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Molecular and Cellular Biology, March 2006, p. 1770-1785, Vol. 26, No. 5
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.5.1770-1785.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Sp1 Deacetylation Induced by Phorbol Ester Recruits p300 To Activate 12(S)-Lipoxygenase Gene Transcription

Jan-Jong Hung, Yi-Ting Wang, and Wen-Chang Chang*

Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China

Received 27 August 2005/ Returned for modification 7 October 2005/ Accepted 8 December 2005


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ABSTRACT
 
We previous reported that Sp1 recruits c-Jun to the promoter of the 12(S)-lipoxygenase gene in 12-myristate 13-acetate-treated cells. We now show that Sp1 that recruited HDAC1 to the Sp1/cJun complex was constitutively acetylated when cells were exposed to phorbol 12-myristate 13-acetate (PMA) (3 h). Prolonged stimulation of the cells with PMA (9 h), however, caused the dissociation of histone deacetylase 1 (HDAC1) and the deacetylation of Sp1, with the latter being able to recruit p300 that in turn caused the acetylation and dissociation of histone 3, thus enhancing the expression of 12(S)-lipoxygenase. We also overexpressed an Sp1 mutant (K703/A, lacking acetylation sites) in the cell and found that cells recruited more p300 and expressed more 12(S)-lipoxygenase. Taken together, our results indicated that Sp1 recruits HDAC1 together with c-Jun to the gene promoter, followed by deacetylation of Sp1 upon PMA treatment. p300 is then recruited to the gene promoter through the interaction with deacetylated Sp1 to acetylate histone 3, leading to the enhancement of the expression of 12(S)-lipoxygenase.


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INTRODUCTION
 
Simian virus 40 promoter factor 1 (Sp1) was one of the first transcription factors purified and cloned from mammalian cells (16, 26). Sp1 recognizes and binds, via three Cys2-His2 zinc finger motifs localized at its carboxyl terminus, GC-rich sites at the simian virus 40 promoter, thus regulating the transcription of target genes (3, 36). Recent studies revealed that both the DNA binding ability and transactivational activity of Sp1 might be influenced by posttranslational modifications of Sp1, including phosphorylation, glycosylation, or acetylation (7, 27). Specifically, phosphorylation of Sp1 by casein kinase II repressed Sp1 activity, and the phosphorylation by DNA-dependent protein kinase or cyclin-dependent kinase 2 regulated the transactivity and enhanced the DNA binding affinity of Sp1 (1, 2, 18, 25). In addition, glycosylation of Sp1 regulates the proteasome-dependent degradation of Sp1 (9, 14, 22). Several studies have also shown, using trichostatin A (TSA) treatment, that Sp1 is acetylated to regulate DNA binding affinity or transactivation (4, 15, 23, 35). However, it is still not known which residue(s) of Sp1 is acetylated. Furthermore, the functional outcome, if any, of the acetylation of Sp1 in the cellular system is still unknown.

Arachidonate 12(S)-lipoxygenase (arachidonate:oxygen 12-oxidoreductase) (EC 1.13.11.31) expressed in the platelet was the first mammalian lipoxygenase discovered (21). It catalyzes the transformation of arachidonic acid into 12(S)-hydroperoxyeicosatetraenoic acid, which is then converted to 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE]. 12(S)-HETE plays a significant role in the pathogenesis of certain forms of epidermal and epithelial inflammation. A high level of 12(S)-HETE may contribute to the inflammatory changes and the abnormal epidermal hyperproliferation in the development of psoriatic plaques. In terms of the promoter activity, two Sp1 binding sites were found at –158 to –150 bp and –123 to –114 bp of the 12(S)-lipoxygenase gene promoter, and Sp1 recruitment is responsible for the basal transcription of lipoxygenase (31). Upon stimulation of cells with phorbol 12-myristate 13-acetate (PMA) or epidermal growth factor (EGF), however, c-Jun can be recruited to the promoter through an interaction with Sp1 to regulate transcription of the 12(S)-lipoxygenase gene (11). Therefore, the interaction between Sp1 and c-Jun, which may be induced by either PMA or EGF treatment, is important for the functional regulation of the human 12(S)-lipoxygenase gene. However, the exact molecular events downstream of the c-Jun recruitment leading to the activation of transcription of 12(S)-lipoxygenase still remain unclear.

Two types of enzymes, the histone deacetylases (HDACs) and histone acetyltransferases (HATs), are known to control the acetylation of histones and other proteins, leading to the chromatin remodeling that modulates the transcription activity of numerous genes (45). Many transcription factors have been shown to associate with HDAC1 to regulate gene transcription. HDAC1 can be recruited to or dissociated from the affected promoter to repress or induce, respectively, the transcription activity of regulated genes (40). For instance, HDAC1 recruited by Sp1 to the promoter of Sp1-regulated genes can cause histone deacetylation, leading to the Ras-induced down-regulation of the metastasis suppressor RECK (10). In analogy, another study proposed a novel mechanism by which STAT5 mediates the deacetylation of C/EBPß, thus allowing transcriptional activation (43). Although it is known that Sp1 can recruit HDAC1 to the promoter of regulated genes (13), it is still unknown whether HDAC1 affects the posttranslational modification of transcription factors. On the other hand, in mammalian cells, several proteins contain HAT activity, including GCN5, PCAF, and p300/CBP (34). The nuclear coactivator p300 is a transcriptional adaptor for many DNA binding activators including nuclear hormone receptors, MyoD, p53, and GATA-1 (8, 12, 17, 19, 20, 34). p300 possesses intrinsic acetyltransferase activity (33), which affects the nucleosomal environment and transcription by chemically modifying histones. However, several nonhistone proteins such as p53, TFIIE, EKLF, and GATA-1 are also acetylated (8, 20, 24, 44). The recruitment of acetyltransferase, an intrinsic activity of p300, to the promoter by transcription factors results in a remodeling of the nucleosomal structure by acetylating histones that, perhaps in concert with nonhistone proteins, allow for the initiation of transcription (29, 42). How p300 interacts with Sp1 to modulate the promoter activity of the 12(S)-lipoxygenase gene, however, remains unclear.

In this report, we studied the acetylation and deacetylation of Sp1 and the effect of the acetylation of Sp1 on the recruitment of HDAC1 and p300 to the promoter of the lipoxygenase gene when A431 cells were stimulated with PMA. We found that Sp1 was constitutively acetylated at Lys703 and that HDAC1 could be recruited by acetylated Sp1 to the promoter of the 12(S)-lipoxygenase gene. Sp1 was then deacetylated by HDAC1, further recruiting p300 to the promoter, thus leading to the induction of 12(S)-lipoxygenase expression by acetylating histones.


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MATERIALS AND METHODS
 
Materials. Polyclonal antibodies against HDAC1 and protein A/G-Sepharose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies against 12(S)-lipoxygenase were purchased from Oxford Biomedical Research Inc. (Oxford, MI). The catalytic domain of p300, protease inhibitor cocktail, monoclonal antibodies against histone 3 and acetyl histone 3, and polyclonal antibodies against Sp1 and acetyl-lysine were purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal antibodies against c-Jun were purchased from BD (San Jose, CA). Biotinylated oligonucleotides were obtained from MD Bio, Inc. (Taipei, Taiwan). The luciferase assay system was from Promega (Madison, WI). Luciferase plasmid pXP-1 was a gift from T. Sakai, Kyoto Prefecture University of Medicine. TRIzol RNA extraction kit, Super-ScriptTMII, Lipofectamine 2000, Dulbecco's modified Eagle's medium, and Opti-MEM medium were obtained from Invitrogen Life Technologies (Grand Island, NY). [14C]acetyl coenzyme A (CoA) (56 mCi/mmol) and [3H]sodium acetate (3.1 Ci/mmol) were purchased from Amersham Bioscience (Baie d'Urfe, Quebec, Canada). The plasmids pGEX6P-1 and pET-28 were purchased from Invitrogen Inc. (Grand Island, NY). pCMV-p300 and pCMV-E1A were kindly provided by H. M. Shih (National Health Research Institutes, Taiwan). Fetal bovine serum was obtained from HyClone Laboratories (Logan, UT). All other chemical reagents used were of the highest purity obtainable.

