Snail associates with EGR-1 and SP-1 to upregulate
transcriptional activation of p15
INK4b
Chi-Tan Hu
1
, Tsu-Yao Chang
2
, Chuan-Chu Cheng
2
, Chun-Shan Liu
2
, Jia-Ru Wu
2
, Ming-Che Li
1
and
Wen-Sheng Wu
2
1 Research Centre for Hepatology, Buddhist Tzu Chi General Hospital and Tzu Chi University, Hualien, Taiwan
2 Institute of Medical Biotechnology, College of Medicine, Tzu Chi University, Hualein, Taiwan
Keywords
EGR-1; p15
INK4b
; Snail; SP-1; transcriptional
regulation
Correspondence
Wen-Sheng Wu, Institute of Medical
Biotechnology, College of Medicine, Tzu Chi
University, No. 701, Chung Yang Rd, Sec 3,
Hualien 970, Taiwan
Fax: +8867 03 8571917

EGR-1 with Snail or SP-1. Furthermore, a double chromatin immunopre-
cipitation assay verified that EGR-1 could form a complex with Snail or
SP-1 on the TPA-responsive element after treatment with TPA for 2–6 h.
Finally, we demonstrated a novel Snail-target region which could be bound
by Snail and was also required for TPA-induced p15–pro228 activation. In
conclusion, Snail associates with EGR-1 and SP-1 to mediate TPA-induced
transcriptional upregulation of p15
INK4b
in HepG2.
Structured digital abstract
 MINT-7384899: Snail (uniprotkb:O95863) physically interacts (MI:0915) with EGR-1 (uni-
protkb:P18146) by anti bait coimmunoprecipitation (MI:0006)
 MINT-7384908: SP-1 (uniprotkb:P08047) physically interacts (MI:0915) with EGR-1 (uni-
protkb:P18146) by anti bait coimmunoprecipitation (MI:0006)
Abbreviations
ChIP, chromatin immunoprecipitaion; EGR-1, early growth response gene 1; MMPs, matrix metalloproteinases; shRNA, short hairpin RNA;
SP-1, stimulatory protein-1; TPA, tetradecanoyl phorbol acetate.
1202 FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS
Introduction
The Snail family of zinc-finger transcription factors
was first described in Drosophila melanogaster [1],
where they were shown to be essential for formation
of the mesoderm [2]. Snail may trigger a phenotypic
change called epithelial mesenchymal transition
required for embryonic development [3,4]. Recent
studies have linked Snail to tumor metastasis because
epithelial mesenchymal transition is a prerequisite for
cell migration and invasiveness [5–8]. Snail genes can
be induced by different growth factors and cytokines,
such as hepatocyte growth factor [9], transforming

required for tetradecanoyl phorbol
acetate (TPA)-induced cell-cycle arrest [17].
Snail family proteins contain a C-terminal tandem
C
2
H
2
zinc finger as a sequence-specific DNA-binding
motif and an N-terminal SNAG repression domain.
The detailed mechanisms by which Snail acts as a tran-
scriptional repressor have been intensively studied.
Snail may bind to a consensus sequence such as
E-box, which is also the binding site for basic helix–
loop–helix transcriptional factors on the target pro-
moter, thus interfering with gene expression. More
recent reports have further shown that Snail may asso-
ciate with polycomb repressive complex 2 or protein
arginine methyltransferase 5 to repress E-cadherin
expression [25,26]. However, how Snail upregulates
gene expression is not yet clear. In a recent report, the
Snail-responsive element(s) on the proximal MMP-9
promoter was identified in MDCK cells. This region
contains the putative binding sites of stimulatory
protein-1 (SP-1) and Ets-1 which are critical for the
transactivation of MMP-9 [23]. However, whether
Snail binds directly to this region and whether it might
cooperate with other transcriptional factors to activate
MMP-9 promoter were not addressed.
Recently, we investigated the mechanisms by which
Snail mediates TPA-induced upregulation of p15

