Báo cáo khoa học: Snail associates with EGR-1 and SP-1 to upregulate transcriptional activation of p15INK4b doc

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

Tel: +8867 03 8565301; ext. 2327
E-mail:
(Received 10 October 2009, revised 10
December 2009, accepted 18 December
2009)
doi:10.1111/j.1742-4658.2009.07553.x
Snail is a multifunctional transcriptional factor that has been described as
a repressor in many different contexts. It is also proposed as an activator
in a few cases relevant to tumor progression and cell-cycle arrest. This
study investigated the detailed mechanisms by which Snail upregulates gene
expression of the CDK inhibitor p15
INK4b
in HepG2 induced by the tumor
promoter tetradecanoyl phorbol acetate (TPA). Using deletion mapping,
the TPA-responsive element on the p15
INK4b
promoter was located between
77 and 228 bp upstream of the transcriptional initiation site, within which
the putative binding regions of early growth response gene 1 (EGR-1) and
stimulatory protein 1 (SP-1) were found. Gene expression of EGR-1, Snail
and SP-1 can be induced by TPA within 0.5–6 h. In addition, basal levels
of SP-1, but not of the other two transcriptional factors, were observed.
Blockade of TPA-induced gene expression of Snail, EGR-1 or SP-1 sup-
pressed activation of the p15–pro228 reporter plasmid harboring the
TPA-responsive element. More detailed deletion mapping and site-directed
mutagenesis further concluded that the overlapping EGR-1/SP-1-binding
site was required for TPA-induced p15–pro228 activation. In an EMSA, a
DNA–protein complex was elevated by TPA, which can be blocked by
antibodies against EGR-1, SP-1 or Snail at 6 h. Immunoprecipitation/
western blotting demonstrated that TPA could trigger the association of

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

growth factor b [10,11] and WNTs [12], that may
trigger tumor progression. However, Snail can also
function as a negative regulator of cell growth [13].
Interestingly, cell division is impaired in Snail-
expressing epithelial cells that have undergone epithe-
lial mesenchymal transition [13–15] and Snail may
trigger invasion while suppressing tumor growth [16].
Our recent report also demonstrated that Snail may
simultaneously trigger both growth inhibition and
cell migration of HepG2 [17].
Conventionally, Snail was known to be a negative
regulator of gene expression and responsible for
diverse cellular effects. Snail was known to repress epi-
thelial markers such as E-cadherin [18,19]. Also, the
Crumbs polarity complex, a key apico-basal polarity
factor, was also found to be suppressed by Snail for
epithelial mesenchymal transition [20]. With regard to
the negative regulation of cell growth, Snail may
repress Cyclin D2 to block the cell cycle in the MDCK
cell line [13]. Recently, the possible role of Snail as a
transcriptional activator was highlighted. For example,
Slug, a Snail-related transcriptional factor, was found
to be capable of activating its own promoter. More-
over, Snail was implicated in the upregulation of
migration- and invasion-related genes including matrix
metalloproteinase 9 (MMP-9) [21–23] and integrin b
subunits [24]. Our recent report also demonstrated that
Snail was responsible for upregulation of the CDK
inhibitor p15
INK4b

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

INK4b
and a TPA-responsive element was identified on the
p15
INK4b
promoter [17]. In this study, we further pin-
point the critical regions by which Snail associates with
other transcriptional factors such as early growth
response gene 1 (EGR-1) and SP-1 to upregulate tran-
scription of p15
INK4b
.
Results
Deletion mapping for the TPA-responsive
element on the p15
INK4b
promoter
Initially, detailed deletion mapping using p15
INK4b
promoter constructs of various lengths was performed
to pinpoint the exact region responsible for promoter
activation (Fig. 1, left). Three constructs, p15–profull,
p15–pro461 and p15–pro228, contain regions encom-
passing 1006, 461 and 228 bp, respectively, upstream
of the translational start site, whereas p15–pro233
contains 233 bp of the distal part of the promoter
within p15–pro461, and p15–pro77 contains 77 bp in
the proximal part of the promoter within p15–pro228.
As demonstrated in Fig. 1 (right), p15–profull, p15–
pro461, p15–pro228 and p15–pro233 exhibited basal
promoter activities which were 4.95-, 4.12-, 1.67- and