Cell culture. Human epidermoid carcinoma A431 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 µg/ml streptomycin sulfate, and 100 units/ml penicillin G sodium at 37°C and 5% CO2 in an incubator. The 90% confluent cells were used for treatment with PMA or TSA.

Western blotting. Whole-cell extracts were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane using a transfer apparatus according to the manufacturer's protocols (Bio-Rad). After incubation with 3% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.5% Tween 20) for 60 min, membranes were washed once with TBST and incubated with antibodies against Sp1 (1:1,000), c-Jun (1:2,000), HDAC1 (1:1,000), p300 (1:2,000), acetyl-lysine (1:1,000), 12(S)-lipoxygenase (1:500), or actin (1:5,000) at room temperature for 12 h. Membranes were washed three times for 10 min and incubated with a 1:3,000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody for 2 h. Blots were washed with TBST three times and developed with the ECL system (Amersham) according to the manufacturer's protocols.

Immunoprecipitation. Cell nuclear extracts were prepared, and an equal amount of protein was used in each experiment. Nuclear extracts were preincubated with protein A/G-Sepharose for 1 h at 4°C and centrifuged to remove the pellets. The supernatants were then incubated with either anti-His or anti-Sp1 antibodies at a dilution of 1:200 for 12 h at 4°C. The immunoprecipitated pellets were subsequently incubated with protein A/G-Sepharose, washed five times with lysis buffer, and separated on a 7% SDS-PAGE gel. After electrophoresis, the gels were processed for immunoblotting with antibodies against Sp1 (1:1,000), c-Jun (1:2,000), HDAC1 (1:1,000), or p300 (1:2,000).

Transfection and reporter gene assay. Cells were transfected with plasmids by lipofection using Lipofectamine according to the manufacturer's instructions, with a slight modification. Cells were replated 24 h before transfection at an optimal cell density in 2 ml of fresh culture in a 3.5-cm dish. For use in transfection, 4 µl of Lipofectamine was incubated with 4 µg of pXP-7-1 luciferase plasmid together with the indicated plasmid as described in each experiment in 0.4 ml of Opti-MEM medium for 30 min at room temperature. The total DNA concentration for each transfection was matched with pXP-1. Cells were transfected by changing the medium with 2 ml of Opti-MEM medium containing the plasmid and Lipofectamine, followed by incubation at 37°C in 5% CO2 for 6 h. After a change of Opti-MEM medium to 2 ml of fresh medium, cells were incubated for additional 18 h. The luciferase activity in the cell lysate was determined as described previously.

RT-PCR. Total RNA of cells was isolated with a TRIzol RNA extraction kit, and 5 µg of RNA was subjected to reverse transcription-PCR (RT-PCR) with SuperScript II. The primers used for PCR for 12(S)-lipoxygenase were 5'- CACCTGTGCTCACTGCCTTA-3' and 5'-AGTTCCTCAATGGTGCCAAC-3', and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5'-CCATCACCATCTTCCAGGAG-3' and 5'-CCTGCTTCACCACCTTCTTG-3'. The PCR products were separated by 1% agarose gel electrophoresis and visualized with ethidium bromide staining.

Immunofluorescence microscopic analysis. A431 cells were seeded onto round glass slides in 24-well plates overnight. After treatment with PMA, cells were fixed in 4% paraformaldehyde at 4°C for 20 min. Cells were rinsed with phosphate-buffered saline (PBS) three times and permeabilized with 1% Triton X-100 for 5 min and then with 0.5% Tween 20 for 15 min. These cells were pretreated with 1% bovine serum albumin in PBS for 60 min at 25°C and incubated with antibodies against Sp1 at a dilution of 1:500 for 1 h; with Cy5-conjugated donkey anti-rabbit immunoglobulin G (IgG) and antibodies against c-Jun, HDAC1, or p300 at a dilution of 1:250 for 1 h; and with fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse IgG for 1 h. These samples were washed with PBS, mounted in 50% glycerol, and analyzed using a Leica TCS SP2 confocal microscope.

Pull-down assay. Glutathione-S-transferase (GST)-Sp1 and GST-c-Jun were expressed in Escherichia coli DH5{alpha} and purified by glutathione-Sepharose affinity beads (Sigma) according to standard protocols. GST-Sp1, acetyl-GST-Sp1, and GST were incubated with the cell nuclear extract in the absence or presence of GST-c-Jun for 2 h at 4°C in 5 ml of binding buffer (20 mM HEPES [pH 7.9], 200 mM NaCl, 1 mM MgCl2, 0.5% NP-40, 1 mM dithiothreitol, and 1 mg/ml of bovine serum albumin). The beads were then washed six times with binding buffer, and the bound proteins were analyzed by SDS-PAGE and recognized with individual antibodies.

DNA affinity precipitation assay (DAPA). The oligonucleotide 5'-TTTGGGCTAGTCTGGGGCGGGG-3', localized –171 to –150 bp within the promoter of 12(S)-lipoxygenase, was biotinylated at 5' termini and then annealed with their complementary strands. The assay was performed by incubating 2 µg of biotinylated DNA probe with 300 µg of nuclear extract that was precleaned with 10 µl of streptavidin-agarose beads and 1 µg of poly(dI-dC) for 1 h and then incubated with 20 µl of streptavidin-agarose in binding buffer [1 µg of poly(dI-dC), 20 mM HEPES (pH 7.9), 0.1 mM KCl, 2 mM MgCl2, 15 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, and 10% (vol/vol) glycerol] for 1 h. Beads were collected and washed three times with binding buffer containing 0.5% NP-40. Proteins bound to the beads were eluted with 2x sample buffer and separated by 7% SDS-PAGE followed by the immunoblot analysis.

Chromatin immunoprecipitation (ChIP) assay. Assays were performed as described previously (6). Cells (1 x 108) were cross-linked in 0.5% formaldehyde in PBS for 15 min at room temperature. After cross-linking, cells were washed three times with PBS, and the cell lysate was collected with lysis buffer. The chromatin was fragmented by sonication to an average size of 500 bp. The samples (1 ml) were precleaned with 10 µl of protein A/G-agarose containing 1 µg of poly(dI-dC) for 1 h and then immunoprecipitated with 5 µg of antibodies against IgG, Sp1, HDAC1, p300, c-Jun, histone 3, and acetyl-histone 3. After 12 h of incubation, the samples were incubated with 20 µl of protein A/G-agarose for 1 h. After six washes, bound proteins were eluted with Tris-EDTA buffer containing 1% sodium dodecyl sulfate. Cross-links were reversed at 65°C for 12 h, and proteins were digested with proteinase K (0.5 mg/ml) for 2 h at 50°C. DNA was purified by phenol-chloroform extraction and ethanol precipitation. Immunoprecipitated DNA was analyzed by PCR and Southern blot. The primer sequences for PCR were as follows: 5'-TTTGGGCTAGTCTGGGGCGGGG-3' and 5'-GGCGCCCCCCAGCAGCTTAGGC-3'.

Acetyltransferase assay. For the in vivo acetylation analysis, A431 cells were washed with PBS and incubated in methionine- and cysteine-free Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum for 1 h at 37°C. Cells were then metabolically labeled with [3H]sodium acetate at 1 mCi/ml for 1 h at 37°C. Whole-cell extracts were prepared with lysis buffer (300 mM NaCl, 50 mM Tris [pH 7.5], 0.5% Triton X-100, 1 mM dithiothreitol) and protein inhibitor cocktail and immunoprecipitated with polyclonal Sp1 antibodies, followed by SDS-PAGE analysis. Gels were fixed in 30% methanol and 10% acetic acid, soaked in Amplify (Amersham), dried, and exposed to X-ray film. The in vitro acetylation assays were performed by incubating the cell extract or the catalytic domain of p300 with GST fusion proteins containing full-length Sp1 or truncated Sp1 mutants with or without lysine site-directed mutagenesis in 20 µl of reaction buffer (50 mM Tris-HCl [pH 8.0], 1 mM dithiothreitol, 0.1 mM EDTA, 50 mM KCl, 5% glycerol, 10 µM sodium butyrate, and protease inhibitor cocktail) and 1 µCi of [14C]acetyl-CoA at 30°C for 1 h. After the reaction, proteins were resolved by SDS-PAGE, and gels were treated as described above.