2.29-fold higher, respectively, than that of the pGL3
vector. After treatment of HepG2 cells with 50 nm
TPA for 24 h, the promoter activity of p15–profull,
p15–pro461 and p15–pro228 increased by 3.8-, 3.7-
and 4.6-fold, respectively, in comparison with that of
untreated HepG2 in each experimental group. It is
worth noting that the TPA-induced promoter activity
of p15–pro228 was slightly higher than that of p15–
profull and p15–pro461, although its basal promoter
activity decreased significantly. Also, the promoter
activity of p15–pro233, which contains the distal part
of p15–pro461, could be induced by TPA by only
C T. Hu et al. Transcriptional activation of p15
INK4b
FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS 1203
1.5-fold, much less than the promoter activity of p15–
pro461 and p15–pro228 (Fig. 1, right). Thus, it
seemed that the TPA-responsive element is mainly
located at the promoter region on p15–pro228, which
belongs to the proximal part of p15–pro461. Further-
more, p15–pro77, which contained the proximal part
of the promoter region in p15–pro228, did not exhibit
basal or TPA-induced promoter activity. This further
narrowed the TPA-responsive element to the region
between 77 to 228 bp upstream of the translational
initiation site.
Snail was required for TPA-induced activation of
p15–pro228
Our previous report showed that Snail was required for
TPA-induced activation of p15–pro461 [17], and we fur-

24 h. Dual luciferase assays were performed and the relative pro-
moter activity of each sample was calculated, taking the data for
pGL3 vector in untreated cells as 1.0. Statistical significance at
*P < 0.05 and **P < 0.005 between the indicated groups.
Fig. 1. Deletion mapping for identification of tetradecanoyl phorbol acetate (TPA)-responsive element for promoter activation of p15
INK4b
.
The full-length p15
INK4b
promoter (p15–profull) and other shorter promoter constructs are shown in the left-hand panel. HepG2 cells were
transfected with pGL3 vector or various p15
INK4b
promoter plasmids coupled with pRL control plasmid, and then untreated (white bar) or
treated with 50 n
M TPA (black bar) for 24 h. Dual luciferase assays were performed. The relative promoter activity of each sample was cal-
culated, taking the data for pGL3 vector in untreated cells as 1.0. The results of 5–7 experiments were averaged with a C.V. of 5.0–8.0%.
The numbers beside the solid bar represent the-folds of induction by TPA for each promoter construct. **Statistical significance (P < 0.005)
between the indicated groups.
Transcriptional activation of p15
INK4b
C T. Hu et al.
1204 FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS
promoter, genomatix software (v. GmbH 1998–2008)
was used to search the putative transcriptional factor-
binding regions within )226 and )80 bp on the TPA-
responsive element (Table S1). Interestingly, we found
binding regions (located between )202 and )169 bp) for
two transcriptional factors, EGR-1 and SP-1, which
according to previous studies could be induced by TPA
[27–29]. Thus we set out to investigate whether these

2–2.5-fold within 1–4 h and returned to the basal level
at 8 h (Fig. 3C). Also, Snail protein was significantly
induced by TPA within 1–2 h, maximally induced by
 2.5-fold at 4 h, and decreased to the basal level at
8 h (Fig. 3C). Collectively, these results indicated that
A
B
C
Fig. 3. Tetradecanoyl phorbol acetate (TPA)-induced gene expres-
sion of EGR-1, Snail and SP-1 in HepG2. HepG2 cells were untreated
(con) or treated with 50 n
M TPA for 0.5, 1, 2, 4 and 8 h (A and C).
Real-time RT/PCR (A) and western blot (C) of EGR-1, SP-1 and Snail
were performed. In (A), the relative mRNA level for EGR-1, SP-1 and
Snail at each time point of TPA treatment was caculated, taking the
basal expression of each gene (con) as 1.0. (B) Real-time PCR for
comparison of the basal levels of the three genes, taking the amount
of EGR-1 as 1.0. In (A) and (B), the results are the average of five
experiments with a C.V. of 5.0–8.5%. (A) Statistical significance at
*P < 0.05 and **P < 0.005 between the results for TPA-treated and
untreated HepG2 (con). (B) Statistical significance at **P < 0.005
between the results for SP-1 and the other two genes. ERK was the
internal control in (C). M, molecular mass marker.
C T. Hu et al. Transcriptional activation of p15
INK4b
FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS 1205
in addition to Snail, EGR-1 and SP-1 can also be
induced by TPA, which may be required for promoter
activation of p15
INK4b