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-

ther investigated whether it was also required for activa-
tion of p15–pro228. For this purpose, a short hairpin
RNA (shRNA) technique was used to observe whether
knockdown of Snail gene expression prevents TPA-
induced activation of p15–pro228. Three combinations
of effective Snail shRNA, namely sh1 (fragments 18 and
20), sh2 (fragments 18 and 19) or sh3 (fragments 19 and
20) prevented TPA-induced activation of p15–pro228 at
24 h by 35, 50 and 40%, respectively, compared with
Lamin A shRNA (used as control shRNA) (Fig. 2). It
appeared that Lamin A shRNA prevented TPA-induced
activation of p15–pro228 by 10–20% (Fig. 2 and data
not shown), probably because of the involvement of
Lamin A in transcriptional regulation. The effects of
Snail shRNAs were verified by western blotting, demon-
strating that TPA-induced Snail protein at 4 h was sup-
pressed by 40–55% by the transfection of sh1, sh2 and
sh3 (Fig. S1A).
Induction of gene expression of Snail, EGR-1 and
SP-1 by TPA
To investigate whether any other transcription factors
cooperate with Snail for activation of the p15
INK4b
Fig. 2. Suppression of tetradecanoyl phorbol acetate (TPA)-induced
p15–pro228 activation by Snail shRNA. HepG2 cells were co-trans-
fected with pGL3 and pRL, or with p15–pro228 and pRL coupled
with combinations of Snail shRNA (sh1, sh2 or sh3 as indicated in
the text) or Lamin shRNA as control. Transfected cells were
untreated (white bar) or treated with 50 n
M TPA (black bar) for

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

candidate transcriptional factors and their correspond-
ing cis-acting recognition sequences are involved in
TPA-induced p15
INK4b
promoter activation.
Initially, the gene expression profiles of these candi-
date transcriptional factors were investigated. Quanti-
tative real-time PCR analysis clearly demonstrated
that, compared with control levels, EGR-1 mRNA was
dramatically induced by 50 nm TPA at 30 min (47.0-
fold), followed by a gradual decrease from 1 to 4 h (to
 10-fold), finally returning to the basal level at 8 h
(Fig. 3A, upper). Also, Snail mRNA was significantly
induced by TPA by  1.5- to 2.3-fold within 30 min to
1 h, maximally induced by 5.0-fold at 2 h, decreased
to  3.0-fold at 4 h, and returned to the basal level at
8 h (Fig. 3A, middle). SP-1mRNA was maximally
induced by 5.2-fold after treatment of TPA for 1h, fol-
lowed by a decrease within 2)4 h (to  2.1- to 2.3-
fold) and returned to the basal level at 8 h (Fig. 3A,
lower). Notably, highly constitutive SP-1 mRNA
expression was observed, which was 5.2- and 5.0-fold
that of EGR-1 and Snail, respectively (Fig. 3B). On
the other hand, using western blot analysis, EGR-1
protein was found to increase dramatically by  5.0-
fold following treatment with TPA for 1 h, gradually
decrease from 2 to 4 h and had disappeared totally at
8 h (Fig. 3C). As seen in the mRNA level (Fig. 3A),
SP-1 protein exhibited constitutive expression. After
TPA treatment, SP-1 protein increased significantly by