Construction of plasmids. The plasmid pCMV-Sp1-His was constructed by using the primers containing the His tag sequence at the C terminus of Sp1 for RT-PCR, and the amplified sequence was then verified. The Sp1 expression vectors pCMV-Sp1-K703/A and pCMV-Sp1-K693/A, carrying a His-tagged dominant stable Sp1 mutated at residues 703 and 693 (lysine to alanine), respectively, were generated and verified by sequencing. GST-Sp1 was generated by cloning PCR-amplified fragments of full-length Sp1 into pGEX-6P-1. All of the truncated Sp1 constructs containing lysine site-directed mutagenesis were generated in pET-21.

Restriction enzyme accessibility assay and DNase I nuclease digestion. Nuclei of A431 cells (5 x 107 cells) were prepared and digested with DNase I (105 units/µl) for 10 min at room temperature or digested with selected restriction enzymes at 37°C for 1 h. DNA was extracted and dissolved in 100 µl of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Twenty micrograms of DNA was further digested with NheI and HindIII for 4 h, and DNA was run in 1% agarose and blotted with a 32P-labeled 400-bp fragment of the 12(S)-lipoxygenase gene promoter region.


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RESULTS
 
Acetylation of Sp1 in cells. At first, we confirmed whether Sp1 in A431 cells was acetylated. Nuclear extract was prepared from cells and then immunoprecipitated with anti-Sp1 or anti-IgG antibodies. The immunoprecipitated pellets were blotted with anti-acetyl-lysine and anti-Sp1 antibodies. As shown in Fig. 1A, one band localized at 90 kDa could be recognized by anti-acetyl-lysine antibodies in the pellet immunoprecipitated by anti-Sp1 antibodies (Fig. 1A, lane 1), but it was not immunoprecipitated by IgG (Fig. 1A, lane 2). In order to confirm the acetylated protein in the immunoprecipitated pellet of anti-Sp1 antibodies, experiments with Sp1 overexpression were performed. Nuclear extracts prepared from cells overexpressing Sp1 and control cells were immunoprecipitated with anti-Sp1 antibodies, and the immunoprecipitated pellets were blotted with anti-acetyl-lysine and anti-Sp1 antibodies. As shown in Fig. 1B, the protein recognized by anti-acetyl-lysine antibodies in Sp1-overexpressed cells was obviously increased compared with that in control cells (Fig. 1B, upper panel, compare lane 2 with lane 1), and the increase was proportional to the expression of Sp1 in cells (Fig. 1B, compare the upper panel with the lower panel). Furthermore, overexpression of Sp1-His in cells was also performed, and the acetylation of Sp1-His was detected by radiolabeling with [3H]sodium acetate in cells. Immunoprecipitation of cell nuclear extracts was then performed by using anti-His antibodies. As shown in Fig. 1C, Sp1-His was significantly acetylated by the detection of tritium labeling (Fig. 1C, upper panel). In order to rule out the possibility that some other proteins coimmunoprecipitated with Sp1 could be recognized by anti-acetyl-lysine antibodies with molecular sizes equal to that of Sp1, the in vitro acetylation assay was performed by using purified GST-Sp1 as a substrate. Under the experimental conditions of using the same amount of GST-Sp1, as shown in Fig. 1D, a significant acetylation on GST-Sp1 determined by [14C]acetyl incorporation incubated with [14C]acetyl-CoA was observed by the reaction with the cell lysate (Fig. 1D, lane 1) and with p300 (Fig. 1D, lane 3). We also used another HAT enzyme, PCAF, in the in vitro acetylation assay, but no acetylation of GST-Sp1 was observed (data not shown). In order to prove that p300 could acetylate Sp1 in vivo, the effect of p300 overexpression on Sp1 acetylation by using radiolabeling with [3H]sodium acetate in cells was studied. Cell nuclear extracts were immunoprecipitated with anti-Sp1 antibodies. As shown in Fig. 1E, overexpression of p300 enhanced the formation of [3H]acetyl-Sp1 in cells. In addition, cells were treated with E1A, a specific inhibitor of p300, to study the acetylation of Sp1. The results shown in Fig. 1F indicated that the acetylation of Sp1 was significantly reduced upon E1A treatment (Fig. 1F, upper panel). Moreover, the stoichiometry of acetylated Sp1 was also studied. At least 14% of Sp1, at least, could be acetylated in A431 cells constitutively (Fig. 1G and H). Taking these in vivo and in vitro results together, we concluded that in A431 cells, Sp1 could be constitutively acetylated and that p300 might play a pivotal role in Sp1 acetylation.


Figure 1
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  		FIG. 1. Sp1 is acetylated by p300. (A) Nuclear extract was prepared from A431 cells, and the immunoprecipitation (IP) was performed by using anti-Sp1 and IgG antibodies. The immunoprecipitated pellets were analyzed by immunoblotting with anti-acetyl-lysine (upper panel) and anti-Sp1 (lower panel) antibodies. (B) Nuclear extracts were prepared from A431 cells with or without Sp1 overexpression. Immunoprecipitation was performed by using anti-Sp1 antibodies. The immunoprecipitated pellets were analyzed by immunoblotting with anti-acetyl-lysine (upper panel) and anti-Sp1 (lower panel) antibodies. (C) The Sp1-His-overexpressed cells were incubated with [3H]sodium acetate for acetyl labeling of cellular proteins. Cell nuclear extracts were prepared, and immunoprecipitation was performed by using anti-His antibodies. The immunoprecipitated pellets were analyzed by immunoblotting with anti-Sp1 antibodies (lower panel), and the tritium labeling of acetylated Sp1 was detected by autoradiography (upper panel) after protein resolution by 7% SDS-PAGE. (D) The in vitro acetylation assay was performed in 20 µl of reaction buffer containing 1 µCi of [14C]acetyl-CoA and 1 µg of GST-Sp1 as a substrate. Either 5 µg of cell nuclear extract or 1 µg of the catalytic domain of p300 was included as an enzyme source. 14C labeling of GST-Sp1 was detected by autoradiography (upper panel), and GST-Sp1 was stained with Coomassie blue as an internal control (lower panel). (E) After transfection with empty vector or the expression vector p300, cells were labeled with [3H]sodium acetate, and cell nuclear extract was then prepared. Immunoprecipitation was performed by using anti-Sp1 antibodies, and proteins in immunoprecipitated pellets were resolved by 7% SDS-PAGE. The overexpressed p300 protein was detected by immunoblotting with anti-p300 antibodies. (F) Cells were transfected with empty vector or expression vector E1A, and nuclear extracts were prepared. Immunoprecipitation was performed by using anti-Sp1 antibodies, and proteins in immunoprecipitated pellets were resolved by 7% SDS-PAGE. The acetylation level of Sp1 was detected by immunoblotting with anti-acetyl-lysine antibodies. (G) Nuclear extracts from A431 cells were immunoprecipitated by 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 µg of anti-acetyl-lysine and then analyzed with anti-Sp1 antibodies. (H) The ratio of acetylated Sp1 was quantified from three independent experiments.