pressed by  60–75% by SP-1 shRNA (fragments 46
or 47) (data not shown). Also, the TPA-induced
increase in Snail was attenuated by Snail shRNA (frag-
ments 18) at 4 h by  50% (data not shown). Taken
together, these results indicated that, in addition to
Snail, both EGR-1 and SP-1 were required for
TPA-induced promoter activation of p15
INK4b
.
Identification of the critical regions in the
TPA-responsive element
We further investigated whether the putative binding
motifs for EGR-1 and SP-1 are critical for activation
of the p15
INK4b
promoter. According to genomatix
software, there is an overlapping EGR-1/SP-1 binding
region ()202 to )186 bp) and an adjacent single SP-1
region ()183 to )169 bp) within the TPA-responsive
element (Table S1). To investigate which is crucial for
TPA-induced p15
INK4b
promoter activation, three dele-
tion constructs of p15–pro228 were employed (Fig. 5A,
left). In one, namely p15–pro228 DE/S-S, both the
overlapping EGR-1/SP-1-binding site and the adjacent
single SP-1 site (the region between )223 and )142 bp)
were deleted. In the other two, namely p15–pro228DE/
S and p15–pro228DS, the region containing the over-
Fig. 4. Prevention of tetradecanoyl phorbol acetate (TPA)-induced

(Fig. 5A, right). By contrast, the TPA-induced activa-
tion of p15–pro228DS (16.38-fold) was the same as
that of the parental p15–pro228, although its basal
activity was slightly reduced. Furthermore, promoter
assays using p15–pro228 with point mutations in the
putative EGR-1- and SP-1-binding sites were per-
formed. As demonstrated in Fig. 5B, TPA-induced
activation of a p15–pro228 mutant (p15–pro228 E/S*)
with three altered nucleotides on the EGR-1/SP-1
overlapping site (GGG fi TAT at )194 to )192) was
reduced by  80% compared with that of wild-type
p15–pro228. By contrast, TPA-induced activation of
the p15–pro228 mutants, namely p15–pro228 SP-1*,
with three altered nucleotides in the single SP-1 region
(TGG fi GAC at )176 to )174), decreased by only
20%. Taken together, these results strongly indicated
that the overlapping EGR-1/SP-1, but not the single
SP-1, binding region was essential for TPA-induced
p15–pro228 activation.
EMSA for in vitro DNA-binding activity of the
candidate transcriptional factors
The critical role of the overlapping EGR-1/SP-1 region
was further investigated by EMSA using nuclear
extract obtained from HepG2 with or without TPA
treatment. The probe p15proE/S contains a sub-
region ()210 to )181 bp) of the TPA-response ele-
ment harboring the overlapping EGR-1/SP-1-binding
site (Fig. 6A). As demonstrated in Fig. 6B, three
mobility-retarded DNA protein complexes, denoted as
SI, SII and SIII, increased in the EMSA of HepG2