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

.
EGR-1 and SP-1 were required for TPA-induced
promoter activation of p15
INK4b
Because gene expression of all these transcriptional
factors can be rapidly induced by TPA, the p15
INK4b
promoter may be activated at an early phase of TPA
treatment. As demonstrated in the time-course experi-
ment (Fig. 4A), promoter activity of p15–pro228 can
be significantly induced by TPA between 4 and 8 h,
followed by a dramatic increase at 12 h (by  20–25-
fold) and sustained until 24 h. We further examined
whether blocking gene expression of the aforemen-
tioned transcriptional factors may prevent TPA-
induced p15–pro228 activation at earlier time points.
As demonstrated in Fig. 4B, TPA-induced promoter
activation of p15–pro228 at 4 and 12 h was greatly
suppressed by shRNA of SP-1 (fragment 46) and
EGR-1 (fragment 36) by 90–95 and 80–95%, respec-
tively, compared with the mock (Lamin A) shRNA. In
comparison, Snail shRNA (fragments 18) prevented
less ( 45–80%) TPA-induced activation of p15–
pro228. The effects of the shRNAs for these transcrip-
tional factors were verified by western blot analysis.
The TPA-induced increase in EGR-1 protein at 1 h
was attenuated by transfection of EGR-1 shRNA
(fragments 33 and 36) by  75–80%, compared with
that of mock (Lamin A) shRNA (Fig. S1B). Similarly,
the TPA-induced increase in SP-1 at 4 h was sup-

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

activation of p15–pro228 by blocking Snail, EGR-1 and SP-1
expression. (A) Time-course analysis of TPA-induced activation
of p15–pro228. HepG2 cells were transfected with pGL3 vector or
p15–pro228 plus pRL control plasmid followed by treatment with
TPA for the times indicated. Dual luciferase assays were performed.
The relative promoter activity of each sample was calculated, taking
the data for pGL3 vector in untreated cells as 1.0. The results are the
average of three experiments with a C.V. of 5.0–8.5%. (B) Knock-
down of Snail, EGR-1 or SP-1 prevented TPA-induced p15–pro228
activation. HepG2 cells were co-transfected with pGL3 and pRL, or
with p15–pro228 and pRL coupled with shRNA of SP-1 (SP46), EGR-
1 (E33), Snail (SN18) or shRNA of Lamin as mock, followed by no
treatment (white bar) or treatment with 50 n
M TPA (black bar) for 4
and 12 h. Dual luciferase assays were performed and the relative
promoter activity of each sample was calculated, taking the data for
pGL3 vector in untreated cells as 1.0. The results are the average of
5–7 experiments with a C.V. of 5.0–8.5%. Statistical significance at
**P < 0.005 between HepG2 cells co-transfected with the indicated
shRNA and with mock shRNA.
Transcriptional activation of p15
INK4b
C T. Hu et al.
1206 FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS
lapping EGR-1/SP-1 site ()223 to )179 bp) and the
single SP-1 site ()176 to )143 bp), respectively, were
deleted. Interestingly, the TPA-induced promoter acti-
vation of p15–pro228DE/S-S (3.17-fold) and p15–
pro228DE/S (3.29-fold) decreased by  80% compared
with those of the parental p15–pro228 (16.28-fold)

(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 TPA for 1, 2 and 6 h. SII increased signi-
ficantly by  2.0-fold after treatment of the cell with
TPA for 1 h and dramatically increased at 2 and 6 h
by  9.5- and 10.0-fold, respectively, compared with
that of untreated HepG2. SI, which migrated more
slowly than SII, increased significantly at 2 and 6 h by
 1.5- to 2.3-fold. SIII, which migrated faster than
SII, increased significantly within 1–6 h by 2.0 to
3.0-fold. In the competition analysis for the sample
from cells treated with TPA for 6 h (Fig. 6B, lanes
6–7), SII could be 90% suppressed by addition of the
unlabeled wild-type EGR-1/SP-1 overlapping fragment
(denoted as E/S competitor in Fig. 6A) which contains
A
B
Fig. 5. Promoter assay of p15–pro228 with
deletions or point mutations on various puta-
tive transcriptional factor binding sites.
HepG2 were co-transfected with pRL control
plasmid and wild type p15–pro228 or pRL
and p15–pro228 with deletion (A) or point
mutations (B) on the EGR-1/SP-1 overlapping
site, the single SP-1 site and the proposed
Snail-binding motif within )202 to )184,
)183 to )169 and )207 to )202 bp upstream
of the translational initiation site, respec-
tively. The map for the sites of deletion and
point mutation on each region are show in
the left-hand panel in (A) and (B). Transfected
cells were either not treated (white bar) or

