Identification and Characterization of the IKK (cid:1) Promoter POSITIVE AND NEGATIVE REGULATION BY ETS-1 AND p53, RESPECTIVELY*

IKK (cid:1) , a subunit of IkB (cid:1) kinase (IKK) complex, has an important role in the activation of nuclear factor-kB (NF-kB), a key regulator of normal and tumor cell proliferation, apoptosis, and response to chemotherapy. However, little is known about the transcriptional regulation of the IKK (cid:1) gene itself. The present study re-vealed that the transcriptional induction of the IKK (cid:1) gene is positively regulated by binding ETS-1, the protein product of the ETS-1 proto-oncogene. Furthermore, ETS-1 mediated activation of IKK (cid:1) is negatively regulated by p53 binding to ETS-1. By analyzing the genomic DNA sequence, we identified the putative IKK (cid:1) promoter sequence in the 5 (cid:1) -flanking untranslated region of the IKK (cid:1) gene. Transfection of EU-4, an acute lymphoblastic leukemia (ALL) cell line, with plasmids containing the IKK (cid:1) 5 (cid:1) -untranslated region sequence upstream of the luciferase reporter showed that this region pos-sessed major promoter activity. Induction or enforced overexpression of p53 represses IKK (cid:1) mRNA and protein expression as well as IKK (cid:1) promoter activity. Deletion and mutation analyses as well as chromatin immunoprecipitation and electrophoretic mobility shift assay the core IKK (cid:1) suggest that of the IKK (cid:1) a by and loss of p53-mediated control over ETS-1-dependent transactivation of IKK (cid:1) may represent a novel pathway for the constitutive activation of NF-kB-mediated gene expression and therapy resistance and HindIII sites. RT-PCR was performed using total RNA extracted from EU-1 cells and primer pairs 5 (cid:2) -GCGCGCTAGCAACTTGCTAC- CATCCCGT-3 (cid:2) and 5 (cid:2) -GCGCAAGCTTTGCCAT-CACTCGTCGGCA-3 (cid:2) that were designed from the ets-1 gene. We also constructed a pSUPER- ETS-1 siRNA plasmid by inserting a 19-nucleotide ETS-1 sequence into expression plasmid pSUPER, provided by Dr. Agami (Netherlands Can- cer Institute), which directs the synthesis of siRNA-like transcripts. A forward primer: 5 (cid:2) -GATCCCCGATATGGAATGTGCAGATGTTCAAG- AGACATCTGCACATTCCATATCTTTTTGGAAA-3 (cid:2) and a reverse primer: 5 (cid:2) -AGCTTTTCCAAAAAGATATGGAATGTGCAGATGTCTCT- TGAACATCTGCACATTCCATATCGGG-3 (cid:2) that contains a 19-nucleo-tide ETS-1 sequence selected from the coding region 100 bp from the start site were synthesized by the Emory University Microchemical Facility. The two 64-nucleotide primers were annealed in the buffer (100 m M potassium, 30 m M HEPES-KOH, pH 7.4, and 2 m M Mg acetate) and phosphorylated in buffer containing T4 PNK. The annealed and phosphorylated oligos were then ligated into pSUPER vector digested with BglII and HindIII. The new construct was then transfected into Escherichia coli for propagation. The plasmid-containing insert was determined by digestion with EcoRI and HindIII. Positive clones have inserts of (cid:1) 360 bp as compared with empty vector (300 bp insert).

NF-kB 1 is a transcription factor that plays an essential role in regulating the balance between cell proliferation and apoptosis, including the response of tumor cells to chemotherapy (1)(2)(3). Activation of the IkB kinase complex (IKK␣/␤/␥) is a critical step in the activation of the NF-kB pathway (4,5). Recent studies have identified an important role for IKK␣ distinct from that of IKK␤ in regulating NF-kB-mediated gene expression (6 -8). Unlike IKK␤, which is localized predominantly in the cytoplasm, IKK␣ can shuttle between the cytoplasm and nucleus (9,10). In the nucleus, IKK␣ is recruited to the promoter region of the NFkB-regulated genes, and activates their expression by interacting with cAMP-response element-binding protein as well as by phosphorylating histone H3, which is critical for the activation of NF-kB-directed gene expression (10,11).
Previous studies have shown that NF-kB in tumor cells is activated by treatment with tumor necrosis factor ␣ (12,13) and certain chemotherapeutic agents (13)(14)(15). Similarly, constitutively activated NF-kB (i.e. activation of NF-kB in cells without stimulation) has been associated with increased cell proliferation and survival in cancer cells, and may be linked to drug resistance in these cells (16 -18). Although the mechanisms by which NF-kB regulates resistance to apoptosis are not completely understood, it is believed that activated NF-kB in the nucleus protects cells against apoptosis through directly activating transcription of NF-kB-dependent genes. These downstream genes include members of the Inhibitor-of-Apoptosis Protein (IAP) family such as cIAP1, cIAP2, XIAP (19 -22), and Bcl-2 family members Bcl-XL and Bfl-1/A1 (23,24).