treated with 50 n
M tetradecanoyl phorbol
acetate (TPA; black bar) for 24 h. Dual lucifer-
ase assays were performed and the relative
promoter activity of each sample was calcu-
lated, taking the data for pGL3 vector in
untreated cells as 1.0. The numbers beside
the black bar indicate the-fold of TPA-induced
promoter activity compared with each
untreated group. The results are the average
of 5–7 experiments with a C.V. of 5.0–7.0.
Statistical significance at **P < 0.005
between the indicated groups.
C T. Hu et al. Transcriptional activation of p15
INK4b
FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS 1207
the region spanning )203 to )184 bp. SII was sup-
pressed by only  40% by a mutant of the E/S com-
petitor with three altered nucleotides (GGG fi TAT
at )194 to )192). However, TPA-induced elevation of
SI was suppressed slightly by the addition of unlabeled
wild-type or mutant E/S competitor. Also, TPA-
induced elevation of SIII was not significantly influ-
enced by either wild-type or mutant E/S competitor at
6 h. Thus, it appeared that among the three TPA-
induced mobility-retarded bands of p15proE/S, SII
was not only the most abundant, but also the most
specific for EGR-1/SP-1-overlapping region. Further-
more, antibody-blocking experiments were performed
to examine which complex contained the candidate

immunopreciptation of Snail coupled with EGR-1
A
B
C
Fig. 6. EMSA for subregions on the tetradecanoyl phorbol acetate
(TPA)-responsive element of the p15
INK4b
promoter. (A) Schematic
representation of subregions in the TPA-responsive element ()228
to )77 bp), including the p15proE/S probe ()210 to )181 bp), the
E/S competitor ()203 to )184 bp) used for EMSA in (B) and (C)
and the p15–proSN probe ()218 to )197 bp) used for EMSA in
Fig. 9. (B) Time-course study for in vitro DNA binding activity.
Nuclear extracts of untreated HepG2 (control) or HepG2 treated
with 50 n
M TPA for 1, 2 and 6 h were incubated with p15proE/S
probe for EMSA. For competition, unlabeled wild-type or mutant E/
S competitor was included in the EMSA reaction using a sample of
HepG2 treated with TPA for 6 h. (C) Detection of the proteins
bound on p15proE/S. Nuclear extracts of untreated HepG2 (lane 1)
and HepG2 treated with 50 n
M TPA for 2 and 6 h were preincubat-
ed with antibodies (2 lg each) against the indicated transcriptional
factors or Raf (used as the negative control antibody) for 30 min,
followed by EMSA reaction. Lane 1 in (B) and (C) are the samples
of probe only. The results are representative of two reproducible
experiments.
Transcriptional activation of p15
INK4b
C T. Hu et al.

fold) for Snail was observed at 6 h, whereas induction
of EGR-1 ( 4.0-fold) was earlier at 2 h, sustained
until 6 h and thereafter decreased. SP-1 exhibited sig-
nificant basal activity, which may be elevated by 2.6-,
3.5- and 2.8-fold by treatment with TPA for 2, 6 and
12 h, respectively. The irrelevant Raf antibody,
employed as mock, did not precipitate Fragment 228
at all. It is worth noting that all three transcriptional
factors exhibited the maximal in vivo DNA-binding
activity at 6 h, which was also the time of maximal
in vitro DNA-binding activity observed in EMSA
A
B
Fig. 8. Binding and interaction of the transcriptional factors on tet-
radecanoyl phorbol acetate (TPA)-responsive element in vivo.
(A) Time-course analysis for single chromatin immunoprecipitaion
(ChIP). HepG2 cells were treated with 50 n
M TPA for 0, 1, 2, 6, 12
and 24 h, ChIP assays for binding of Snail, EGR-1 and SP-1 on Frag-
ment 228 were performed. Ab, antibody; IP, immunoprecipitation.
(B) HepG2 cells were treated with 50 n
M TPA for 0, 2 and 6 h, dou-
bled ChIP assays were performed using the indicated antibodies
for first immunoprecipitation (left) and second immunoprecipitation
(right). Raf antibody was used as MOCK antibody for the first
immunoprecipitation in both experimental groups. In both (A) and
(B), histone antibody was used to precipitate the promoter region
of GAPDH as the positive control group. In (A), PCR products of
Fragment 228 from each sample are shown as the Input. These
results are representative of three reproducible experiments.