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

transcriptional factors induced by TPA. As demon-
strated in Fig. 6C, TPA-induced elevation of complex
SII at 2 h was greatly reduced by preincubation of the
nuclear extracts with antibodies of SP-1 and EGR-1
(lanes 6 and 8), but decreased only slightly if Snail
antibody was used (lane 7). At the 6 h time point, SII
was greatly reduced by preincubation of the nuclear
extract with antibodies against each of the three tran-
scription factors (lanes 9–11). Raf antibody (as the
control antibody) did not block TPA-induced elevation
of complex SII at either time point (lanes 12 and 13).
By contrast, TPA-induced elevations of both complex
SI and SIII were not significantly blocked by any anti-
bodies. Thus, SII, but not SI and SIII, is the most
important DNA–protein complex that contains the
candidate transcriptional factors induced by TPA.
Taken together, by examining the pattern of SII we
suggest that the in vitro DNA-binding activity of all
three transcriptional factors toward p15proE/S could
be elevated within 2–6 h following treatment with
TPA.
Immunoprecipitation/western blotting for
TPA-induced association of the candidate
transcriptional factors
Thus far, Snail, EGR-1 and SP-1 appeared to act in
concert for TPA-induced p15
INK4b
promoter activa-
tion. We further investigated whether they may
interact with each other during this process. By

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.

1208 FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS
western blot analysis (Fig. 7, upper), EGR-1 could be
detected in the immunopreciptates of Snail from cells
treated with TPA at 6.0 h. By immunoprecipitation of
SP-1 coupled with EGR-1 western blot (Fig. 7, lower),
EGR-1 (indicated by large arrow) could be abundantly
detected in the immunopreciptates of SP-1 from cells
treated with TPA at the 2.0 h time point. A nonspe-
cific band (indicated by small arrow) below the
EGR-1-specific band can be detected in all samples
analyzed. In addition, SP-1 could not be detected in
the immunopreciptate of Snail from TPA-treated
HepG2 (data not shown). Taken together, it appeared
that TPA could induce the association of EGR-1 with
both Snail and SP-1, but not the association of Snail
with SP-1.
Chromatin immunoprecipitaion assay for in vivo
DNA-binding activity of the candidate
transcriptional factors
To further examine whether TPA may induce DNA
binding of the candidate transcriptional factors toward
the p15
INK4b
promoter in vivo, chromatin immunopre-
cipitaion (ChIP) assays were performed. As shown in
Fig. 8A, the DNA-binding activity of all three
transcriptional factors toward the promoter fragment
encompassing TPA-responsive element ()228 to )1 bp,
denoted as Fragment 228) could be induced by TPA.
The maximal TPA-induced binding activity ( 3.0-

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.

Fig. 7. Immunoprecipitation (IP)/western blotting for the associa-
tion of Snail, EGR and SP-1. HepG2 cells were untreated (con) or
treated with 50 n
M tetradecanoyl phorbol acetate (TPA) for 0.5, 2, 6
and 8 h. Eight hundred micrograms of protein for each sample was
used for immunopreciptation followed by western blotting. The
antibodies used for immunopreciptation and western blotting are
indicated on the right and left of the gel, respectively. Positions of
EGR-1 and nonspecific bands are indicated by an arrow. Western
blots of ERK were performed to monitor equal amounts of protein
in each sample used for immunopreciptation. The results are repre-
sentative of 2–3 reproducible experiments.
C T. Hu et al. Transcriptional activation of p15
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FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS 1209
(Fig. 6C). This implied that 6 h is the most critical
time point for TPA-induced binding of the transcrip-
tional factors on the p15
INK4b
promoter.
Double ChIP assay for TPA-induced interaction
of the critical transcriptional factors on the
promoter region
Using both ChIP assay (Fig. 8A) and immunoprecipi-
tation/western analysis (Fig. 7) we found that TPA
may induce both interaction of EGR-1 with Snail or
SP-1 with TPA-responsive element of the p15
INK4b
promoter, and also protein–protein association of
EGR-1 with Snail or SP-1. Thus, it is intriguing to