In contrast to the role of NF-kB in protecting cells from apoptosis, the p53 tumor suppressor gene plays an important role in inducing apoptosis in response to various types of stress including chemotherapeutic drug treatment. p53 functions as a transcription factor that can either positively or negatively regulate transcription of a particular gene promoter (25). Previous studies have shown that p53 induces apoptosis by activating apoptosis-promoting genes such as Bax, DR5, and Fas (26 -28), or by repressing apoptosis-inhibiting genes such as MDR, Bcl-2, and survivin (29 -32).
A recent study using microarrays representing over 33,000 individual human genes aimed at identifying differentially expressed genes in response to p53-induced apoptosis has found that a total of 1501 genes (4.4%) responded to p53, and ϳ80% of these were repressed by p53 (33). In activating gene expression, p53 functions via DNA sequence-specific binding (34). The specific p53 consensus sequence consists of two copies of a decamer motif separated by 0 to 13 bp of random nucleotide (35). Transcriptional repression mediated by p53 is complex and may occur via multiple mechanisms. A few genes are suppressed by p53 through direct DNA binding (36 -39), whereas most genes repressed by p53 lack p53 binding (40). Repression of the latter genes by p53 involves interactions of p53 with other transcriptional factors such as TBP, the TATA box-binding protein (41), Sp-1 (42), and ETS-1 (43,44).
Both NF-kB activation and loss of wild-type (wt) p53 function are involved in progressive development of many cancers and resistance to chemotherapy; thus these two events may be associated. Previous studies have shown that WT p53 and the p65 (RelA) subunit of NF-kB mutually repress each others ability to activate transcription through competitive binding to the cAMP-response element-binding protein (45,46). Furthermore, we have found that WT p53 can directly repress p65 promoter activity (47).
In the present study, we demonstrate an additional mechanism for the interaction of p53 and NF-kB. Our studies showed that p53 represses the expression of IKK␣, and p53-repression of IKK␣ occurs at the transcriptional level. Because IKK␣ is important in regulating a novel NF-kB activation pathway, loss of p53 function by point mutation or overexpression of MDM2 may contribute to uncontrolled transactivation of IKK␣ in cancer cells resulting in increased survival and resistance to apoptosis because of enhanced NF-kB activation.

EXPERIMENTAL PROCEDURES
Cell Lines-Three cell lines, EU-1, EU-4, and EU-8, established from children with B-cell precursor ALL were used in this study. The EU-1 line expresses WT p53, whereas EU-4 and EU-8 lack p53 expression (48). The cell lines were grown in standard culture medium (RPMI 1640 containing 10% fetal bovine serum, 2 mmol/liter L-glutamine, 50 units of penicillin, and 50 g/ml streptomycin) at 37°C in 5% CO 2 in air.
Identification and Construction of Full-length IKK␣ Promoter Plasmid-To date, there have been no studies of the IKK␣ gene promoter reported. We have searched the 5Ј-flanking untranslated DNA sequence of the IKK␣ gene by navigating the Human Genome web site (ncbi.nlm.nih.gov/Genomes/index.html). A 1010-bp fragment from Ϫ940 to ϩ70 in this region was generated by PCR using primers 5Ј-ATGAACTACTGTGCTGGC-3Ј and 5Ј-GCTCCATGGGGCGGGAGG-3Ј, and cloned into the promoterless luciferase vector pGL3 basic (Promega) at XhoI and HindIII sites to produce the pLuc-1010 construct.
Generation of IKK␣ Promoter Deletion and Mutation Constructs-In addition to the full-length IKK␣ promoter plasmid, various 5Ј-3Ј or 3Ј-5Ј deletion constructs and site-directed mutants were made. For construction of the putative IKK␣ promoter, deletion constructs pLuc-508, pLuc-168, pLuc-120, pLuc-110, pLuc-100, pLuc-70, and pLuc-890 and PCR primer pairs were determined from the corresponding sites in the sequence of the 1010-bp fragment, and then used to make different constructs. Mutant pLuc-168mm construct was made using mutated nested primers at the Cys and Gly residues within the 10-bp motif, and mutant pLuc-1010m was made using mutated primers at the ETS-1 binding motif with a GGAA to TTAA substitution. Mutated promoter fragments were made by site-directed mutagenesis using in vitro sitedirected mutagenesis system (Promega). Constructs including different deleted or mutated fragments were then ligated to the pGL3 basic vector. DNA sequencing was performed to confirm that the sequences of the PCR products were correct as compared with the sequence of the 5Ј-flanking UTR of the IKK␣ gene obtained from the Human Genome.
