regarding the regulation of its expression was available,
experiments were designed to clone, define and characterize the promoter
region of the human dipeptidyl-peptidase III (DPP-III) gene. In this study,
we cloned a 1038 bp 5¢-flanking DNA fragment of the human DPP-III
gene for the first time and demonstrated strong promoter activity in this
region. Deletion analysis revealed that as few as 45 nucleotides proximal to
the transcription start site retained 40% of the activity of the full-length
promoter. This promoter lacked the TATA box but contained multiple GC
boxes and a single CAAT box. Similarly, two Ets-1 ⁄Elk-1-binding motifs
are present in the first 25 nucleotides from the transcription start site. Bind-
ing of Ets-1 ⁄Elk-1 proteins to these motifs was visualized by electropho-
retic mobility shift and chromatin immunoprecipitation assays. Mutations
of these binding sites abolished not only binding of the Ets protein, but
also the intrinsic promoter activity. Increased DNA-binding activity of
Ets-1 ⁄Elk-1 by v-Ha-ras also augmented the mRNA level and promoter
activity of this gene. Similarly, co-transfection of DPP-III promoter–repor-
ter constructs with Ets-1 expression vector led to a significant increase in
promoter activity. From these results, we conclude that Ets-1 ⁄Elk-1 plays a
critical role in transcription of the human DPP-III gene.
Abbreviations
ChIP, chromatin immunoprecipitation; CLR, Chang liver Ras cells; CLDR, Chang liver DRas cells; DPP-III, dipeptidyl-peptidase III; EMSA,
electrophoretic mobility shift assay; ERK, extracellular regulated kinase; Inr, initiator element; MEK, mitogen-activated protein kinase kinase.
FEBS Journal 277 (2010) 1861–1875 ª2010 The Authors Journal compilation ª2010 FEBS 1861
peptidase in elevating the level of plasma angiotensin
hydrolysing activity during pregnancy has been
described. Similarly, it exhibits high affinity for Leu-
enkephalins [5]. These features suggest a potential role
for DPP-III in the regulation of blood pressure [10]
and in pain modulation [14]. Human DPP-III has been
shown to increase with the histological aggressiveness
of human ovarian primary carcinomas [15]. Because
levels of DPP-III alter in several physiological and
pathological conditions it must necessarily be amena-
ble to regulated expression. However, no systematic
study has been carried out to elucidate the regulatory
molecular mechanisms associated with its expression.
Therefore, this study was designed to clone and char-
acterize the human DPP-III promoter in order to eluci-
date the transcriptional regulation of the gene. In this
regard, we identified the region that plays an impor-
tant role in determining the basal promoter activity of
the gene. Furthermore, with the help of binding assays
and site-directed mutagenesis, we established that
Ets-1 ⁄Elk-1 play a key role in the regulation of DPP-III
transcription in human glioblastoma cells.
Results
PCR amplification, sequence analysis and
demonstration of promoter activity in the
5¢upstream region of the human DPP-III gene
Using primer designs based upon the DPP-III gene
located on chromosome 11q 12 fiq13.1 of the human
genome sequence (accession number NT_033903.7), we
were able to amplify a single 1038 bp DNA fragment
by PCR (data not shown). This fragment was cloned
Fig. 1. Nucleotide sequence of the 5¢-flank-
ing region of the human DPP-III gene. The
transcriptional initiation site determined by
5¢-RACE in U87MG cells is denoted as +1.
Different primers were used for amplifica-
tion of the 1038 bp full-length promoter and
its deletion fragments, 5¢-RACE and ChIP
assays are shown by arrows. The most 5¢-
end base of the full-length promoter and the
different deletion constructs are shown in
bold and indicated by arrows ( fi) with
respect to the transcription initiation site
(; +1). The translation initiation codon ATG is
underlined. Potential cis-element regulatory
motifs are in italics and marked by dashed
arrows. Two AT-rich sequences present at
positions )24 and )29 are written in bold.
The intronic sequences are written in lower
case. Primers used for the amplification
of DPP-III promoter sequence are also
underlined.
Ets-1 ⁄Elk-1 key regulators of DPP-III transcription A. A. Shukla et al.