examine whether these proteins interact with each
other on the TPA-responsive element. To address the
issue, a double ChIP assay using Fragment 228 was
performed. As shown in Fig. 8B (left), after the first
and second immunoprecipitation by antibodies of
EGR-1 and Snail, respectively, significant levels of
Fragment 228 can be detected in chromatin from cells
treated with TPA for 2 h, and this further increased by
2.5-fold at 6 h. In the reverse double ChIP using Snail
and EGR-1 antibody for the first and second immuno-
precipitations, respectively, a similar pattern of TPA-
induced binding with Fragment 228 was observed.
Also, after the first and second immunoprecipitation
by antibodies of EGR-1 and SP-1, respectively, signifi-
cant levels of Fragment 228 could be detected at 2 h
and this further increased by 3.0-fold at 6 h. In the
reverse double ChIP using SP-1 and EGR-1 antibody
for the first and second immunoprecipitations, respec-
tively, a basal level of Fragment 228 could be detected,
which increased slightly at 2 h and greatly (by 5.0-fold)
at 6 h. No PCR product of Fragment 228 could be
detected if mock antibody was used in the first immu-
noprecipitation in either experimental group. This
result confirmed that TPA may induce the association
of EGR-1 with Snail or SP-1 on the TPA-responsive
element of the p15
INK4b
promoter.
A proposed Snail target site involved in
TPA-induced p15–pro228

was involved in TPA-induced p15–pro228 activation.
Binding of Snail with the proposed Snail target
site
To examine whether the proposed Snail target region
can be bound by Snail, we performed EMSA using a
probe denoted as p15–proSN ()218 to )197 bp) which
contains this region (Fig. 6A). As shown in Fig. 9A,
two of the mobility-shifted bands (SNI and SNII)
increased significantly by 1.5–2.0-fold in EMSA using
nuclear extract from HepG2 treated with TPA for 1 h
compared with that from untreated HepG2. Both
bands further increased by 6.0- to 8.0-fold at 2 h and
decreased at 6 h. Another band, SNIII, had a rather
abundant basal level, increased by 2.5- and 5.0-fold at
1 and 2 h, respectively, followed by a decrease at 6 h.
In the competition group, SNII and SNIII were totally
abolished at 2 and 6 h by the addition of 200-fold
unlabeled p15–proSN, whereas SNI was not sup-
pressed at 2 h. We further examined whether alter-
ation in the 5-bp consensus sequence motif (TCACA)
of the proposed Snail target region may influence the
pattern of EMSA. As shown in Fig. 9B, the TPA-
induced elevation of SNII and SNI at both 2 and 6 h
decreased by 55–65% in the EMSA using p15–proSN
mutant as the probe (p15–proSN* with CAC fi GTG
at )206 to )204), compared with that using wild-type
probe (compare lanes 3 and 4 with lanes 7 and 8). In
addition, SNIII decreased dramatically in all the sam-
ples using p15–proSN* as the probe (compare lanes 2
and 4 with lanes 6 and 8). We further examined

either HepG2 treated with TPA or Snail overexpress-
ing HepG2, SNII is the most specific DNA–protein
complex which may contain the proposed Snail target
fragment bound by Snail.
Further, a ChIP assay was performed to investigate
whether Snail may bind to the proposed target region
in vivo. The target DNA was an 84-bp promoter frag-
ment ()200 to )284 bp) denoted as p15–proSN-ChIP,
which contains the proposed Snail target region
upstream of the EGR-1/SP-1 overlapping site
(Fig. 10A). As shown in Fig. 10B, slight basal binding
activity of Snail toward p15–proSN-ChIP was
observed in untreated HepG2, which was further
increased in HepG2 treated with TPA for 2 and 6 h by
 4.5-fold compared with the basal level. As a nega-
tive control, the binding of Raf with p15–proSN-ChIP
was not increased in TPA-treated HepG2.
Snail may enhance basal and TPA-induced
p15–pro228 activation
Thus far, we have found that Snail is not only associ-
ated with EGR-1 and SP-1 on the EGR-1/SP-1-over-
lapping region (Figs 7 and 8B), but is also capable of
binding to the proposed Snail target site (Figs 9 and
10). Both regions were required for TPA-induced
p15
INK4b
promoter activation (Fig. 5B). In addition,
we have previously shown that in HepG2 stably over-
expressing Snail, the promoter activity of p15
INK4b