Generation of ETS-1 and p53 Expression Plasmids-The ETS-1 expression plasmid was generated by inserting a cDNA fragment synthesized by RT-PCR into the pcDNA3.1ϩ vector (Invitrogen) at the NheI and HindIII sites. RT-PCR was performed using total RNA extracted from EU-1 cells and primer pairs 5Ј-GCGCGCTAGCAACTTGCTAC-CATCCCGT-3Ј and 5Ј-GCGCAAGCTTTGCCAT-CACTCGTCGGCA-3Ј that were designed from the ets-1 gene. We also constructed a pSUPER-ETS-1 siRNA plasmid by inserting a 19-nucleotide ETS-1 sequence into expression plasmid pSUPER, provided by Dr. Agami (Netherlands Cancer Institute), which directs the synthesis of siRNA-like transcripts. A forward primer: 5Ј-GATCCCCGATATGGAATGTGCAGATGTTCAAG-AGACATCTGCACATTCCATATCTTTTTGGAAA-3Ј and a reverse primer: 5Ј-AGCTTTTCCAAAAAGATATGGAATGTGCAGATGTCTCT-TGAACATCTGCACATTCCATATCGGG-3Ј that contains a 19-nucleotide ETS-1 sequence selected from the coding region 100 bp from the start site were synthesized by the Emory University Microchemical Facility. The two 64-nucleotide primers were annealed in the buffer (100 mM potassium, 30 mM HEPES-KOH, pH 7.4, and 2 mM Mg acetate) and phosphorylated in buffer containing T4 PNK. The annealed and phosphorylated oligos were then ligated into pSUPER vector digested with BglII and HindIII. The new construct was then transfected into Escherichia coli for propagation. The plasmid-containing insert was determined by digestion with EcoRI and HindIII. Positive clones have inserts of ϳ360 bp as compared with empty vector (300 bp insert).
Expression plasmids for WT p53 and four mutant p53 (p53-143, p53-175, p53-248, p53-273) as well as MDM2 were kindly provided by Dr. B. Vogelstein (Johns Hopkins University School of Medicine). The p53-143 has mutant and WT conformations at 37.5 and 32.5°C, respectively (49). Transfection and Luciferase Activity Assay-Both transient and stable gene transfections were performed to analyze p53 induction and its effects on IKK␣ expression. For stable gene transfection, EU-4 cells in exponential growth were transfected with p53-143 or an empty control vector by electroporation at 290 V, 950 microfarads using a Gene Pulser II System (Bio-Rad). The cells were seeded 48 h post-transfection into culture dishes for the selection of G418-resistant colonies. Colonies were grown in methylcellulose medium containing G-418 for 2-3 weeks, and clones are picked and grown in RPMI medium with or without G-418 for the duration of the experiments. For gene reporter assay, cells were transiently co-transfected by electroporation with the IKK␣ promoter-luciferase constructs as described above and different forms of p53 (WT and mutants), MDM2, ETS-1, and ETS-1 siRNA expression plasmids. Briefly, 1 ϫ 10 7 cells in exponential growth were mixed with the corresponding IKK␣ promoter-luciferase constructs plus different doses of p53, MDM2, ETS-1, and ETS-1 siRNA plasmids and electroporated as described above. Transfected cells were resuspended in 10 ml of RPMI containing 10% fetal bovine serum. At 48 h post-transfection, cell extracts were prepared with 1ϫ lysis buffer, and then 20-l aliquots of the supernatant were mixed with 100 l of luciferase assay reagent (Promega) and analyzed on a Microplate Luminometer (Turner Designs). Luciferase activity was normalized to ␤-galactosidase activity as an internal control.
Western Blot Assay-Cells were lysed in a buffer composed of 150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM EDTA, 1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 20 g/ml aprotinin, and 25 g/ml leupeptin for 30 min at 4°C. After clarification, equal amounts of protein extracts were resolved by SDS-PAGE and transferred to nitrocellulose paper. After blocking with buffer containing 20 mM Tris-HCl (pH 7.5) and 500 mM NaCl, 5% nonfat milk for 1 h at room temperature, the filter was incubated with specific antibodies for 2 h at room temperature, followed by horseradish peroxidase-labeled secondary antibody. Blots were developed using a chemiluminescent detection system (ECL, Amersham Biosciences). After stripping, the filter was reprobed with an anti-actin antibody (Calbiochem) as a control for equal protein loading and protein integrity.
RT-PCR-RT-PCR was performed to analyze the effect of p53 on IKK␣ mRNA expression. Total RNA was prepared from EU-4/p53-143 cells and control cells shifted to 32.5°C for different times with the Ultraspec Reagent (Biotecx). RT-PCR was conducted using an Access RT-PCR Kit (Promega) according to the manufacturer's protocols. The oligonucleotide primers used to analyze IKK␣ transcripts were: forward, 5Ј-CAGTATCTGGCCCCAGAGCT-3Ј and reverse, 5Ј-CAGCCCA-CACTTTACGCAGC-3Ј. ␤-Actin was used as an internal control. The PCR products were visualized with ethidium bromide staining under UV light after electrophoresis on 1.5% agarose gel.