Deacetylation of Sp1 upon PMA stimulation. We found that Sp1 was constitutively acetylated in A431 cells (Fig. 1). Thus, we examined whether PMA treatment could change Sp1 acetylation. Cells were treated with 1, 5, and 10 nM PMA for 9 h, and the nuclear extracts of cells were then immunoprecipitated by anti-Sp1 antibodies, followed by immunoblot analyses with anti-Sp1 and anti-acetyl-lysine antibodies, which have been shown to be highly specific for protein acetylation in our previous study (data not show). As shown in Fig. 2A, no significant change in the protein level of Sp1 in nuclear extracts was observed (Fig. 2A, panel b), but the degree of acetylation of Sp1 was decreased in a dose-dependent manner upon PMA treatment (Fig. 2A, panels a and c). PMA at a concentration of 10 nM induced a more than 90% decrease of acetylated Sp1. The deacetylation of Sp1 was time dependent (Fig. 2B). Cells treated with 10 nM PMA for 3 h showed no changes in the level of acetylated Sp1. However, treatment of cells with 10 nM PMA for 4, 6, and 9 h resulted in more than 40, 70, and 90% deacetylation of Sp1, respectively. In addition, cells were treated with PMA for an extended period of time to study the acetylation level of Sp1 and the recruitment level of acetylated Sp1 to the promoter of the 12(S)-lipoxygenase gene in A431 cells. The results indicate that the acetylation level of Sp1 was reversed after 18 h of treatment with PMA in A431 cells (Fig. 2B). In order to confirm these results, overexpression of Sp1-His in cells was performed. The Sp1-His-overexpressed cells were treated with 10 nM PMA for 9 h, and the cell nuclear extracts were then immunoprecipitated with anti-His antibodies. As shown in Fig. 2C, cells transfected with the Sp1-His vector expressed much more Sp1-His (Fig. 2C, panel b). Treatment of Sp1-His-overexpressed cells with PMA induced a more than 90% attenuation of Sp1-His in the acetylated form (Fig. 2C, panels a and c). We previously reported that cells treated with PMA showed an enhanced level of 12(S)-lipoxygenase expression in 9 h (31). We then studied whether there was a correlation between the deacetylation of Sp1 and the induction of 12(S)-lipoxygenase gene expression with PMA treatment. An inhibitor of HDAC1, TSA, which has been proven to enhance the acetylation of Sp1 (23), was therefore employed for the study. The results are summarized in Fig. 2D with GAPDH as an internal control for gene expression (Fig. 2D, panel b). Treatment of cells with TSA attenuated the level of PMA-induced 12(S)-lipoxygenase mRNA in a dose-dependent manner (Fig. 2D, panels a and e). TSA at 100 ng almost completely blocked the PMA action in inducing the expression of 12(S)-lipoxygenase. The TSA effects on the acetylation level of Sp1 were also examined. The PMA-induced deacetylation of Sp1 was blocked by TSA treatment in a dose-dependent manner (Fig. 2D, panel c). TSA at 100 ng completely blocked the deacetylation of Sp1 caused by PMA as evidenced by the fact that the level of acetylated Sp1 remained at the basal level in the presence of 100 ng of TSA. These results indicate that there is a negative correlation between the level of acetylated Sp1 and the gene expression of 12(S)-lipoxygenase in A431 cells with PMA treatment.


Figure 2
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  		FIG. 2. Deacetylation of Sp1 in A431 cells exposed to PMA. (A) Cells were treated with 1, 5, and 10 nM PMA for 9 h, and cell nuclear extracts were prepared. Immunoprecipitation (IP) was performed by using anti-Sp1 antibodies, and the pellets were analyzed by immunoblotting with anti-acetyl-lysine (a) and anti-Sp1 (b) antibodies. The relative level of acetyl-Sp1 was quantified from three independent experiments (c). (B) Cells were treated with 10 nM PMA for 3, 4, 6, 9, 18, and 24 h. Nuclear extracts were then immunoprecipitated with anti-Sp1 and then analyzed with anti-acetyl-lysine and anti-Sp1 antibodies (a). The relative level of acetylated Sp1 was quantified from three independent experiments (b). (C) The Sp1-His-overexpressed cells were treated with 10 nM PMA, and cell nuclear extracts from control and PMA-treated cells were then prepared. Immunoprecipitation was performed by using anti-His antibodies, and the pellets were analyzed by immunoblotting with anti-acetyl-lysine (a) and anti-His (b) antibodies. The relative level of acetyl-Sp1-His was quantified from three independent experiments (c). (D) The effect of TSA on PMA-induced gene expression of 12(S)-lipoxygenase was studied. Cells cultured in 1 ml of medium were treated with 1, 10, and 100 ng of TSA in the absence or presence of 10 nM PMA for 9 h, and total RNA and cell nuclear extracts were prepared separately. The mRNA levels of 12(S)-lipoxygenase (a) and GAPDH (b) as an internal standard were quantified by RT-PCR. Cell nuclear extracts were immunoblotted with anti-acetyl-lysine (c) and anti-Sp1 (d) antibodies. The relative levels of acetyl-Sp1 and 12(S)-lipoxygenase were quantified within the linear range of the PCR dose curve from three independent experiments (e). The linear range of the PCR dose curve is shown in the upper right corner.

Interaction of Sp1 with c-Jun, HDAC1, and p300 in cells exposed to PMA. Our previous studies revealed that when A431 cells are stimulated with PMA, c-Jun can be recruited to the promoter of the 12(S)-lipoxygenase gene through an interaction with Sp1 (11). It was also reported that Sp1 could interact with HDAC1 and p300 to regulate gene expression (11, 37). Thus, herein, we studied the relation and dynamic interactions between c-Jun, Sp1, HDAC1, and p300 after cells were exposed to PMA treatment by using immunocoprecipitation and immunofluorescence microscopic analyses. We found that treatment of cells with PMA significantly induced in a time-dependent manner the recruitment of p300 to Sp1 as seen in increments of p300 in anti-Sp1 immunoprecipitants. The recruitment of p300 to Sp1 was only slightly enhanced 3 h after PMA treatment but greatly increased 9 h after treatment (Fig. 3A, panels a and f). The immunofluorescence microscopic analysis also revealed intensified colocalizations between Sp1 and p300 in nuclei of cells treated with PMA for 9 h (Fig. 3B). Immunocoprecipitation also showed that HDAC1 recruitment to Sp1 increased 3 h after PMA treatment. However, HDAC1 recruitment was dramatically decreased when examined at the 9-h time point (Fig. 3A, panels b and f). In accordance with this result, confocal microscopic data indicated that colocalization between Sp1 and HDAC1 was slightly more apparent at 3 h than at 9 h (Fig. 3C). On the other hand, the presence of c-Jun in anti-Sp1 immunoprecipitants was unchanged at both time points (Fig. 3A, panels c and f). Again, confocal images in Fig. 3D confirm this notion. Under experimental conditions thus carried out, an attenuation of Sp1 in the acetylated form was also observed in a time-dependent manner (Fig. 3A, panels d and f). Taken together, these results suggest that at an early stage (3 h) of PMA treatment, Sp1 interacts with HDAC1, whereas in a later stage (9 h), Sp1 interacts with p300. The recruitment of c-Jun to Sp1 remains unaltered throughout the examined period with PMA stimulation.


Figure 3
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  		FIG. 3. Interaction of Sp1 with c-Jun, HDAC1, and p300 in A431 cells. (A) Cells were treated with 10 nM PMA for 3 and 9 h, and the cell nuclear extracts from control and PMA-treated cells were prepared. Immunoprecipitation (IP) was performed by using anti-Sp1 antibodies, and the pellets were analyzed by immunoblotting with anti-p300 (a), anti-HDAC1 (b), anti-c-Jun (c), anti-acetyl-lysine (d), and anti-Sp1 (e) antibodies. The relative level of proteins was quantified from three independent experiments (c). (B) Cells were treated with 10 nM PMA for 3 and 9 h. After PMA treatment, cells were fixed and reacted with anti-p300 and anti-Sp1 antibodies, followed by staining with secondary antibodies conjugated with FITC or Cy5, respectively. The protein localization of p300 and Sp1 in cells upon PMA treatment was analyzed by confocal microscopy. (C) Cells were treated with 10 nM PMA for 3 and 9 h. After PMA treatment, cells were fixed and reacted with anti-HDAC1 and anti-Sp1 antibodies, followed by staining with secondary antibodies conjugated with FITC or Cy5. The protein localization of HDAC1 and Sp1 in cells upon PMA treatment was analyzed by confocal microscopy. (D) Cells were treated with 10 nM PMA for 3 and 9 h. After PMA treatment, cells were fixed and treated with anti-c-Jun and anti-Sp1 antibodies, followed by staining with secondary antibodies conjugated with FITC or Cy5. The protein localization of c-Jun and Sp1 in cells upon PMA treatment was analyzed by confocal microscopy.