C T. Hu et al. Transcriptional activation of p15
INK4b
FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS 1211
it appears that Snail may act as a transcriptional
activator in TPA-induced p15
INK4b
gene expression.
Because Snail is conventionally known to be a major
transcriptional repressor, this needs to be investigated
further. Therefore, we examined whether overexpres-
sion of Snail may influence p15–pro228 promoter acti-
vation. To our surprise, after transient transfection of
HepG2 with Snail-expressing plasmid for 36 h, the
TPA-induced p15–pro228 promoter activity increased
by 2.1-fold, and the basal p15–pro228 promoter was
elevated by 3.2-fold compared with that transfected
with pcDNA3 vector in each group (Fig. 11). This
result suggested that Snail may both enhance the TPA-
induced p15
INK4b
promoter activity (which might be
achieved via cooperation of EGR-1 and SP-1), and
also activate the p15
INK4b
promoter by itself.
Discussion
Previously, the mechanisms by which Snail upregu-
lates gene expression were not explored in as much
depth as those by which Snail downregulates gene
expression. Our study demonstrated the first time

ence or absence of tetradecanoyl phorbol acetate (TPA). HepG2
cells were transfected with Snail expressing plasmid or control
pcDNA3 vector followed by treatment with TPA (solid bar) for 12 h
or were left untreated (hollow bar). Dual luciferase assays were
performed and the relative ratio of Firefly/Renilla luciferase activity
of each sample was calculated, taking the data for HepG2 trans-
fected with pcDNA3 and without TPA treatment as 1.0. The results
are the average of 5–7 experiments with a C.V. of 5.0–7.0. Statisti-
cal significance at **P < 0.005 between the results of HepG2
transfected with Snail and pcDNA3 vector in either the presence or
absence of TPA.
A
B
Fig. 10. Chromatin immunoprecipitaion (ChIP) of the proposed
Snail binding region. (A) Scheme indicating the position of the pro-
moter fragment (p15–proSN-ChIP) for ChIP, which is located
between )200 and )284 bp containing the Snail target region
upstream of EGR-1/SP-1. (B) HepG2 cells were untreated or treated
with 50 n
M tetradecanoyl phorbol acetate (TPA) for 2 and 6 h, ChIP
assay for binding of Snail on promoter fragment p15–proSN-ChIP
were performed. Raf antibody was used as the negative control
antibody. Acetylated (Lys9/14) histone H3 antibody was used to
precipitate the promoter region of GAPDH as internal control group.
The PCR product of the target promoter fragment from chromo-
some of each sample is shown as the Input. The results are repre-
sentative of three reproducible experiments. Ab, antibody; IP,
immunoprecipitation.
Transcriptional activation of p15
INK4b

EGR-1 was also suggested by Grotegut et al., which con-
tradicts our observation. This may be because the experi-
mental systems used in the studies were different. In
Grotegut’s [9] report, an inducible system was used to
overexpress exogenous Snail, which may prevent hepato-
cyte growth factor-induced EGR-1. By contrast, we used
a shRNA technique to block the TPA-induced endoge-
nous Snail, resulting in the prevention of TPA-induced
EGR-1. This issue was further addressed by transfection
of the cells with exogenous Snail, followed by examining
whether TPA-induced EGR-1 mRNA is influenced.
Overexpression of Snail was verified by real-time PCR in
which Snail mRNA in cells transfected with Snail plas-
mid increased by 20.0- and 10.0-fold at 2 and 4 h, respec-
tively, compared with that in cells transfected with
pcDNA3 vector (Fig. S3B, upper). Interestingly, overex-
pression of Snail in HepG2 did not significantly suppress
TPA-induced EGR-1 at 2 h, although it greatly sup-
pressed TPA-induced EGR-1 at 4 h (by 80%) (Fig. S3B,
lower). This indicated that at high concentrations Snail
may prevent TPA-induced EGR-1 expression at a later
stage, consistent with that observed in Grotegut’s study.
Taken together, it can be suggested that Snail has dual
effects on EGR-1 expression, i.e. stimulatory and sup-
pressive at low and high concentrations, respectively.
The detailed mechanism for this phenomenon is worthy
of further investigation.
Interaction of EGR-1 with SP-1
The involvement of SP-1 in the transcriptional upregu-
lation of p15