CHIP Assay-The CHIP assay as described previously (47) was performed to analyze the DNA binding activity of p53 and ETS-1 protein to IKK␣ promoter. EU-4 cells were co-transfected with 10 g of IKK␣ promoter construct pLuc-508 and 10 g of either p53 or ETS-1 expression plasmids by electroporation as described above. After 48 h, formaldehyde was added at 1% to the culture media and cells were incubated at room temperature for 10 min with mild shaking to cross-link ETS-1 and p53 protein to the IKK␣ promoter. Then, cells (1 ϫ 10 6 ) were washed twice with cold phosphate-buffered saline and resuspended in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) with 1 mM phenylmethylsulfonyl fluoride, 1 g/ml aprotinin, and 1 g/ml pepstatin A. After a brief sonication, lysates were cleared by centrifugation and were diluted 10-fold with dilution buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, and 167 mM NaCl) containing protease inhibitors as above. Anti-p53 and anti-ETS-1 or control antibodies were added at 4°C overnight with rotation. Immunoprecipitated complexes were collected by protein A/G plus-agarose. Precipitants were sequentially washed with low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and LiCl wash buffer (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), once, respectively, followed by two washes with 1ϫ TE. After the final wash, 250 l of elution buffer (1% SDS, 0.1 M NaHCO 3 ) was added, and the precipitants were incubated at room temperature for 15 min with rotation. Then 5 M NaCl was added to reverse the formaldehyde cross-linking by heating at 65°C for 4 h. After precipitation with ethanol, the pellets were resuspended and treated with proteinase K. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. Pellets were resuspended in TE buffer and subjected to PCR amplification using forward and reverse primers (5Ј-GTGGTTCCGT-TCAGCCCT-3Ј and 5Ј-TGCTCGCGCGTCTTTG-3Ј) to the pLuc-508 fragment. The resulting product was 188 bp and contained the last two putative p53 consensus sequences as well as the ETS-1-binding motif in the IKK␣ promoter. The PCR product was separated by agarose gel electrophoresis.
EMSA-Sequence-specific DNA binding activity of ETS-1 to IKK␣ promoter was assayed by EMSA. Nuclear protein extraction was prepared using a kit (NE-PER TM from Pierce). Nuclear protein (5 g) from EU-1 and EU-4 cells was incubated for 15 min in a binding buffer (10 mM Tris-HCl, 50 mM NaCl, 0.5 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 7.5 mM MgCl) plus 0.1 g of poly(dI-dC) carrier, and 32 Plabeled oligonucleotide probe 5Ј-GTTACCGGAAGTGACGCATT-3Ј spanning the Ϫ50/Ϫ31 IKK␣ promoter that contains the ETS-1 consensus (CCGGAAGT). A mutated probe of 5Ј-GTTACCTTAAGTGACG-CATT-3Ј with changes of the ETS-1 core consensus from GGAA to TTAA (underlined) served as control. Anti-ETS-1 and rabbit IgG antibodies as additional controls were used to supershift the specific complexes of interest by pretreating the extract for 1 h at 4°C with antibodies. The samples were electrophoresed on a 5% polyacrylamide gel, dried, and developed with an intensifying screen at Ϫ70°C.
Co-immunoprecipitation-EU-1 or EU-4 cells transiently transfected with p53 were lysed in a buffer composed of 50 mM Tris (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 10 mM sodium phosphate, 10 mM NaF, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin. After centrifugation, 30 g of the clarified cell lysate was incubated with 15 l of protein G plus/protein A-agarose and 1 g of p53, MDM2, or ETS-1 antibodies, respectively. After 24 h incubation, the agarose was centrifuged, washed four times with ice-cold lysis buffer, suspended in electrophoresis sample buffer, and boiled for 5 min. The immunoprecipitated protein was further analyzed by Western blotting as described above.

RESULTS
Identification of the Proximal 5Ј-Flanking Region of the IKK␣ Gene-By navigating the Human Genome, we found the nucleotide sequence of the proximal 5Ј-flanking UTR of the IKK␣ gene. The DNA sequence 5Ј to the start site that represents a known transcription start site of the IKK␣ coding sequence is shown in Fig. 1. The DNA sequence of the 5Ј-flanking UTR of the IKK␣ gene contains three potential p53 response consensus Three putative p53-binding sites are underlined that are highly associated with a canonical p53 consensus sequence, i.e. the three regions contain 4-, 1-, and 10-nucleotide (italics) spacers, respectively, between the two decamer "half-sites" of the p53 consensus elements (bold). The potential core promoter region contains a consensus for binding transcription factors ETS-1 (bold and lowercase). A vertical arrow marks the cDNA start site and a bent arrow indicates the first codon.
FIG. 2. A, differential effect of WT p53 and mutant p53 on IKK␣ promoter activity following transient transfection. EU-4 cells were cotransfected with 5 g of the IKK␣ promoter-luciferase construct (pLuc-1010) and either 10 g of various p53 expression plasmids (WT and mutations in codons 143, 175, 248, and 273) or control plasmid (neo), respectively. Electroporation was performed at 300 V, 950 microfarads. At 48 h post-transfection, cell extracts were analyzed for luciferase activity. Data represent the mean of three independent experiments normalized to ␤-galactosidase activity; bars, ϮS.D., -fold induction represents the difference between luciferase activity in test samples containing the indicated plasmids and the neo vector control. B, dose response of WT p53-mediated repression of the IKK␣ promoter, which is reversed by MDM2. EU-4 cells were cotransfected with 5 g of pLuc-1010 and either with increasing amounts (2.5, 5, and 10 g) of WT p53 (lanes 3-5) or with increasing amounts (2.5, 5, and 10 g) of MDM2 in the presence of a fixed amount (10 g) of WT p53 (lanes 6 -8). EU-4 cells were transfected with 5 g of either pGL3 basic vector (lane 1) or pLuc-1010 (lane 2) alone as controls. The total amount of plasmid was adjusted to 25 g per transfection with an empty neo plasmid. Electroporation and analyses of luciferase activity were performed as described above. Titrations of the cellular extracts from plasmidtransfected cells were analyzed for p53, MDM2 by Western blot analysis (inset). sites located between nucleotides Ϫ345 to Ϫ322, Ϫ98 to Ϫ78, and Ϫ29 to ϩ1. These putative p53 response elements are associated with a canonical p53 consensus sequence that consists of two copies of a decamer motif 5Ј-RRRCWWGYYY-3Ј separated by 0 to 13 bp of random nucleotides, in the motif, R ϭ G or A, W ϭ T or A, Y ϭ C or T (34). Moreover, an ETS-1binding site (Ϫ46 to Ϫ39) was found in the most likely core promoter region of the IKK␣ gene.