Recruitment of Sp1, c-Jun, HDAC1, and p300 to the promoter of 12(S)-lipoxygenase in cells treated with PMA. Cells were treated with PMA for 3, 6, and 9 h, fixed, and then sonicated. ChIP assays were performed to study the recruitment of Sp1, c-Jun, HDAC1, and p300 to Sp1 binding sites on the promoter of the 12(S)-lipoxygenase gene in cells treated with PMA. The kinetic change in the recruitment of transcription factors was quantified (Fig. 4A). Since the number of PCR cycles was still within the linear range of the PCR dose curve (Fig. 4G), DNA recruited by Sp1, c-Jun, HDAC1, p300, or acetyl-H3 was quantified accordingly. As shown in Fig. 4B, there was no change in the recruitment of Sp1 to the gene promoter of 12(S)-lipoxygenase with PMA treatment at 3 and 9 h. This result agreed with our previous report indicating no change in Sp1 binding to DNA in the presence of PMA as determined by a gel mobility shift assay (22). The recruitment of c-Jun to the gene promoter increased at 3 h, reached the maximum at 6 h, and then slightly declined at 9 h after PMA treatment (Fig. 4C). The profile of this kinetic change was similar to that seen in c-Jun protein biosynthesis induced by PMA (39). Recruitment of HDAC1 was significantly increased at 3 h but then decreased even below the basal level 6 h after the PMA stimulation (Fig. 4D). On the other hand, the recruitment of p300 to the gene promoter was not apparent at 3 h but was enhanced significantly after cells were treated with PMA for 9 h (Fig. 4E). Profiles of these kinetic changes in the recruitment of HDAC1 and p300 to the gene promoter after PMA treatment were quite similar to those seen with the immunoprecipitation studies (Fig. 3A, panels a and b). Acetyl-histone 3 recruitment to the gene promoter, however, was increased in a time-dependent manner upon PMA stimulation (Fig. 4F). Furthermore, the result of a double chromatin immunoprecipitation assay revealed that the recruitment level of acetylated Sp1 to the promoter of the 12(S)-lipoxygenase gene was reduced specifically upon PMA treatment (Fig. 4H and I).


Figure 4
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  		FIG. 4. PMA treatment induces dynamic changes in the recruitment of transcription factors to the promoter of the 12(S)-lipoxygenase gene in A431 cells. (A) Cells were treated with 10 mM PMA for 3, 6, and 9 h. After PMA treatment, cells were fixed and sonicated. A ChIP assay was then performed with antibodies against Sp1, c-Jun, HDAC1, p300, and acetyl-histone 3, respectively. Changes in the recruitment of Sp1 (B), c-Jun (C), HDAC1 (D), p300 (E), and acetyl-histone 3 (F) to the gene promoter of 12(S)-lipoxygenase were quantified from three independent experiments. The input sample is within the linear range of between 28 and 34 cycles of the PCR (G). (H) Cells were treated with 10 nM PMA for 3, 4, 6, 9, 18, and 24 h and then fixed. The ChIP was performed with anti-Sp1 antibodies (c). ChIP for the DNA recruited by anti-Sp1 was again performed with anti-acetyl-lysine antibodies (a) and IgG (b). (I) The relative level of DNA recruited by acetyl-Sp1 was quantified from three independent experiments. hrs, hours.

Acetylation of Sp1 at Lys703. Although Sp1 is known to be acetylated, whether it is the acetylated form or the deacetylated form that interacts with HDAC1 and p300, respectively, remains inconclusive and gains support only from indirect evidence. In Fig. 1, we have provided evidence that the acetylation of Sp1 might be required for the induction of 12(S)-lipoxygenase in the PMA treatment paradigm by using TSA to block the deacetylating enzyme. However, TSA might have inhibited the deacetylation of other proteins and not only Sp1. Therefore, it was absolutely necessary to identify the acetylated residue on Sp1 to prove unequivocally the functional role of acetylated Sp1, or the deacetylated Sp1, for the same matter. Thus, we constructed two truncated Sp1 expression vectors, pET21-Sp1(1-615) containing 6 lysine residues and pET21-Sp1(616-778) containing 14 lysine residues (Fig. 5A), and expressed these two truncated proteins in E. coli. We studied then the acetylation of these two truncated proteins by the in vitro acetylation assay. Our results revealed that full-length Sp1 and N-terminally truncated Sp1 at residues 616 to 778 [Sp1(616-778)] could be acetylated in the presence of [14C]acetyl-CoA, while C-terminally truncated Sp1(1-615) could not (Fig. 5B, lanes 1, 2, and 3). Next, we performed a single site-directed mutation on 14 lysine residues within the region of residues 616 to 778 (Fig. 5A) and expressed them in E. coli. Acetylation of these mutant proteins was again studied by the in vitro acetylation assay. Results indicated that all the mutant proteins except Sp1(616-778)(K703/A) could be acetylated (Fig. 5B). Therefore, we concluded that Lys703 on Sp1 could be acetylated by the in vitro acetylation assay. The full-length expression vectors pCMV-Sp1, pCMV-Sp1(K693/A), and pCMV-Sp1(K703/A) were then constructed and overexpressed in cells. Cell nuclear extracts were then prepared and immunoprecipitated with anti-Sp1 antibodies. Western blotting of the immunoprecipitated pellet was performed by using anti-acetyl-lysine (Fig. 5C, upper panel) and anti-Sp1 (Fig. 5C, lower panel) antibodies. The results revealed that, indeed, Sp1(K703/A) was not constitutively acetylated in cells. Taken together, the results showed that Lys703 was the acetylation site on Sp1.


Figure 5
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  		FIG. 5. Lys703 of Sp1 is acetylated. (A) Expression vectors carrying various truncated Sp1 mutants and those with site-directed mutations were constructed. (B) All the proteins were expressed in E. coli and then purified. The in vitro acetylation assay for Sp1 and its mutant proteins was then performed with p300 in the presence of [14C]acetyl-CoA. (C) Expression vectors of Sp1, Sp1(K703/A), and Sp1(K693/A) were overexpressed in A431 cell. Cell nuclear extracts were prepared. Immunoprecipitation (IP) was performed by using anti-Sp1 antibodies, and the pellets were analyzed by immunoblotting with anti-acetyl-lysine (upper panel) and anti-Sp1 (lower panel) antibodies.

Enhancement of transcription activity of 12(S)-lipoxygenase by Sp1(K703/A) overexpression. We then studied the effect of Sp1 deacetylation on the transcription activity of the 12(S)-lipoxygenase gene by using the expression vector Sp1(K703/A). Cells were transfected with pcDNA3.0, pcDNA3.0-Sp1, or pcDNA3.0-Sp1(K703/A) together with luciferase reporter vector pXP-7-1 inserted within the promoter region of 12(S)-lipoxygenase containing three Sp1 binding sites. After 18 h of incubation, the effect of Sp1 overexpression on the expression of the luciferase reporter was analyzed. As shown in Fig. 6A, overexpression of wild-type Sp1 and mutant Sp1(K703/A) activated the luciferase reporter activity in a dose-dependent manner. However, mutant Sp1(K703/A) was much more potent than wild-type Sp1. Overexpression with 1.5 µg of wild-type Sp1 induced a twofold stimulation, but overexpression with 1.5 µg of mutant Sp1(K703/A) induced a ninefold stimulation. The stimulatory effect of wild-type Sp1 and mutant Sp1(K703/A) on promoter activity was blocked by E1A that inhibits p300 (Fig. 6B). Treatment of cells with 1 to 10 µg of E1A not only completely inhibited the stimulatory effect of wild-type Sp1 and mutant Sp1(K703/A) but also attenuated the basal promoter activity of the 12(S)-lipoxygenase gene in A431 cells. Furthermore, the effect of the overexpression of wild-type Sp1 on the protein level of 12(S)-lipoxygenase in the cell was compared to that of mutant Sp1(K703/A). As shown in Fig. 6C, overexpression of either wild-type Sp1 or mutant Sp1(K703/A) induced the expression of 12(S)-lipoxygenase in a dose-dependent manner (Fig. 6C, panel b). Overexpression of mutant Sp1(K703/A), however, induced a significantly higher level of 12(S)-lipoxygenase expression than that of wild-type Sp1 (Fig. 6C, panels a and d).