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

Because there is no putative binding region of Snail
such as the E-box on the TPA-responsive element,
whether Snail binds on an unidentified region around
the EGR-1/SP-1 overlapping site for activation of the
p15
INK4b
promoter is an intriguing issue to be
explored. Using genomatix software, there is a 5-bp
consensus sequence motif (TCACA) upstream of the
EGR-1/SP-1 overlapping site on promoters of
p15
INK4b
(at )207 to )203), which is also found on the
MMP-9 promoter, another Snail-upregulated gene [21–
23]. It is tempting to speculate that the sequence
around this motif is the potential Snail target site for
p15
INK4b
promoter activation (see Discussion).
To investigate whether the proposed Snail target
region was required for TPA-induced p15
INK4b
pro-
moter activation, a p15–pro228 mutant denoted as
p15–pro228SN* with three nucleotides altered in this
region (CAC fi GTG at )206 to )204) (Fig. 5B, left)
was employed. As shown in Fig. 5B (right), TPA-
induced promoter activity of p15–pro228SN*
decreased by 45% compared with that of wild-type
p15–pro228. Thus, the proposed Snail target region

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

whether Snail protein could be contained within the
band shifts. As shown in Fig. 9C, SNII and SNIII
were suppressed by 95 and 65% at 2 h if the EMSA
reaction mix was preincubated with Snail antibody,
but not Raf antibody (the control antibody), for
30 min (compare lanes 4 and 8). At 6 h, the blocking
effect of the Snail antibody was less prominent
because the amount of both complexes had already
Transcriptional activation of p15
INK4b
C T. Hu et al.
1210 FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS
decreased at this time (compare lanes 5 and 9). By
contrast, the amount of SNI was not significantly
influenced by Snail antibody at any time. To further
validate the specificity of band shifts with regard to
Snail, HepG2 was transiently transfected with a Snail-
expressing plasmid for 36 h, followed by EMSA. The
Snail mRNA in the Snail-transfected cell increased by
 16.0-fold, as detected by real-time RT/PCR
(Fig. S2). Interestingly, SNI and SNII (but not SNIII)
increased by 3.0–3.5-fold in the EMSA using nuclear
extract from HepG2 transfected with Snail, compared
with the cell transfected with pcDNA3 vector
(Fig. 9D, compare lanes 1 and 2). Moreover, the
amount of SNII (but not SNI) in EMSA for HepG2
overexpressing Snail decreased dramatically (by 90%)
if p15–proSN* was used as the probe instead of wild-
type p15–proSN (Fig. 9D, compare lanes 1,2 with
3,4). Taken together, it appeared that in EMSA for

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

was higher than in the parental cell [17]. Thus
CD
AB
Fig. 9. EMSA of the proposed Snail target
site. Nuclear extracts of untreated HepG2
(lane 2 in A, C and lanes 2 and 6 in B),
HepG2 treated with 50 n
M tetradecanoyl
phorbol acetate (TPA) for 1, 2 or 6 h (lanes
3–7 in A and 3–9 in C, as indicated) or 2 and
6 h (lanes 3–4 and 7–8 in B), and HepG2
transfected with Snail overexpressing plas-
mid or pcDNA3 vector (C) were incubated
with p15–proSN wild type probe (all lanes in
A and C, lanes 1–4 in B and lane 1–2 in D)
or p15–proSN* mutant probe (lanes 5–8 in
B and lanes 3–4 in D) followed by EMSA.
For competition analysis (lanes 6–7 in A),
unlabeled wild-type p15–proSN was
included in the EMSA using a sample of
HepG2 treated with TPA for 2 and 6 h. For
antibody blocking analysis (lanes 6–9 in C),
nuclear extracts from HepG2 treated with
TPA for 2 and 6 h were preincubated with
antibodies (2 lg each) against Snail or Raf
(as the negative control antibody) followed
by EMSA. ‘Probe only’ in (A), (B) and (C)
represents the sample without nuclear
extract. The results are representative of
2–3 reproducible experiments.