Repression of IKK␣ Promoter Activity by WT p53-To determine the promoter activity of the sequence located 5Ј to the IKK␣ gene, a fragment of DNA spanning nucleotides Ϫ940 to ϩ70 was placed 5Ј to the promoterless luciferase vector pGL3 basic. The level of luciferase activity was examined upon transient transfection into EU-4 cells. Substantial levels of luciferase activity were detected if the transfected reporter construct contained the Ϫ940 to ϩ70 sequence in the sense but not in the antisense orientation (data not shown). To investigate whether the IKK␣ promoter is regulated by p53, the IKK␣ promoterluciferase construct and plasmids containing WT p53 and various forms of mutant p53 were co-transfected into EU-4 cells, respectively. From the data presented in Fig. 2A, it can be seen that WT p53 produced a 70 -80% decrease in IKK␣ promoter activity (relative to the control plasmid CMV-neo), whereas p53 mutations in codon 143, 175, 248, and 273 increased (2.5-4.5fold) the IKK␣ promoter activity. Repression of IKK␣ promoter activity by WT p53 was dose-dependent, and this inhibitory effect was reversed by co-transfection with MDM2 (Fig. 2B). Titrations of transfected p53 and MDM2 in cellular extracts were determined by Western blot assay (Fig. 2B, inset).
Inhibition of IKK␣ Expression by WT p53-To further evaluate whether p53 regulates IKK␣ expression, the p53-null ALL cell line EU-4 was utilized for either stable transfection with a temperature-sensitive p53 allele encoding alanine at codon 143 or transient transfection with a p21 promoter-luciferase construct. The p53 protein in p53-143 transfected EU-4 (EU-4/ p53-143) cells selected from a single clone exists in a mutant conformation at 37.5°C, and temperature shift to 32.5°C induces a WT conformation of p53. As shown in Fig. 3A, the promoter activity of the p53 target gene p21 was induced in a time-dependent manner in EU-4/p53-143 cells cultured at 32.5°C. Moreover, Western blot assay showed that expression of endogenous p21 and MDM2, also p53 targets, was increased in EU-4/p53-143 cells at 32.5°C (Fig. 3B), indicating that a functional WT p53 was induced in EU-4 transfected with p53-143 mutant at 32.5°C. Under the same experimental conditions, however, expression of IKK␣ was down-regulated in EU-4/p53-143 cells, whereas the levels of IKK␤, IKK␥, and the housekeeping gene actin remained unchanged (Fig. 3C). To further characterize whether IKK␣ mRNA is repressed by p53, we performed RT-PCR analysis in EU-4/p53-143 cells. As shown in Fig. 3D, the expression of IKK␣ mRNA was rapidly and strongly decreased in EU-4/p53-143 cells cultured at 32.5°C. The expression of IKK␣ mRNA remained unchanged in EU-4 cells transfected with a control plasmid (CMV-neo) at 32.5°C.
IKK␣ Promoter Activity Is Induced Primarily by ETS-1-To determine the core promoter region of the IKK␣ gene, we generated a series of deleted constructs of the IKK␣ promoter and performed transfection and luciferase activity assays. Our results demonstrated that the core promoter region resides between Ϫ50 and Ϫ1 as shown in Fig. 4. Construct pLuc-120 (Ϫ50 to ϩ70) expressed maximum luciferase activity similar to that of the full-length promoter pLuc-1010, and both 3Ј-5Ј deleted constructs pLuc-890 (Ϫ940 to Ϫ51) and 5Ј-3Ј deleted construct pLuc-70 (ϩ1 to ϩ70) showed no promoter activities as compared with control (transfection of pGL3-basic vector only). Interestingly, a narrow region (Ϫ50 to Ϫ30, 20 bp) containing the ETS-1 binding site expressed the majority of promoter activity, because activity of construct pLuc-100 (Ϫ30 to ϩ70)

FIG. 4. Deletion and mutation analysis of the IKK␣ promoter activity.