Figure 6
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  		FIG. 6. Deacetylated Sp1 enhances the expression of 12(S)-lipoxygenase. (A) Cells cultured in 2 ml of medium on a 3.5-cm dish were transfected with expression vectors of Sp1 or Sp1(K703/A) together with 1 µg of the luciferase reporter pXP-7-1 carrying the promoter of the 12(S)-lipoxygenase gene for 18 h, and the luciferase activity was assayed. Values represent the means ± standard errors for three independent experiments. (B) Cells cultured in 2 ml of medium on a 3.5-cm dish were transfected with 1 µg of expression vectors of Sp1 or Sp1(K703/A) together with 1 µg of the luciferase reporter pXP-7-1 for 18 h. In order to study the functional role of p300 on gene expression, the expression vector of E1A was cotransfected, and the luciferase activity was then assayed. (C) The effect of overexpression of Sp1 and Sp1(K703/A) on gene expression of 12(S)-lipoxygenase in cells was studied. Cells cultured in 10 ml of medium on a 10-cm dish were transfected with 4 µg of expression vectors of Sp1 or Sp1(K703/A) for 24 h. Cell lysates were then prepared, and an immunoblotting assay was performed with antibodies against 12(S)-lipoxygenase (a), Sp1 (b), and actin as an internal standard (c). The relative level of acetyl-Sp1 was quantified from three independent experiments (d).

Interaction of deacetylated Sp1 with p300 and HDAC1. We then studied the effect of Sp1 deacetylation on the interaction of Sp1 with p300 and HDAC1, respectively, by overexpressing mutant Sp1(K703/A) in the cell. Cells were transfected with vectors Sp1-His and Sp1(K703/A)-His. Cell nuclear extracts were then immunoprecipitated with anti-His antibodies, and the effect of Sp1 deacetylation on the interaction of Sp1 with p300 and HDAC1 was studied. Results are shown in Fig. 7A. Apparently, p300 interacted more with mutant Sp1(K703/A) than with wild-type Sp1 (Fig. 7A, panels a and f), whereas HDAC1 interacted more with wild-type Sp1 than with mutant Sp1(K703/A) (panels b and g). In order to address whether the acetylated form of Sp1 might attenuate the interaction between p300 and Sp1, GST-Sp1 was acetylated in vitro, and GST-Sp1 or the resultant acetyl-GST-Sp1 was incubated with the nuclear extracts. The results indicate that GST-Sp1 coimmunoprecipitated with p300 more than acetyl-GST-Sp1 did (Fig. 7B). To confirm that the effect of HDAC1 on 12(S)-lipoxygenase expression is through Sp1, we mutated all of the Sp1 binding sites within the promoter of the 12(S)-lipoxygenase gene and then studied its transcription by luciferase activity. The results indicate that the luciferase activity was increased by overexpression of HDAC1, but this effect was reversed when the GC-rich sites of the promoter were mutated in A431 cells (Fig. 7C). Furthermore, the interaction of HDAC1 with Sp1 was increased in the presence of c-Jun (Fig. 7D), and the recruitment of HDAC1 to the promoter of the 12(S)-lipoxygenase gene was also increased by PMA treatment and overexpression of HDAC1 (Fig. 7E). In contrast, the recruitment of HDAC1 was obviously decreased by a knockdown of c-Jun by small interfering RNA in cells (Fig. 7G). Interestingly, when Sp1-HA and Sp1(K703/A)-HA were expressed in the cells, the colocalization of p300 and Sp1(K703/A)-HA was more than that with Sp1-HA (Fig. 7F). Taken together, these results clearly indicated that acetylated or deacetylated Sp1 interacts differentially with p300 and HDAC1 and that c-Jun also plays a functional role in the interaction of HDAC1 with Sp1.


Figure 7
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  		FIG. 7. Sp1 interacts with c-Jun, HDAC1, and p300. (A) Cells cultured in 10 ml of medium on a 10-cm dish were transfected with 4 µg of expression vectors of Sp1-His or Sp1(K703/A)-His for 24 h. Cell nuclear extracts were prepared. Immunoprecipitation (IP) was performed by using anti-His antibodies, and the pellets were analyzed by immunoblotting with antibodies against p300 (a), HDAC1 (b), c-Jun (c), Sp1 (d), and acetyl-lysine (e), and the relative levels of p300 (f), HDAC1 (g), and c-Jun (h) were quantified from three independent experiments. (B) The interaction of p300 with Sp1 or acetylated Sp1 in vitro was studied. Purified GST-Sp1 (1 µg) was acetylated with 1 µg of p300 in the presence of 5 µM acetyl-CoA in 20 µl of reaction buffer and then purified again. Both Sp1 and acetyl-treated Sp1 were incubated with 100 µg of nuclear extract and then pulled down by 20 µl of glutathione beads. The pellets were analyzed by immunoblotting with antibodies against p300 (a), GST (b), and acetyl-lysine (c). The relative level of p300 was quantified from three independent experiments (d). (C) Cells cultured in 2 ml of medium on a 3.5-cm dish were transfected with 0.1, 0.5, and 1.0 µg of expression vectors of HDAC1 together with 1 µg of luciferase reporter pXP-7-1 for 18 h, and the luciferase activity was then assayed. (D) Cells cultured in 10 ml of medium on a 10-cm dish were transfected with 4 µg of expression vectors of HDAC1 and then treated with 10 nM PMA. Treated cells were fixed and sonicated, and a ChIP assay was then performed. Recruitment of transcription factor proteins to Sp1 binding sites on the gene promoter of 12(S)-lipoxygenase was analyzed by ChIP with antibodies against HDAC1 (a), c-Jun (b), Sp1 (c), and IgG (d). The input is an internal control (e) that is within the linear range of the PCR dose curve (g). The relative level of DNA was quantified from three independent experiments. (E) Cells were cultured in 10 ml of medium on a 10-cm dish, and nuclear extracts were then prepared for immunoprecipitation with anti-Sp1 antibodies in the presence of 0, 1, 5, and 10 µg of purified c-Jun and then analyzed with immunoblotting by anti-HDAC1 (a), anti-Sp1 (b), and anti-c-Jun (c) antibodies. The relative level of proteins was quantified from three independent experiments. (F) Cells were transfected with 1 µg of pcDNA3.0-Sp1-HA or pcDNA3.0-Sp1(K703/A)-HA for 18 h. Cells were fixed and treated with anti-HA and anti-p300 antibodies, followed by staining with secondary antibodies conjugated with FITC or Cy5. The protein localization of p300 and expressed Sp1-HA or Sp1(K703/A)-HA in cells were analyzed by confocal microscopy. (G) The small interfering RNAs of c-Jun (sense, 5'-GGAAAAAGUGAAAACCUUGtt-3'; antisense, 5'-CAAGGUUUUCACUUUUUCCTC-3') purchased from Ambion Inc. were transfected into the cells with siPORT Amine for 2 days to knock down the level of c-Jun (a). Nuclear extracts were prepared for immunoprecipitation with anti-Sp1 antibodies, followed by immunoblotting with anti-HDAC1, anti-c-Jun, and anti-Sp1 (b) antibodies. The relative level of proteins was quantified from three independent experiments (c). RNAi, RNA interference.