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

that Snail cooperates with other transcriptional fac-
tors such as EGR-1 and SP-1 to upregulate gene
expression using TPA-induced p15
INK4b
as a model.
This molecular event occurred in a time-dependent
manner, in which SP-1, EGR-1 and Snail may be
recruited successively to their target regions on the
TPA-responsive element and interact with each other.
The time course for TPA-induced gene expression of
these transcriptional factors correlated well with
the timing of protein–protein and protein–DNA
interactions.
Interaction of Snail with EGR-1
Previously, EGR-1 has been found to be involved in
TPA-induced gene expression of tyrosine hydroxylase in
PC12 cells [30] and MDR1 in hematopoietic cell K562
[31]. Our results further demonstrated that EGR-1 acted
with Snail for TPA-induced promoter activation of
p15
INK4b
. It is worth noting that the TPA-induced gene
expression of EGR-1 was earlier than that of Snail
(Fig. 3A,C), which was also observed during hepatocyte
growth factor-induced cell migration of HepG2 [9].
Consistent with this, ChIP assays demonstrated that
TPA-induced binding of EGR-1 with the TPA-respon-
sive element Fragment 228 was earlier at 2 h and was
sustained until 6 h, whereas that of Snail was induced at
Fig. 11. Effects of Snail on activation of p15–pro228 in the pres-

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

C T. Hu et al.
1212 FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS
6 h. This is in accord with the result of both immunopre-
cipitation/western analysis (Fig. 7) and double ChIP
assays (Fig. 8B) demonstrating that TPA-induced maxi-
mal association of EGR-1with Snail occurred at 6 h.
One more interesting issue to be addressed is the regu-
lation of Snail and EGR-1 gene expression by each other.
Previously, Grotegut et al. [9] demonstrated that EGR-1
was required for hepatocyte growth factor-induced tran-
scription of Snail. According to the time course experi-
ment (Fig. 3A,C), TPA-induced maximal expression of
EGR-1 mRNA and protein occurred earlier than that of
Snail, and it is possible that induction of Snail may be
mediated by EGR-1. As expected, quantitative real-time
RT/PCR analysis showed that TPA-induced Snail
mRNA expression at 2 h can be suppressed by EGR-1
shRNA (fragment E33) by 68% compared with TPA-
induced Snail mRNA expression in cells transfected with
mock (Lamin) shRNA (Fig. S3A, upper left). To our sur-
prise, Snail shRNA (fragment S18) can also suppress
TPA-induced EGR mRNA expression at the 4 h time
point by 42% (Fig. S3A, lower right). These results dem-
onstrated that EGR-1 gene expression induced by TPA
at the earlier stage was required to trigger gene expression
of Snail at 2 h, which was then used to sustain TPA-
induced EGR-1 mRNA expression at later period (i.e.
4 h). This implied that both transcriptional factors may
be upregulated by each other in a positive feedback man-
ner. However, a negative feedback effect of Snail on

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

INK4b
was investigated decades ago. Early
reports demonstrated that a SP-1 consensus site was
required for transforming growth factor b-induced
p15
INK4b
promoter activation [32]. Furthermore, the
cooperation of SP-1 with Smad2, Smad3 and Smad4
was required for this transcriptional event [33]. In our
results, the role of SP-1 in TPA-induced p15
INK4b
pro-
moter activation was also essential, as evidenced by
basal and TPA-induced SP-1 gene expression
(Fig. 3A), SP-1 binding on Fragment 228 (Fig. 8A)
and the association of SP-1 with EGR-1 (Fig. 7).
Furthermore, the cis-acting element of SP-1 for TPA-
induced p15
INK4b
promoter activation was also clari-
fied in this study. There are two SP-1-binding motifs
on the TPA-responsive element, one overlaps with that
of EGR-1 at )202 to )186 bp, the other is the adja-
cent single SP-1 site at )183 to )169 bp (Table S1).
Strikingly, deletion of or point mutation in the EGR-
1/SP-1 overlapping site, but not the single SP-1 site,
abolished TPA-induced activation of p15–pro228
(Fig. 5A,B). Therefore, the possibility that SP-1
(together with EGR-1) may bind on the overlapping
EGR-1/SP-1 region rather than on the adjacent single