A, schematic representation of IKK␣ promoter-luciferase reporter plasmids: pLuc-1010 containing the full-length promoter with three putative p53 response elements (Ⅺ) and an ETS-1 binding site (E); pLuc-508, pLuc-168, pLuc-120, pLuc-110, pLuc-100, pLuc-70, and pLuc-890 containing a series of 5Ј-3Ј or 3Ј-5Ј deleted promoters; pLuc-168mm containing a deleted promoter with the second and third putative p53 response elements mutated by site-directed mutagenesis (f). The deleted pLuc-110 construct contains a truncated ETS-1 binding site (•), and the pLuc-1010m contains a mutated ETS-1 binding site (•). B, transient transfection and luciferase assay for the effect of WT p53 on differentially deleted or mutated IKK␣ promoter activity. EU-4 cells were co-transfected with 5 g of each IKK␣ promoter construct as shown in A and 10 g of either WT p53 expression plasmid or empty neo vector as control. Controls also included transfection of 5 g of PGL3 basic vector plus WT p53 or neo. Electroporation and luciferase assays were performed as described in the legend to Fig. 2. Data represent the mean Ϯ S.D. of three independent experiments normalized to ␤-galactosidase activity.
was approximately one-seventh of that of pLuc-120. Furthermore, there were approximately two-thirds reductions in the activity of deleted promoter pLuc-110 (Ϫ40 to ϩ70) with a partial deletion (truncation) of the ETS-1 binding site compared with the activity of pLuc-120 with an intact ETS-1 binding site, and the full-length promoter with a mutated ETS-1 binding site (pLuc-1010m) showed significant reduction of promoter activity, suggesting that the transcription factor ETS-1 is an important regulator for IKK␣ expression.
To further confirm that ETS-1 regulates the IKK␣ promoter, we evaluated whether co-transfection of the ETS-1 expression plasmid would increase the activity of the IKK␣ promoter in cell line EU-8 with no ETS-1 expression. Furthermore, we examined whether blockage of endogenous ETS-1 by siRNA would decrease IKK␣ promoter activity in EU-4 with high levels of ETS-1 expression. We have generated a pSUPER/ ETS-1 plasmid containing a 19-nucleotide siRNA sequence specific for targeting ETS-1 (Fig. 5A). Transfection of this plasmid into EU-4 cells significantly suppressed expression of the endogenous ETS-1 protein (Fig. 5B). Co-transfection of ETS-1 siRNA plasmid remarkably reduced IKK␣ promoter activity in a dose-dependent manner (Fig. 5C). As we expected, co-transfection of ETS-1 expression plasmid into EU-8 cells significantly increases IKK␣ promoter activity (Fig. 5D), although the transfection efficiency in EU-8 cells was lower than in EU-4 cells as tested for activity of transfected ␤-galactosidase (data not shown).
Repression of IKK␣ Promoter Activity by p53 Does Not Require Direct DNA Binding-As also shown in Fig. 4, the activity of the deleted IKK␣ promoter construct pLuc-168mm that contains mutations of the second and third putative p53 response consensus was similarly repressed by WT p53 as com-FIG. 5. ETS-1 activates IKK␣ promoter activity. A, agarose gel electrophoresis of pSUPER vector and pSUPER-containing ETS-1 siRNA insert digested with EcoRI and HindIII. Plasmid pSUPER/ETS-1 has a cleaved fragment around 360 bp as compared with empty vector that has ϳ300 bp of cleaved fragment. B, inhibition of endogenous ETS-1 in EU-4 cells by transfection of pSUPER/ETS-1 plasmid, and enforced expression of ETS-1 in EU-8 cells by transfection of the ETS-1 expression plasmid. The expression of ETS-1 was examined by Western blot assay, and actin served as control. Controls also contained transfection of empty vectors pSUPER in EU-4 and neo in EU-8. C, co-transfection and luciferase assay for the effect of ETS-1 siRNA on IKK␣ promoter activity in EU-4 cells with a high level of ETS-1 expression. Cells were co-transfected with 5 g of pLuc-1010 construct with either 10 g of pSUPER vector as control (lane 2) or increasing amounts (2.5, 5, and 10 g) of pSUPER/ETS-1 siRNA (lanes 3-5). Controls also include transfection of pLuc-1010 only (lane 1). The total amount of plasmid was adjusted to 15 g/transfection using empty vectors. D, co-transfection and luciferase assay for the effect of enforced ETS-1 expression on IKK␣ promoter activity in EU-8 cells with no endogenous ETS-1 expression. Cells were co-transfected with 5 g of pLuc-1010 construct with either 10 g of neo vector as control (lane 2) or increasing amounts (2.5, 5, and 10 g) of ETS-1 plasmid (lanes 3-5). Transfection and luciferase activity assays in both C and D were performed as described in the legend to Fig. 2. pared with other IKK␣ promoter constructs such as pLuc-168, suggesting that the potential p53-binding sites are dispensable for response to p53-regulated repression. To further investigate the role of the putative p53-binding sites in the IKK␣ promoter in regulating the promoter activity, we performed EMSA as well as CHIP analyses to evaluate the binding capacity of p53 to the IKK␣ promoter. Results from both CHIP (Fig. 6A) and EMSA (data not shown) indicated that p53 did not bind to IKK␣ promoter. In contrast, ETS-1 induced IKK␣ promoter activity by directly binding to the promoter as detected by the CHIP assay (Fig. 6A). Results from EMSA further proved that ETS-1 specifically binds to the DNA sequence spanning Ϫ50/ Ϫ31 of the IKK␣ promoter containing ETS-1 response element (Fig. 6B).