Role of acetylated Sp1 in recruiting p300 and HDAC1 to the promoter of the lipoxygenase gene. We then studied whether the recruitment of p300 and HDAC1 to Sp1 binding sites on the promoter of the 12(S)-lipoxygenase gene was dependent on the level of acetylated Sp1. Cells were transfected with the expression vector, wild-type Sp1, or mutant Sp1(K703/A), and the binding of nuclear proteins to Sp1 binding sites on the promoter of the 12(S)-lipoxygenase gene was examined by in vitro DAPA and in vivo ChIP assays. Overexpression of mutant Sp1(K703/A) recruited more p300 to the promoter than wild-type Sp1, as seen from the results of the two binding studies (Fig. 8A, panels a and f, and B, panels a and g). On the other hand, overexpression of wild-type Sp1 recruited more HDAC1 to the promoter than mutant Sp1(K703/A) (Fig. 8A, panels b and g, and B, panels b and h). In addition, both wild-type Sp1 and mutant Sp1(K703/A) expression could recruit c-Jun to the promoter of the 12(S)-lipoxygenase gene (Fig. 8A, panels c and h, and B, panels c and i). Furthermore, we also studied whether p300 had any effect on the recruitment of histone 3 to the promoter. Cells were treated with E1A to inhibit the recruitment of p300, and the recruitment of histone 3 to the promoter was then examined. The results shown in Fig. 8C clearly indicate that in the presence of E1A, recruitment of p300 to the promoter (Fig. 8C, panels b and g) was reduced, and, concomitantly, the recruitment of acetyl-histone 3 (panels c and h) and the recruitment of histone 3 (panels d and i) were also decreased. However, there was no change in the level of Sp1 (Fig. 8C, panels a and f). In addition, the recruitment of p300 to the promoter of the 12(S)-lipoxygenase gene was decreased with TSA treatment in A431 cells (Fig. 8D). Finally, in order to confirm this conclusion, we expressed Sp1 and Sp1(K703/A) in cells to study the promoter sensitivity of DNase I and restriction enzymes. The results shown in Fig. 8E indicate that the promoter of the 12(S)-lipoxygenase gene was more sensitive to DNase I digestion (Fig. 8E, lanes 1 to 6) and more accessible to SmaI (lanes 7 to 12) under the expression of Sp1(K703/A) than that of Sp1 (Fig. 8E).


Figure 8
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  		FIG. 8. Deacetylated Sp1 increases recruitment of p300 to the promoter of the 12(S)-lipoxygenase gene. (A) Cells cultured in 10 ml of medium on a 10-cm dish were transfected with 4 µg of expression vectors of Sp1 or Sp1(K703/A) for 24 h. Cell nuclear extracts were prepared and then immunoprecipitated with biotinylated promoter DNA of 12(S)-lipoxygenase. Samples were then analyzed by immunoblotting with antibodies against p300 (a), HDAC1 (b), c-Jun (c), and Sp1 (d) and with the internal control (e). The relative levels of p300 (f), HDAC1 (g), c-Jun (h), and Sp1 (i) were quantified from three independent experiments. (B) Cells cultured in 10 ml of medium on a 10-cm dish were transfected with 4 µg of expression vectors of Sp1 or Sp1(K703/A), fixed, and sonicated, and a ChIP assay was then performed. Recruitment of transcription factor proteins to Sp1 binding sites on gene promoter of 12(S)-lipoxygenase was analyzed by immunoprecipitation with antibodies against p300 (a), HDAC1 (b), c-Jun (c), Sp1 (d), and IgG (e). The input was an internal control (f). The relative levels of DNA recruited by p300 (g), HDAC1 (h), c-Jun (i), and Sp1 (j) were quantified from three independent experiments. The number of PCR cycles was still within the linear range of the dose curve (k). (C) In order to study the effect of E1A on the recruitment of Sp1, p300, and acetyl-histone 3 to Sp1 binding sites on the gene promoter of 12(S)-lipoxygenase, cells were transfected with 4 µg of expression vector of E1A for 24 h. A ChIP assay was then performed, and the recruitment of transcription factor proteins was detected by immunoprecipitation with antibodies against Sp1 (a), p300 (b), acetyl histone 3 (c), and histone 3 (d). The input was an internal control (e). The relative levels of DNA recruited by Sp1 (f), p300 (g), acetyl histone 3 (h), and histone 3 (i) were quantified from three independent experiments. The number of PCR cycles was still within the linear range of the dose curve (j). (D) Cells were treated with 20, 100, or 200 ng TSA for 9 h. A ChIP assay was then performed, and the recruitment of transcription factor proteins was detected by immunoprecipitation with antibodies against p300 (a) and IgG (b). The input was an internal control (c). The relative level of DNA recruited by p300 (d) was quantified from three independent experiments. The number of PCR cycles was still within the linear range of the dose curve (d, upper right corner). (E) Cells cultured in 10 ml of medium on a 10-cm dish were transfected with 4 µg of expression vectors of Sp1 or Sp1(K703/A). Nuclear extracts were extracted for the DNase I sensitivity assay and restriction enzyme accessibility assay of the 12(S)-lipoxygenase gene. Lanes 1 to 6, nuclei of A431 cells were digested with 1 and 5 µg of DNase I for 5 min at room temperature. Lanes 7 to 12, nuclei of A431 cells were treated with SmaI for 1 h at 37°C, and Southern blot analysis was then performed with a 32P-labeled 400-bp fragment of the 12(S)-lipoxygenase gene promoter as a probe. wt, wild type.


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DISCUSSION
 
In this study, several pieces of evidence that support the notion that acetylation/deacetylation of Sp1 regulates the gene transcription of 12(S)-lipoxygenase were obtained.