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–

pro228 and in vitro DNA-binding activity toward the
TPA-responsive element were enhanced in HepG2 over-
expressing Snail (S7) compared with that of the parental
HepG2. Moreover, in the EMSA result (Fig. 6C), we
found that Snail antibody can suppress TPA-induced
mobility shift of p15–proES containing the EGR-1/SP-1
binding region at the 6 h time point. However, whether
there are Snail-specific target regions in the p15
INK4b
promoter has not been clarified. Investigation of this
issue can be initiated by comparing the promoter
sequence of p15
INK4b
with that of other genes known to
be upregulated by Snail. Recently, we found that gene
expression of MMP-9 can also be induced by TPA in an
EGR-1-, SP-1- and Snail-dependent manner (unpub-
lished results), implying that both p15
INK4b
and MMP-9
can be upregulated by similar transcriptional apparatus.
Furthermore, there is a 5-bp consensus sequence motif
(TCACA) upstream of the EGR-1/SP-1-binding site on
both the p15
INK4b
and MMP-9 promoter. Interestingly,
alteration of three nucleotides in this region partially
suppress (by  45%) TPA-induced activation of p15–
pro228 (Fig. 5B). Moreover, using EMSA with a probe
containing this proposed Snail-binding region (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

promoter in a time-dependent
manner as follows (Fig. 12). Before TPA treatment,
SP-1 is constitutively bound on the EGR-1/SP-1-over-
lapping region. After 1–2 h of TPA treatment, EGR-1
is recruited to the same region and associates with
SP-1. Subsequently (within 4–6 h of TPA treatment),
Snail is also recruited. Finally, Snail may bind with
both EGR-1 and the region (TCACA) upstream of the
EGR-1/SP-1 overlapping site simultaneously. Thus a
tri-component transcriptional complex may be formed
to trigger p15
INK4b
promoter activation.
Materials and methods
Cell culture, chemicals and plasmid
The cultured condition for HepG2 cells was as described in
our previous report [17]. Tetradecanoyl phorbol acetate was
purchased from Sigma-Aldrich (Poole, UK). Plasmid with a
full-length Snail coding sequence under the CMV promoter
was a gift from C. C. Chang (Tzu Chi University, Hualien,
Taiwan).
Construction of deletion mutants of the p15
INK4b
promoter
The reporter plasmids p15–profull, p15–pro461 and p15–
pro228 were as used in the previous report [17]. Plasmid
p15–pro233 was derived from p15–pro461 by double diges-
tion with SmaI/KpnI followed by ligation of the digested
small fragment into pGL3. Plasmids p15–pro77, p15–
pro228DE/S-S, p15–pro228DE/S and p15–pro228DS were

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,

USA). The sequences of the oligonucleotides for mutagene-
sis were 5¢-ATCACAGCGGACAGG
TATCGGAGCCTAA
GGGGG-3¢ (on the EGR-1/SP-1-overlapping site); 5¢-GG
GCGGAGCCTAAGGGGG
GACGGAGACGCCGGC-
CCC-3¢ (on the adjacent SP-1-binding site); 5¢-TGGCCTC
CCGGCGAT
GTGAGCGGACAGGGG (on the proposed
Snail-binding site). Underlined letters indicate the changed
nucleotides. The mutagenesis reaction began with annealing
of the selection oligonucleotides and the mutagenic oligonu-
cleotides to the DNA template, followed by synthesis of the
mutant strand with T4 DNA polymerase and T4 DNA
ligase. The heteroduplex DNA was then transformed into
the repair minus Escherichia coli strain BMH71-18 mutS,
and the cells were grown in selective media to obtain clones
containing the mutant plasmid. Plasmids resistant to the
novel GeneEditorÔ Antibiotic Selection Mix were then iso-
lated and transformed into the final host strain, TOP10,
using the same selection conditions.
Promoter activity assay
Various truncated formats of pGL3-based reporter plasmids
of p15
INK4b
promoter were transfected with or without vari-
ous shRNAs into HepG2 cells using the effectene transfec-
tion system (Invitrogen Ltd, Renfrew, UK). After the
required treatments, cells were harvested and assayed for
dual luciferase activity, as described in the manual protocol