p53 and ETS-1 Interact Physically and Functionally to Regulate IKK␣ Promoter-Previous studies have shown that some genes are repressed by p53 via direct DNA binding, whereas most genes repressed by p53 lack DNA binding. Repression of the latter genes involves interactions of p53 with other transcriptional factors. Our results showed that p53 does not bind to the IKK␣ promoter. Because the IKK␣ promoter is strongly activated by ETS-1, we evaluated whether p53 interacts with ETS-1 and represses IKK␣ promoters through inhibiting the ETS-1 activation. Co-immunoprecipitation assay showed that p53 physically binds to ETS-1 (Fig. 7A), which is consistent with the result reported by Kim et al. (44). We additionally demonstrated that the presence of WT p53 reduced binding of ETS-1 to the IKK␣ promoter, whereas mutant p53 enhanced this binding (Fig. 7B). Furthermore, we compared the promoter activity between construct pLuc-120 with an intact ETS-1 re-sponse consensus and construct pLuc-110 with deletion of the ETS-1 core consensus GGAA in co-transfection with the WT p53 expression plasmid. As shown in Fig. 7C, the promoter activity of pLuc-120 is significantly inhibited by p53, whereas the promoter activity of pLuc-110 is not repressed by p53. DISCUSSION In this study we report the identification and analysis of the promoter region of the IKK␣ gene and the regulation of its expression, hoping to gain insight into the properties of IKK␣ in regulating NF-kB activation in tumor cells. By searching the Human Genome, we identified the nucleotide sequence of the proximal 5Ј-flanking UTR of the IKK␣ gene. When cloned into a luciferase vector and transfected into EU-4 ALL cells, the DNA sequence of 5Ј-flanking UTR of the IKK␣ gene showed considerable promoter activity. By deletion mapping, the core promoter region of the IKK␣ gene was found between Ϫ50 to ϩ1, which was predominantly activated by ETS-1 and negatively regulated by p53. The negative regulation of IKK␣ expression by p53 was further proven by the evidence that induction of WT p53, using a temperature-sensitive p53 mutant transfected in EU-4 cells, repressed both mRNA and protein expression of IKK␣.
It has been known that many genes that are negatively regulated by p53 lack p53 response elements in their promoter. Although the IKK␣ gene promoter has several potential p53 response consensus sequences that are associated with classical p53-binding sites, our results showed that mutations of the putative p53 response elements in the core promoter region did not interfere with p53 repression of IKK␣ promoter activity. Further-FIG. 6. Analyses for binding capacity of p53 and ETS-1 to IKK␣ promoter. A, agarose gel electrophoresis shows the PCR results from each CHIP assay. The IKK␣ promoter construct pLuc-508 and either WT p53 or ETS-1 expression plasmids were cotransfected into EU-4 cells and precipitated with anti-p53 or anti-ETS-1 antibodies (lanes 2 and 5, respectively). For negative control, immunoprecipitation was performed either using a normal mouse or rabbit IgG or in the absence of antibody (no) in each experiment (lanes 3, 6, and 4, 7, respectively). Lane 1 shows DNA size markers. The PCR product (188 bp) in lane 5 contains the ETS-1 binding site in the IKK␣ promoter. B, EMSA to examine the binding of ETS-1 to IKK␣ promoter. Nuclear extracts from EU-1 and EU-4 cells were incubated in binding reactions with 32 P-labeled WT or mutant (mut) probes containing the CCGGAAGT sequence spanning Ϫ46/Ϫ39 of the IKK␣ promoter. Samples were run on a nondenaturing 5% polyacrylamide gel and imaged by autoradiography. Lane 1, labeled mut probe with the EU-1 nuclear extract; lanes 2-5, labeled WT probe with nuclear extracts of EU-1; lanes 6 -8, labeled WT probe with nuclear extracts of EU-4 cells. In reactions depicted in lane 2, 25-fold molar excess of non-labeled WT probe was added. In reactions depicted in lanes 4 and 7, cell extracts were preincubated with 2 g of rabbit IgG for 1 h at 4°C before probes were added. In reactions depicted in lanes 5 and 8, cell extracts were preincubated with 2 g of rabbit anti-ETS-1 antibody. The specific protein-DNA complexes and supershift with antibodies are indicated. more, neither CHIP analysis nor EMSA detected binding of p53 to the IKK␣ promoter. These results indicate that the putative p53 response consensus sequences are dispensable for p53mediated repression of the IKK␣ promoter activity, which is consistent with many previous observations that p53 negatively regulates a gene promoter in the absence of direct DNA binding (40). The mechanism is generally ascribed to sequestration of components of the basal transcription machinery by p53 through protein-protein interaction in the absence of DNA binding (42).
