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

Using deletion mapping, the TPA-responsive element on the p15INK4bpromoter was located between 77 and 228 bp upstream of the transcriptional initiation site, within which the putative bi

transcriptional activation of p15INK4b

Chi-Tan Hu1, Tsu-Yao Chang2, Chuan-Chu Cheng2, Chun-Shan Liu2, Jia-Ru Wu2, Ming-Che Li1and Wen-Sheng Wu2

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: wuwstcu1234@yahoo.com.tw

(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 p15INK4bin HepG2 induced by the tumor promoter tetradecanoyl phorbol acetate (TPA) Using deletion mapping, the TPA-responsive element on the p15INK4bpromoter 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 p15INK4bin 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.

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 p15INK4b required for tetradecanoyl phorbol

acetate (TPA)-induced cell-cycle arrest [17]

Snail family proteins contain a C-terminal tandem

C2H2 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 p15INK4b and a TPA-responsive element was identified on the p15INK4b 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 p15INK4b

Results

Deletion mapping for the TPA-responsive element on the p15INK4bpromoter Initially, detailed deletion mapping using p15INK4b 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

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 whefur-ther 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 p15INK4b

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 p15INK4bpromoter (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.

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-actcorrespond-ing recognition sequences are involved in TPA-induced p15INK4bpromoter 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.

in addition to Snail, EGR-1 and SP-1 can also be

induced by TPA, which may be required for promoter

activation of p15INK4b

EGR-1 and SP-1 were required for TPA-induced promoter activation of p15INK4b

Because gene expression of all these transcriptional factors can be rapidly induced by TPA, the p15INK4b 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 p15INK4b

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 p15INK4b 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 p15INK4bpromoter activation, three dele-tion constructs of p15–pro228 were employed (Fig 5A, left) In one, namely p15–pro228DE/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 (SNEGR-18) 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.

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.

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 p15INK4b 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 p15INK4bpromoter (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 indicatpreincubat-ed 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.

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 p15INK4b 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.

(Fig 6C) This implied that 6 h is the most critical

time point for TPA-induced binding of the

transcrip-tional factors on the p15INK4bpromoter

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 p15INK4b

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 p15INK4bpromoter

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

p15INK4b 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

p15INK4b(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 p15INK4bpromoter activation (see Discussion)

To investigate whether the proposed Snail target region was required for TPA-induced p15INK4b 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

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 p15INK4b promoter activation (Fig 5B) In addition,

we have previously shown that in HepG2 stably over-expressing Snail, the promoter activity of p15INK4b was higher than in the parental cell [17] Thus

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.