First, Sp1 was constitutively acetylated at Lys703. Second, HDAC1 was recruited to the promoter, consequently causing the deacetylation of Sp1. Third, deacetylated Sp1 recruits p300 to the promoter and enhances gene transcription, perhaps through the acetylation of histone 3. Sp1 has been recently found to be acetylated, and the acetylated form may be involved in gene regulation by affecting the DNA binding or protein interactions (9, 23, 35). In order to study the role of acetylation in Sp1-regulated gene transcription, we first confirmed that Sp1 could be acetylated in A431 cells by using the in vivo acetylation assay. The acetylated Sp1 in cells was recognized by anti-acetyl-lysine antibodies in immunoprecipitation experiments and by radiolabeling with [3H]sodium acetate (Fig. 1). The in vivo results thus obtained were consistent with results of previous studies indicating that Sp1 can be acetylated in the cell (9). In this study, we also presented evidence to support the notion that p300 might manifest the functional role of acetylated Sp1 in A431 cells. Overexpression of p300 enhanced the level of acetylated Sp1 in cells (Fig. 1E). In addition, treatment of cells with E1A to inhibit p300 could reduce the acetylated level of Sp1 (Fig. 1F). These results indicated that p300 is critical for regulating the level of the acetylation of Sp1, which is thus the functional manifestation of the latter. However, we cannot rule out the possibility that other proteins containing HAT activity may also regulate Sp1 acetylation. One of the major findings in this study was the identification of the acetylation site of Sp1 in the cell. With the site-directed mutation approach, we demonstrated that Sp1 was acetylated at lysine 703 residing at the C terminus (Fig. 5C). Previous studies revealed that the C terminus of Sp1 is the domain that interacts with the promoter of the lipoxygenase gene as well as with p300 (37). In our present study using A431 cells, PMA treatment caused a decrease in the level of acetylated Sp1 (Fig. 2). The degree of acetylation of Sp1, however, did not apparently have any effect on Sp1 binding to the promoter because no change in Sp1 binding to the Sp1 binding sites on the promoter was observed, as determined by the ChIP assay and the DNA affinity precipitation assay (Fig. 4) and by studying the binding of overexpressed Sp1(K703/A) to the promoter (Fig. 8). These results were consistent with our previous findings using a gel mobility shift assay showing no alteration in the Sp1 binding to the promoter of the 12(S)-lipoxygenase gene before and after cells were treated with PMA (31). However, the change in the acetylation level of Sp1 upon PMA stimulation did affect the recruitment of p300 to the promoter (Fig. 7). Our previous studies indicated that the induction of c-Jun expression and its interaction with Sp1 were prerequisites for PMA- or EGF-induced gene expression of 12(S)-lipoxygenase in A431 cells (11). The temporal profile of c-Jun expression, after cells received PMA, showed a maximum induction of c-Jun at 3 h after PMA treatment that persisted up to the 9-h time point. The same temporal profiles were seen in the interaction between c-Jun and Sp1 (Fig. 3) and in the binding of the c-Jun/Sp1 complex to the Sp1 binding domain on the promoter (Fig. 4). Taken together, these results indicated that as soon as c-Jun was induced, c-Jun interacted with Sp1 almost immediately. Sp1 thus functioned as an anchor carrying c-Jun to the promoter 3 h after PMA treatment. However, the activation of the promoter activity and the transcription of the 12(S)-lipoxygenase gene were not significantly increased 3 h after PMA treatment. They became significantly increased only until 9 h after the PMA treatment. Apparently, there was a 6-h delay between the formation of the c-Jun/Sp1 complex and the activation of gene expression. In this study, we found that during this 6-h period, Sp1 was deacetylated and p300 was recruited to the lipoxygenase promoter. In order to provide a link between the deacetylation of Sp1 and the expression of the 12(S)-lipoxygenase gene induced by PMA, we used TSA as an HDAC inhibitor to treat the cells. The results revealed that TSA inhibited the transcription of the 12(S)-lipoxygenase gene induced by PMA and reversed the PMA-induced deacetylation of Sp1 (Fig. 2D). Since deacetylated Sp1 apparently enhanced the recruitment of p300 to the promoter (Fig. 4 and 8), deacetylation of Sp1 in this 6 h of the observation period may play a pivotal role in inducing the transcription of the 12(S)-lipoxygenase gene. Recently, several studies reported that TSA is able to increase or decrease the expression levels of several genes. The gene expression of p21 and transforming growth factor ß receptors is increased by TSA treatment, but that of cyclooxygenase 2 is attenuated (13, 38, 40). TSA may do so by affecting the posttranslational acetylation of transcription factors or coactivators. It is known that the HDAC family containing the deacetyltransferase activity is an important regulator for gene transcription through its ability to deacetylate histones, which leads to chromatin packaging (33). Some studies revealed that several nonhistone proteins can also be deacetylated to regulate the transcription of target genes. For example, HDAC1 is able to deacetylate C/EBPß and, as a result, activates the target genes of the latter (43). In this study, we found that the recruitment of HDAC1 to the promoter was seen in the early phase (3 h) of PMA treatment, immediately followed by the deacetylation of Sp1 in the cell (Fig. 2B and 4D). These results suggest that the recruitment of HDAC1 to the promoter might play a functional role in the deacetylation of Sp1. Furthermore, we also found that Sp1 in the acetylated form is essential for recruiting HDAC1 to the promoter because HDAC1 interacts less with mutant Sp1(K703/A) that lacks the acetylation site in the cell (Fig. 7A). In fact, the colocalization of HDAC1 with Sp1 in the nucleus was apparent after cells were treated with PMA for 3 h. Six hours later, however, the colocalization was largely reduced (Fig. 3C). These results suggest that the level of acetylated Sp1 might also play a role in the recruitment of HDAC1 to the promoter of the 12(S)-lipoxygenase gene. The HAT activity-containing proteins such as p300, PCAT, and GCN5 can regulate gene transcription by acetylating histones as well as some nonhistone transcription factors, which in turn leads to chromatin remodeling and the initiation of transcription (33). In this study, p300 was shown to interact with deacetylated Sp1 via several different lines of evidence. The immunoprecipitation experiments (Fig. 3A), the immunofluorescence analysis (Fig. 3B), and the ChIP assay (Fig. 4B and E) demonstrated that p300 interacted with Sp1 before being recruited to the promoter of the 12(S)-lipoxygenase gene at the late stage (9 h) of PMA treatment, when Sp1 was deacetylated (Fig. 2B). Coimmunoprecipitation of p300 with Sp1 was attenuated when Sp1 was acetylated (Fig. 7B). p300 interacted with mutant Sp1(K703/A) more than with wild-type Sp1 (Fig. 7A). In the in vitro DAPA and in vivo ChIP assays, p300 was seen to be recruited more to the promoter of the 12(S)-lipoxygenase gene of mutant Sp1(K703/A) than to that of wild-type Sp1 (Fig. 8A and B). Our results reported in this study also indicated that the recruitment of p300 to the promoter was critical for the expression of 12(S)-lipoxygenase. The p300 protein was recruited to the promoter (Fig. 4A) before the transcription activity of the 12(S)-lipoxygenase gene could be observed in the presence of PMA (Fig. 2D). E1A, an inhibitor of p300, attenuated the transcription of the lipoxygenase gene (Fig. 6B). Thus, our results support the notion that the recruitment of p300 can activate the transcriptional activity of target genes (30). Our results also indicate that treatment of cells with E1A not only attenuated the recruitment of p300 but also decreased the level of acetylated histone 3 at the promoter of the 12(S)-lipoxygenase gene (Fig. 8C). The p300 protein apparently regulates the acetylation of histone 3, leading chromatin remodeling to activate the transcription activity of 12(S)-lipoxygenase in A431 cells (Fig. 8E). A scheme illustrating the mechanistic model of PMA-induced 12(S)-lipoxygenase gene transcription is shown in Fig. 9. Three steps in response to PMA stimulation are proposed. According to the results of immunoprecipitation experiments (Fig. 3) and the chromatin immunoprecipitation assay (Fig. 4), the interaction of HDAC1 with Sp1 including its free form and DNA-bound form to deacetylate Sp1 was increased with PMA treatment. Two additional pieces of evidence support the notion that Sp1 primarily acts as an anchor protein to recruit HDAC1. First, Sp1 always binds to the promoter region in most genes containing a GC-rich region. Second, the recruitment of HDAC1 is increased quickly after PMA treatment. In cells receiving PMA for 3 h, HDAC1 begins to interact with the c-Jun/Sp1 complex, with the latter in the constitutively acetylated form at the promoter of the 12(S)-lipoxygenase gene (step 1). Sp1 is then deacetylated by HDAC1, and the resultant deacetylated Sp1 recruits p300 to the promoter between 6 and 9 h after PMA treatment (step 2). The p300 protein thus recruited to the promoter can then acetylate histone 3, leading to a chromatin remodeling that induces the transcription activity of the 12(S)-lipoxygenase gene (step 3). Sp1 recruiting of c-Jun to the promoter as an important initial step in gene transcription has been demonstrated first by us in A431 cells for the expression of 12(S)-lipoxygenase and more recently in growth factor- and/or phorbol ester-induced expression of genes including nicotinic acetylcholine receptor ß4 (32), cytosolic phospholipase A2 (5), p21WAF1/CIP1 (28), and human keratin 16 (41). Our results indicate that the activation of a promoter may require several transcription factors acting in a highly coordinated, temporal-spatial manner. Our results also suggest that transcription factors, as limited in numbers as they are found in nature, can perform chromatin IP-arrays to screen the candidate genes. It will be of great interest to see if the molecular mechanism elucidated from this study may also be seen in many other Sp1-regulated gene transcription systems.


Figure 9
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  		FIG. 9. Scheme illustrating the mechanistic model of PMA-induced activation of 12(S)-lipoxygenase gene transcription.


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ACKNOWLEDGMENTS
 
We thank the National Cheng-Kung University Bio-imaging Optical Core Laboratory for the assistance in confocal microscopic analysis. Thanks are also due to Tsung-Ping Su and Wai-Ming Kan for their critical reviewing of the manuscript.

This work was supported by the Ministry of Education Program for Promoting Academic Excellent of University under grant number 91-B-FA09-1-4 and grant NSC 94-2320-B006-094 of Taiwan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, Republic of China. Phone: 886-6-2353535, ext. 5496. Fax: 886-6-2749296. E-mail: wcchang{at}mail.ncku.edu.tw. Back


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Molecular and Cellular Biology, March 2006, p. 1770-1785, Vol. 26, No. 5
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.5.1770-1785.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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