(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

with the bound proteins, immunoprecipitation of the tran-
scriptional factors was performed using polyclonal antibod-
ies against Snail, EGR-1 and SP-1. Raf-1 is used as a mock
antibody. As a positive control, antibody of acetylated
(Lys9/14) histone H3 (Ac-histone H3) (Santa Cruz Biotech-
nology) was used to precipitate the histone-bound GAPDH
promoter. The recovered DNA was analyzed by PCR with
primers flanking the putative transcriptional factor binding
sites as indicated. For quantitation, the gels were scanned
and the intensity of each PCR band was estimated with gel-
digitizing software un-scan-it gel v. 5.1. Primers used for
the p15
INK4b
promoter (Fragment 228), p15–proSN-ChIP
and GAPDH promoter fragment were designed using
primer 3 software as follows: Fragment 228, 230 bp:
(F)5¢-TACTAGCGGTTTTACGGGCG-3¢, (R)5¢-TCGAA
CAGGAGGAGCAGAGAGCGA-3¢; p15–proSN-ChIP,
84 bp: (F)5¢-ATGCGTCCTAGCATCTTTGG, (R)5¢-TGT
CCGCTGTGTCGCCG; GAPDH promoter fragment,
150 bp: (F)5¢-TAGCCGGGCCTGGCCTCC-3¢, (R)5¢-GG
ATTGCTTCTGGGAAA-3¢.
Quantitative RT-PCR
Real-time PCR of EGR-1 and Snail were performed by
QuantiTect SYBR PCR kit (Qiagen, Crawley, UK) using
ABI 7300 real-time PCR system (Applied Biosystems, Fos-
ter City, CA, USA). The primer sequences used were Snail:
5¢-TCCAGAGTTTACCTTCCAGCA-3¢ (forward) and
5¢-CTTTCCCACTGTCCTCATCTG-3¢ (reverse); EGR-1:
5¢-CTCTTAGGTCAGATGGAGGTTCTC-3¢ (forward)

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

antibodies bound on protein A–Sepharose beads on roller
at 4 °C for 2 h. After extensive washing, proteins were
eluted from the beads with loading buffer followed by
western blot of the indicated proteins.
shRNA technology
Lentiviral plasmids each encoding shRNA targeting differ-
ent regions of the indicated mRNA were obtained from
RNAi Core Laboratory (Academia Sinica, Taiwan). Cells
at 60% confluence were transfected with 0.26 ngÆ l L
)1
shRNA either alone or in combination, using Effectene
transfection reagent (Invitrogen) according to the manufac-
turer’s protocol. Lentiviral plasmid encoding human Lamin
A shRNA was used as mock shRNA. The shRNA frag-
ments targeting different regions of Snail, EGR-1 and SP-1
mRNA are given in Table S3.
Statistical analysis
Data were analyzed using Student’s t-test in excel. All the
quantitative studies were performed at least in triplicate, as
appropriate. Differences were considered significant at
P < 0.05. Statistical significance between groups is indi-
cated by *P < 0.05 and **P < 0.005.
Acknowledgements
We thank National Science Council in Taiwan
and Research Centre of Hepatology in Buddhist
Tzu Chi General Hospital for financial support, and
Ms Sindy Wang and Mr Joseph Long for technical
assistance.
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C T. Hu et al. Transcriptional activation of p15
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FEBS Journal 277 (2010) 1202–1218 ª 2010 The Authors Journal compilation ª 2010 FEBS 1217
Supporting information
The following supplementary material is available:
Fig. S1. Effective shRNA of EGR and Snail prevented
TPA-induced EGR-1 and Snail.
Fig. S2. Overexpression of Snail mRNA in HepG2
transfected with plasmid encoding Snail.
Fig. S3. Quantitative RT/PCR for cross-regulation of
EGR-1 and Snail.
Fig. S4. Restriction map on the TPA responsive
element.
Table S1. The putative transcriptional factor binding
regions on the TPA-responsive element (between )226
and )80 bp).
Table S2. The raw data of pRL activity.
Table S3. shRNA targeting sequences of Snail, EGR-1

and SP-1 used for knockdown approach.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and read-
ers, this journal provides supporting information
supplied by the authors. Such materials are peer-
reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Techni-
cal support issues arising from supporting informa-
tion (other than missing files) should be addressed to
the authors.
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