1862 FEBS Journal 277 (2010) 1861–1875 ª2010 The Authors Journal compilation ª2010 FEBS
into a TA cloning vector and sequenced. Analysis of
its nucleotide sequence showed 100% homology to the
upstream region and part of the reported 5¢-end of
DPP-III mRNA. These results indicated that the
amplified region is physically linked to exon 1 of the
DPP-III gene. Nucleotide sequence analysis of
the cloned 1038 bp human DPP-III promoter revealed
that it contained 63.19% G + C nucleotides, no
detectable TATA box, a single CAAT box and several
GC boxes (Sp1-binding sites). Multiple putative tran-
scription factor binding sites were identified in this
region using the motif finder program (http://motif.
genome.jp/) with high-stringency parameters for core
similarity of 0.85 and matrix similarity of 0.85. As
shown in Fig. 1, these motifs include NF-jB, USF,
C⁄EBP, CREB, NF-1 and multiple binding sites for
the Sp1 and Ets family of transcription factors, sug-
gesting that the amplified region is a potential
promoter. To demonstrate promoter activity in the
amplified fragment, we cloned it upstream of the lucif-
erase reporter gene in the pGL3-Basic vector. Trans-
fection of the resulting construct (pAAS-1) into
U87MG, Caov-2, Chang liver, Panc1 and NIH 3T3
cells yielded 800-, 200-, 100-, 80- and
25-fold higher luciferase activity respectively, com-
pared with the pGL3-Basic transfected cells (Fig. 2).
These results established that the cloned fragment is a
functional DPP-III promoter.
Mapping of the transcriptional start site
As a first step towards characterization of the human
DPP-III promoter, the transcriptional start site in
U87MG cells was mapped using 5¢-RACE. Resolution
of RACE products on agarose gel revealed the amplifi-
cation of a single 200 bp fragment (Fig. 3). This
fragment was cloned into a TA cloning vector and six
representative clones were subjected to double-stranded
DNA sequencing. All clones exhibited 100% homology
to DPP-III mRNA and contained the same nucleotide
(G) corresponding to the 33rd nucleotide upstream of
the translation initiation codon in the reported mRNA
sequence (accession number NM_005700). These
results suggested that this nucleotide is the transcrip-
tion initiation site (marked +1 in Fig. 1).
0
100
200
300
400
500
600
700
800
900
1000
U87MG Caov-2 Chang Liver Panc1 NIH3T3
Luciferase activity
fold increase over pGl3-basic
x796.23
x212.53
x102.28 x77.88
x23.42
Fig. 2. Demonstration of promoter activity in the 5¢flanking region
of the human DPP-III gene in different cell lines. The PCR-amplified
5¢flanking region of human DPP-III gene (1038 bp) was cloned
upstream of the luciferase reporter gene in promoter-less plasmid
pGL3-Basic. The resulting construct (pAAS-1) was transfected into
U87MG (human glioblastoma grade III), Caov-2 (ovarian carcinoma),
Chang liver (human liver), Panc1 (human pancreatic carcinoma) or
NIH 3T3 (mouse fibroblast) cells. After 48 h of transfection, cells
were washed three times with ice-cold NaCl ⁄P
i
, lysed and lucifer-
ase activity was assayed in the cell lysates. Cells transfected with
pGL3-Basic were processed in an identical way and served as the
negative control. Values are the mean ± SE of at least three inde-
pendent experiments performed in triplicate. Other details are
given in Materials and methods.
(T)nTTTV
(A)nAAA
Oligo dT-anchor primer
A
B
PCR anchor
Primer
AAS2
AAS1
DL100
PCR negative
control
RACE product
~200 bp
1 2 3
500 bp
100 bp
Fig. 3. Mapping of the transcription initiation site of the DPP-III
gene. (A) Schematic diagram showing the location of different
primers used for 5¢-RACE on U87MG cDNA. (B) Total cellular RNA
isolated from U87MG cells was reverse transcribed using a gene-
specific primer (AAS-2) and used in 5¢-RACE to map the transcrip-
tion initiation site. PCR performed without a template served as the
negative control. RACE products were resolved on 2% agarose gel
and stained with ethidium bromide. The prominent 200 bp frag-
ment (indicated on the right-hand side) was excised, cloned and
subjected to double-strand DNA sequencing. DL100 corresponds to
a 100 bp DNA ladder (MBI Fermentas, Vilnius, Lithuania).
A. A. Shukla et al. Ets-1 ⁄Elk-1 key regulators of DPP-III transcription
FEBS Journal 277 (2010) 1861–1875 ª2010 The Authors Journal compilation ª2010 FEBS 1863
Deletion analysis of human DPP-III promoter
In order to define the minimal promoter region and
identify the functional transcription factor binding
motif(s) in this region of the DPP-III gene, a series of
promoter–reporter constructs with varying lengths for
the 5¢-region were generated. The 5¢-ends of these
constructs are marked ( ) in Fig. 1. These constructs
were transfected into human glioblastoma cells
(U87MG) followed by estimation of the luciferase
activity. The construct pAAS-2 ()781 ⁄+5), which
lacks first 252 bases from the 5¢-end of the full-length
promoter, retained 92% of the promoter activity
(740-fold promoter activity over pGL3-Basic vector)
(Fig. 4A). Further deletion of 548 or more bases
from the 5¢-end resulted in a significant reduction in
promoter activity. Constructs which lacked 548 bp
()485 ⁄+5, pAAS-3), 803 bp ()230 ⁄+5; pAAS-4),
909 bp ()123 ⁄+5; pAAS-5), 940 bp ()93 ⁄+5; pAAS-
6), 980 bp ()53 ⁄+5; pAAS-7) and 993 bp ()40 ⁄+5;
pAAS-8) from the 5¢-end, retained 51, 48, 39, 42, 44
and 40% promoter activity, respectively, compared
with the full-length promoter (Fig. 4A). All these con-
structs exhibited > 300-fold promoter activity com-
pared with pGL3-Basic. Thus, 45 nucleotides (minimal
promoter) from the 3¢-end of the DPP-III promoter
retained 40% of the full-length promoter (pAAS-1)
activity (320-fold over pGL3-Basic). Sequence analysis
of the minimal promoter region ()40 ⁄+5) revealed no
obvious TATA or CAAT boxes. However, two perfect
Luciferase activity
(fold increase over pGL3-basic)
100
200
300
400
500
600
700
800
900
AB
**
**
*
*
x816.25
x741.11
x418.59 x390.93
x312.07 x337.31
x352.61
x319.48
–1033 Luc
Luc
Luc
Luc
Luc
Luc
Luc
Luc
–781
–485
–230
–124
–93
–53 +5
–40 +5
+5
+5
+5
+5
+5
+5
pAAS–1
pAAS–2
pAAS–3
pAAS–4
pAAS–5
pAAS–6
pAAS–7
pAAS–8
0
Luciferase activity
(fold increase over pGL3-basic)
x824.23
x312.07
0
100
200
300
400
500
600
700
800
900
x4.22
b
a
–1033
Luc
+5
pAAS–1
Luc
–124 +5
pAAS–5
Luc
–124 –21
pAAS–9
Fig. 4. Functional analysis of deletion constructs of the human DPP-III gene. (A) A series of DNA fragments were PCR amplified using full-
length promoter fragment as the template. Seven fragments with different 5¢-ends (nucleotides )781, )485, )230, )124, )93, )53 and
)40) and a common 3¢-end (nucleotide +5) were cloned upstream of the luciferase reporter gene in promoter-less plasmid pGL3-Basic to
generate constructs pAAS-2, pAAS-3, pAAS-4, pAAS-5, pAAS–6, pAAS-7 and pAAS-8, respectively. U87MG cells were transiently co-trans-
fected with test plasmid and pRL-TK internal control plasmid. Values significantly different from pAAS-1 are marked by *. (B) A DNA frag-
ment lacking 25 bases from the 3¢end of pAAS-5 was amplified using DPP-III F-124 and DPP-III R-21 as the sense and antisense primers.
pAAS-1 was used as a template for the PCR. The 99 bp amplified fragment was digested with XhoI and HindIII and cloned into the pGL3-
Basic to generate pAAS-9 ()124 ⁄)21). U87MG cells were transiently transfected with pAAS-9. pAAS-1 and pAAS-5 were also transfected in
separate experiments. Luciferase activity in the cell lysates was measured 48 h after transfection. Each transfection was performed in tripli-
cate and the results are expressed as the mean ± SE of three independent experiments. aSignificantly higher compared with pAAS-9
(P< 0.001); bsignificantly higher compared with pAAS-9 (P< 0.001). Statistical analysis was performed using a paired two-tailed Student’s
t-test.
Ets-1 ⁄Elk-1 key regulators of DPP-III transcription A. A. Shukla et al.
1864 FEBS Journal 277 (2010) 1861–1875 ª2010 The Authors Journal compilation ª2010 FEBS
consensus Ets-1 ⁄Elk-1 core binding motifs (GGAA
GCAGGAA) separated by three bases were present in
this region (at positions )6 and )13). To elucidate the
role of these motifs, we deleted 25 bases from the 3¢-
end of the construct pAAS-5, thus generating pAAS-9
()124 ⁄)21). This construct, which lacked 5 bases of
exon 1 and 20 bases of the promoter region, including
Ets-1 ⁄Elk-1-binding motifs, exhibited no promoter
activity (Fig. 4B). These results established that nucleo-
tides between )20 and +5 are essential for DPP-III
promoter activity.