In the present study, we identified that the p53 repression of the IKK␣ gene promoter is through regulation of ETS-1. ETS-1 is a DNA-binding protein that regulates transcription by specific binding to sequence containing a GGAA core usually found in the ETS-1-regulated promoter (50). A previous study found that the DNA sequence CCGGAAGT (ETS1-3) was the most efficient binding motif for purified ETS-1 protein (51). Intriguingly, the IKK␣ core promoter region contains one such sequence (Ϫ46 to Ϫ39) as shown in Fig. 1. Our promoter studies and analysis of ETS-1 DNA binding in vitro indicate that ETS-1 binds to the region containing the ETS1-3 sequence in the IKK␣ promoter and strongly acti-vates IKK␣ promoter activity. Previous studies have demonstrated that ETS-1 and p53 are closely associated proteins. ETS-1 is required for the formation of a stable p53-DNA complex under physiological conditions in UV-induced apoptosis (52). Other studies have reported that p53 inhibits transcriptional activation of thromboxane synthase by binding to the gene promoter and physically interacting with ETS-1 (44), whereas p53 represses the human presenilin-1 gene by interacting with ETS-1 but without direct DNA binding (43). Consistent with the latter study, our results showed that p53 does not bind to IKK␣ promoter and represses IKK␣ promoter activity by physically interacting with ETS-1 and inhibiting ETS-1-mediated activation.
The critical interaction between ETS-1 and p53 in regulating promoter activity is well characterized in our study, in which p53 significantly inhibits the binding of ETS-1 to the IKK␣ promoter and the activity of the IKK␣ promoter construct containing intact ETS-1 binding site but not the construct with deletion of the core GGAA for ETS-1 binding. It has been demonstrated that the tumor suppressor p53 and the protooncogenic factor ETS-1 are important regulators in neoplastic FIG. 7. p53 and ETS-1 physically and functionally interact to regulate the IKK␣ promoter. A, binding of ETS-1 to either endogenous p53 or transfected p53 in ALL cells. Cell lysates from EU-1 treated with IR (10 gray) for 3 h and EU-4 transfected with WT p53 (EU-4/p53) were immunoprecipitated (IP) with antibodies as indicated. Normal mouse or rabbit antibodies served as control (Con). Proteins in immune complexes were separated on denaturing gels, transferred to filters, and detected by Western blotting with anti-MDM2, anti-p53, and anti-ETS-1 antibodies. Antibodies for Western blotting were from different species than those used in IP. B, effect of p53 (both WT and mutant) on ETS-1 binding to the IKK␣ promoter detected by EMSA. The 32 P-labeled oligo-probe from the IKK␣ promoter as described in Fig. 6B was incubated in binding reactions with nuclear extracts from parental EU-4 cells (lane 1), and EU-4 transfected with control plasmid (neo, lane 2) and increasing amounts (1, 5, and 10 g) of mut-p53 (lanes [3][4][5] or WT p53 (lanes 6 -8) expression plasmids. EMSA was performed as described in the legend to Fig. 6B. C, effect of p53 on ETS-1-dependent IKK␣ promoter activity. EU-4 cells were co-transfected with 5 g of either pLuc-120 or pLuc-110 plus increasing amounts (1, 5, and 10 g) of WT p53 expression plasmid (lanes 2 and 6, 3 and 7, 4 and 8, respectively), or without addition of WT p53 (lanes 1 and 5). The total amount of plasmid was adjusted to 15 g/transfection using an empty vector. Transfection and luciferase activity assay were performed as described in the legend to Fig. 2. transformation and cancer progression. The co-regulation of IKK␣, an important activator of NF-kB, by p53 and ETS-1 may represent one of the mechanisms for p53-and ETS-1-mediated cancer formation and progression.
In our study, the inhibitory effect of p53 on IKK␣ promoter activity was abrogated by co-transfection of the p53-inhibitory oncogene MDM2. Furthermore, transfection of mutant p53 genes activated IKK␣ promoter activity. The molecular mechanism by which mutant p53 is able to directly up-regulate expression of a number of genes in contrast to the down-regulation of these genes by WT p53 has not been completely understood. Our results showed that enforced expression of mutant p53 increased ETS-1 DNA binding to the IKK␣ promoter, suggesting that mutant p53 proteins may gain a function to activate IKK␣ via interacting with ETS-1. These results further support the involvement of p53 regulation of IKK␣ expression in tumorigenesis and in development of resistance to chemotherapy-induced apoptosis. Constitutive NF-kB activation frequently occurs in cancer and leukemia cells, which is not always associated with increased degradation of IkB. We were prompted to study the p53 regulation of IKK␣ expression by our preliminary observation that several ALL cell lines with the p53 mutation expressed constitutive NF-kB, whereas ALL lines with the WT p53 phenotype usually lacked constitutive NF-kB activation. 2 We hypothesized that p53 may have a role in controlling NF-kB activation by regulating IKK␣. Results from this study support this hypothesis.
Our present study demonstrates that WT p53 represses IKK␣ expression at the transcriptional level. Loss of p53 function either by mutation or by overexpression of MDM2, which frequently occurs in cancer and leukemia, will release the repression of IKK␣, ultimately resulting in constitutive NF-kBmediated gene activation. Thus, transcriptional induction of IKK␣ may represent a novel pathway through which loss of p53 may contribute to tumorigenesis.