SP-1 was much higher. Interestingly, simultaneous
binding of EGR-1 and SP-1 to adjacent DNA-binding
sites was also shown to be required for maximal activ-
ity of the interleukin-2 receptor b-chain promoter sug-
gesting synergistic functions of SP-1 and EGR-1 [34].
The time course for the interaction of SP-1 with EGR-
1 is another issue to be addressed. Notably, SP-1, but
not EGR-1, exhibited a basal level of gene expression
and DNA-binding activity in untreated HepG2, as
shown in Figs 3B and 8A. Furthermore, the double
ChIP assay (Fig. 8B) indicated that association of the
SP-1/EGR-1 immunocomplex with the TPA-responsive
element was observed after TPA treatment for 2–6 h.
It may be proposed that in untreated HepG2, SP-1
may constitutively bind on the overlapping EGR-1/
SP-1 region in p15
INK4b
promoter. After TPA treat-
ment for 2 h, EGR-1 may be recruited to its target site
on the same regions to interact with SP-1.
Potential Snail binding region on TPA-responsive
element
In our previous report [17], the critical role of Snail in
transcriptional regulation of the p15
INK4b
promoter was
C T. Hu et al. Transcriptional activation of p15
INK4b
FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS 1213
studied using EMSA. The promoter activity of p15–

proSN), we demonstrated that Snail antibody may
block the formation of DNA–protein complexes (espe-
cially SNII) in the TPA-treated sample (Fig. 9C). On
the other hand, overexpression of Snail also increased
the formation of SNII (Fig. 9D). Furthermore, using a
ChIP assay we demonstrated that after treatment with
TPA, the binding of Snail with its proposed target
region greatly increased in HepG2 (Fig. 10). Taken
together, we suggest that Snail protein may bind on the
region upstream of the EGR-1/SP-1-binding site for
TPA-induced activation of the p15
INK4b
promoter.
Finally, we demonstrated that overexpression of Snail
not only enhanced the TPA-induced p15–pro228 pro-
moter activity, but also elevated the basal promoter
activity (Fig. 11). It is possible that the Snail-induced
basal p15
INK4b
promoter activation might be achieved
by Snail itself via binding to the proposed Snail target
region. Alternatively, some other transcriptional tran-
scriptional factors in addition to Snail may be required
and other unknown Snail-binding regions might be
involved in this process. This interesting issue is worthy
of further investigation.
We propose the model for TPA-induced sequential
binding of SP-1, EGR-1 and Snail on the TPA-respon-
sive element of p15
INK4b

derived from p15–pro228 by double digestion with MluI/
XhoI, KpnI/PvuII, SauI/NheI and SauI/PvuII, respectively,
followed by filling in the restriction site overhangs by
Klenow enzyme. Subsequently, the digested DNA fragments
Fig. 12. Proposed model for tetradecanoyl phorbol acetate
(TPA)-induced sequential binding of SP-1, EGR-1 and Snail on the
TPA-responsive element in p15
INK4b
promoter. (Upper) At time zero,
SP-1 may be constitutively bound on the EGR-1/SP-1-overlapping
region (red). (Middle) After treatment with TPA for 1–2 h, EGR-1 is
recruited to the constitutively bound SP-1. (Lower) At 4–6 h, Snail
may associate simultaneously with the preformed EGR-1/SP-1
complex via EGR-1 and the sequence upstream of EGR-1/SP-1-over-
lapping region. The adjacent signal SP-1 site and the proposed Snail-
binding region are given in blue and black, respectively. The proposed
binding motif (TCACA)of Snail is marked as italic.
Transcriptional activation of p15
INK4b
C T. Hu et al.
1214 FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS
were self-ligated. The restriction enzyme map of the TPA-
responsive element within p15–pro228 is shown in Fig. S4.
Site-directed mutagenesis on TPA-responsive
element of p15
INK4b
promoter
The reporter plasmid p15–pro228 was used as a template
for site-directed mutagenesis using a GeneEditorÔ in vitro
site-directed mutagenesis system (Promega, Madison, WI,