Site-directed mutagenesis
To further corroborate our results, we mutated these
two Ets-1 ⁄Elk-1 binding motifs sequentially in pAAS-1
using site-directed mutagenesis and assessed the pro-
moter activities of the resulting constructs (Fig. 5).
pAAS-1Mut1 (harbouring a mutated Ets-1 ⁄Elk-1 motif
at position )6) and pAAS-1Mut2 (having Ets-1 ⁄Elk-1
mutated motifs at both position )6 and position )13),
were transfected in U87MG cells. Mutations in the
motif at )6 (pAAS-1Mut1) resulted in an 81% loss of
basal promoter activity compared with the full-length
construct (pAAS-1). Whereas mutations in both
Ets-1 ⁄Elk-1-binding motifs (pAAS-1Mut2) resulted in
a 96% loss of basal promoter activity compared with
the full-length construct (pAAS-1, Fig. 5). Abolition of
the promoter activity confirmed that Ets-1 ⁄Elk-1 bind-
ing motifs are essential for transcription of the human
DPP-III gene. Because mutation of the motif present
at the )6 position alone resulted in an 81% loss of
promoter activity, we conclude that Ets-1 ⁄Elk-1-bind-
ing motifs are critical for DPP-III gene expression.
Binding of transcription factors to the minimal
promoter region (-40 ⁄+5)
To show the in vivo binding of Ets-1 and Elk-1 tran-
scription factors with the human DPP-III minimal
promoter region, we performed chromatin immunopre-
cipitation (ChIP) assays using Ets-1 and Elk-1 antibod-
ies. The immunoprecipitated chromatin was subjected
to PCR using primers flanking the binding motifs pres-
ent at positions )6 and )13 in the DPP-III promoter.
Amplification of a specific 81 bp DNA fragment was
observed when the DNA was immunoprecipitated
using Ets-1 or Elk-1 antibodies. However, no amplifi-
cation of any DNA fragment was evident when DNA
was precipitated using mouse IgG (negative control)
(Fig. 6). The identity of the amplified products as part
+ Anti mouse IgG
antibody
+ Anti Ets-1 antibody
DL100
+ Anti Elk-1 antibody
81 bp
200 bp
Fig. 6. In vivo analysis of binding of Ets-1 and Elk-1 to the DPP-III
promoter by ChIP assay. U87MG cells were fixed with 1% formal-
dehyde to cross-link the existing in vivo proteins–DNA complex.
Nuclei of the cross-linked cells were isolated and subjected to soni-
cation to shear the DNA. Anti-Ets-1 and anti-Ek-1 IgG were used to
immunoprecipitate DNA bound to these proteins. PCR was
performed using DPP-III F-45 and ChIP R as the sense and
antisense primers to specifically amplify 81 bp DPP-III promoter
region including the Ets-1 ⁄Elk-1-binding motifs present at
positions )6 and )13. PCR using the same primers was also
performed with DNA immunoprecipitated using mouse IgG as
template and served as a negative control. DL100 corresponds to a
100 bp DNA ladder and a single fragment of 200 bp is indicated by
an arrow.
100
200
300
400
500
600
700
800
900
pAAS-1 pAAS-1
Mut1
pAAS-1
Mut2
a
b
Luciferase activity
fold increase over pGl3-basic
0
x816.25
x152.48
x35.94
Fig. 5. Functional relevance of Ets-1 ⁄Elk-1-binding motifs in DPP-III
promoter activity. U87MG cells were transiently transfected with
either wild-type promoter, construct pAAS-1 or promoter constructs
containing one ()6; pAAS-1Mut1) or both ()6 and )13; pAAS-
1Mut2) mutated Ets-1 ⁄Elk-1-binding motifs. Luciferase activity was
measured 48 h after transfection and is plotted in the left-hand
panel. Each transfection was performed in triplicate and the results
are expressed as the mean ± SE of three independent experi-
ments. (a) Significantly higher compared with pAAS-1Mut1
(P< 0.001); (b) significantly higher compared with pAAS-1Mut2
(P< 0.001). Statistical analysis was performed using a paired two-
tailed Student’s t-test.
A. A. Shukla et al. Ets-1 ⁄Elk-1 key regulators of DPP-III transcription
FEBS Journal 277 (2010) 1861–1875 ª2010 The Authors Journal compilation ª2010 FEBS 1865