(Promega, Madison, WI, USA). The pRL vector expressing
Renilla luciferase activity was co-transfected with experimen-
tal reporter plasmid as an internal control. The normalized
promoter activity was calculated as: Firefly luciferase activity
divided by Renilla luciferase activity. In general, the activity
of the control vector pRL in each sample was stable and not
significantly influenced by TPA. The raw data of one dual
luciferase assay are given in Table S2. The coefficient of var-
iation of pRL activity of each sample was within 2.0–9.0%
by statistical analysis of triplicate data (n = 3).
Nonradioactive EMSA
A DNA probe was biotinylated using a Biotin 3¢-end DNA
labeling kit (Pierce, Rockford, IL, USA). The LightShift
Chemiluminescent EMSA Kit (Pierce) was used for the
DNA–protein binding reaction, as described in the manual.
Subsequently, the binding reaction mixture was separated by
nondenaturing gel electrophoresis using 6.0–8.0% acrylam-
ide. The gel was transferred to a nitrocellulose membrane fol-
lowed by UV cross-linking. Membranes were then incubated
with streptavidin–horseradish peroxidase coupled with Lumi-
nol substrate for chemiluminescent detection of the protein–
DNA complex. To verify the transcriptional factors bound on
DNA, antibodies raised against zinc-finger motif (amino acids
602–610) of SP-1, the C-terminus of EGR-1 or the middle
region (amino acids 21–150) of Snail (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA, USA) was incubated separately with
nuclear extracts prior to EMSA reaction.
ChIP assay
A ChIP assay was performed using a modification of the
standard protocol. Briefly, after cross-linking chromosome

and 5¢-GGCATATGATGGAGATGATACTGA-3¢ (rev-
erse). SP-1: 5¢-TAGGAGAGATTGGGAGAAATCATC-3¢
(forward) and 5¢-AAGATACCAGAAGGTCGAGAGA
GA-3¢ (reverse). GAPDH was included as internal control
to correct for equal RNA amounts. HotStar Taq DNA
polymerase was used for primer extension. PCR mixtures
were preincubated at 95 °C for 15 min to activate the poly-
merase. Each of the 40 PCR cycles consisted of 16 s of
C T. Hu et al. Transcriptional activation of p15
INK4b
FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS 1215
denaturation at 94 °C, annealing of primers for 30 s
at 55 °C and 15 s of extension at 72 °C. The relative
amounts of mRNA were calculated using 7300 system sds
Software.
Western blot
Western blot was performed as described in our previous
report [17]. Briefly, the cells were lyzed and equal amount
of proteins in each sample were separated by 12% SDS/
PAGE and transferred to a poly(vinylidene difluoride)
membrane. Membranes were blocked in 5% dry milk and
probed with antibodies against molecules of interest. Fol-
lowing incubation with alkaline phosphatase-conjugated
secondary antibody, proteins were visualized with NBT/
BCIP for color development. For quantitation, the intensity
of each specific band was estimated with gel digitizing
software un-scan-it gel v. 5.1.
Immunoprecipitation/western blot
Immunoprecipitaion was performed by standard method.
Briefly, 800 lg of protein in cell lysate was incubated with

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Transcriptional activation of p15
INK4b
C T. Hu et al.
1218 FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS