A Splice Variant of Stress Response Gene ATF3 Counteracts NF-κB-dependent Anti-apoptosis through Inhibiting Recruitment of CREB-binding Protein/p300 Coactivator
2005; Elsevier BV; Volume: 281; Issue: 3 Linguagem: Inglês
10.1074/jbc.m508471200
ISSN1083-351X
AutoresBayin Hua, Mimi Tamamori‐Adachi, Yang Luo, Kiyoshi Tamura, M. Morioka, Mizue Fukuda, Yujiro Tanaka, Shigetaka Kitajima,
Tópico(s)RNA regulation and disease
ResumoActivating transcription factor (ATF) 3 plays a role in determining cell fate and generates a variety of alternatively spliced isoforms in stress response. We have reported previously that splice variant ATF3ΔZip2, which lacks the leucine zipper region, is induced in response to various stress stimuli. However, its biological function has not been elucidated. By using cells treated with tumor necrosis factor-α and actinomycin D or cells overexpressing ATF3ΔZip2, we showed that ATF3ΔZip2 sensitizes cells to apoptotic cell death in response to tumor necrosis factor-α, at least in part through suppressing nuclear factor (NF)-κB-dependent transcription of anti-apoptotic genes such as cIAP2 and XIAP. ATF3ΔZip2 interacts with a p65 (RelA)-cofactor complex containing CBP/p300 and HDAC1 at NF-κB sites of the proximal promoter region of the cIAP2 gene in vivo and down-regulates the recruitment of CBP/p300. Our study revealed that ATF3ΔZip2 counteracts anti-apoptotic activity of NF-κB, at least in part, by displacing positive cofactor CBP/p300 and provides insight into the mechanism by which ATF3 regulates cell fate through alternative splicing in stress response. Activating transcription factor (ATF) 3 plays a role in determining cell fate and generates a variety of alternatively spliced isoforms in stress response. We have reported previously that splice variant ATF3ΔZip2, which lacks the leucine zipper region, is induced in response to various stress stimuli. However, its biological function has not been elucidated. By using cells treated with tumor necrosis factor-α and actinomycin D or cells overexpressing ATF3ΔZip2, we showed that ATF3ΔZip2 sensitizes cells to apoptotic cell death in response to tumor necrosis factor-α, at least in part through suppressing nuclear factor (NF)-κB-dependent transcription of anti-apoptotic genes such as cIAP2 and XIAP. ATF3ΔZip2 interacts with a p65 (RelA)-cofactor complex containing CBP/p300 and HDAC1 at NF-κB sites of the proximal promoter region of the cIAP2 gene in vivo and down-regulates the recruitment of CBP/p300. Our study revealed that ATF3ΔZip2 counteracts anti-apoptotic activity of NF-κB, at least in part, by displacing positive cofactor CBP/p300 and provides insight into the mechanism by which ATF3 regulates cell fate through alternative splicing in stress response. Alternative splicing of pre-mRNAs encoding transcription factors is one of the common mechanisms for generating the complexity and diversity of gene regulation (1Lopez A.J. Annu. Rev. Genet. 1998; 32: 279-305Crossref PubMed Scopus (534) Google Scholar). A variety of functionally distinct isoforms is generated from a single gene by use of different combinations of splice junctions, and may play role in coordinating gene regulation in response of cells to various environmental stimuli. However, the altered gene regulatory function and the biological implication of spliced variants still remain elusive.Activating transcription factor (ATF) 3The abbreviations used are: ATFactivating transcription factorTNF-αtumor necrosis factor-αCBPcyclic AMP-response element-binding protein (CREB)-binding proteinHDAChistone deacetylasep/CAFCBP-associated factorChIPchromatin immunoprecipitationGFPgreen fluorescence proteinPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideGSTglutathione S-transferaseDAPI4′,6-diamidino-2-phenylindole dihydrochlorideGFPgreen fluorescent proteinm.o.i.multiplicity of infectionGAPDHglyceraldehyde-3-phosphate dehydrogenaseRTreverse transcriptionsiRNAsmall interfering RNANF-κBnuclear factor-κB. 3The abbreviations used are: ATFactivating transcription factorTNF-αtumor necrosis factor-αCBPcyclic AMP-response element-binding protein (CREB)-binding proteinHDAChistone deacetylasep/CAFCBP-associated factorChIPchromatin immunoprecipitationGFPgreen fluorescence proteinPBSphosphate-buffered salinePMSFphenylmethylsulfonyl fluorideGSTglutathione S-transferaseDAPI4′,6-diamidino-2-phenylindole dihydrochlorideGFPgreen fluorescent proteinm.o.i.multiplicity of infectionGAPDHglyceraldehyde-3-phosphate dehydrogenaseRTreverse transcriptionsiRNAsmall interfering RNANF-κBnuclear factor-κB. 3 is a member of the ATF/CREB family of basic leucine zipper-type transcription factors. It is induced upon exposure of cells to a variety of physiological and pathological stimuli (2Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expr. 1999; 7 (and references therein): 321-335PubMed Google Scholar), and it is thought to have cell-detrimental effects, such as cell cycle arrest and apoptosis (2Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expr. 1999; 7 (and references therein): 321-335PubMed Google Scholar, 3Yin T. Sandhu G. Wplfgang C.D. Burrier A. Webb R.L. Rigel D.F. Hai T. Whelan J. J. Biol. Chem. 1997; 272: 19943-19950Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 4Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar, 5Kang Y. Chen C.R. Massague J. Mol. Cell. 2003; 11: 915-926Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). Ectopic expression of ATF3 in heart, liver, and pancreatic β-cells causes cardiac enlargement, liver cell dysfunction, and diabetes, respectively (6Okamoto Y. Chaves A. Chen J. Kelley R. Jones K. Weed H.G. Gardner K.L. Gangi L. Yamaguchi M. Klomkleaw W. Nakayama T. Hamlin R.L. Carnes C.A. Altschuld R.A. Bauer J.A. Hai T. Am. J. Pathol. 2001; 159: 639-650Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 7Allen-Jennings A.E. Hartman M.G. Kociba G.J. Hai T. J. Biol. Chem. 2001; 276: 29507-29514Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 8Hartman M.G. Lu D. Kim M.L. Kociba G.J. Shukri T. Buteau J. Wang X. Frankel W.L. Guttridge D. Prentki M. Grey S.T. Ron D. Hai T. Mol. Cell. Biol. 2004; 24: 5721-5732Crossref PubMed Scopus (253) Google Scholar), supporting cytopathic activity of ATF3. On the other hand, ATF3 is also rapidly induced in the regenerating liver (9Hsu J.-C. Laz T. Mohn K.L. Taub R. Proc. Natl. Acad. Sci. U. S. 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Tanaka Y. Nakabeppu Y. Sunamori M. Sedivy J.M. Kitajima S. EMBO J. 2005; 24: 2590-2601Crossref PubMed Scopus (90) Google Scholar). Thus, ATF3 may function differently depending on the cellular context.ATF3 is composed of 181 amino acids, and the basic region and leucine zipper domain from 88 to 147 amino acids are required for dimer formation and specific DNA binding (14Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar, 15Hsu J.-C. Bravo R. Taub R. Mol. Cell. Biol. 1992; 12: 4654-4665Crossref PubMed Scopus (157) Google Scholar). The homodimer of ATF3 represses transcription from various promoters with ATF sites (14Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar, 15Hsu J.-C. Bravo R. Taub R. Mol. Cell. Biol. 1992; 12: 4654-4665Crossref PubMed Scopus (157) Google Scholar, 16Wolfgang C.D. Chen B.P.C. Martindale J.L. Holbrook N.J. Hai T. Mol. Cell. Biol. 1997; 17: 6700-6707Crossref PubMed Scopus (140) Google Scholar), whereas heterodimers with c-Jun or JunB activate transcription (15Hsu J.-C. Bravo R. Taub R. Mol. Cell. Biol. 1992; 12: 4654-4665Crossref PubMed Scopus (157) Google Scholar). In addition to the heteromeric complexity, various spliced isoforms of ATF3 may further generate functional diversity in different cellular context. ATF3ΔZip has been isolated in serum-stimulated HeLa cells (14Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar), whereas ATF3ΔZip2c and ΔZip3 were identified in amino acid-deprived cells (17Pan Y. Chen H. Siu F. Kilberg M.S. J. Biol. Chem. 2003; 278: 38402-38412Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Another isoform, ATF3b, is implicated in mediating cAMP signaling of proglucagon transcription in pancreatic α-cells (18Wang J. Cao Y. Steiner D.F. J. Biol. Chem. 2003; 278: 32899-32904Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). We have isolated previously ATF3ΔZip2a and -b from cells treated with various stimuli such as A23187, TNF-α, endoplasmic reticulum stress, or oxidative stress (19Hashimoto Y. Zhang C. Kawauchi J. Imoto I. Adachi M.T. Inazawa J. Amagasa T. Hai T. Kitajima S. Nucleic Acids Res. 2002; 30: 2398-2406Crossref PubMed Scopus (68) Google Scholar). These two isoforms encode the C-terminally truncated protein of 135 amino acids, which shares the N-terminal 116 amino acids with the full-length ATF3 but contains novel 19 amino acids at the C terminus. ATF3ΔZip2 lacks the leucine zipper domain and thus is capable of DNA binding. It is localized in the nucleus and counteracts the transcriptional regulation by full-length ATF3 in a reporter assay (19Hashimoto Y. Zhang C. Kawauchi J. Imoto I. Adachi M.T. Inazawa J. Amagasa T. Hai T. Kitajima S. Nucleic Acids Res. 2002; 30: 2398-2406Crossref PubMed Scopus (68) Google Scholar). However, the functional role and biological significance of this spliced isoform in stress response are unknown.Nuclear factor-κB (NF-κB) is a transcription factor that plays a critical role in the expression of genes involved in immune and inflammatory responses (20Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5541) Google Scholar, 21Karin M. Ben-Nerish Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4043) Google Scholar). It is located in the cytoplasm of nonstimulated cells as a form bound to IκB, but it is released by degradation of IκBα and enters the nucleus in response to stimuli. There are five known members of the mammalian NF-κB/Rel family: p65 (RelA), c-Rel, RelB, p50, and p52. p65, RelB, and c-Rel are transcriptionally active, whereas p50 and p52 function as the DNA-binding subunits. 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NF-κB has also been shown to inhibit apoptosis by DNA-damaging agents, including cancer therapeutics (29Wang C.-Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2499) Google Scholar, 30Baldwin A.S. J. Clin. Investig. 2001; 107: 241-246Crossref PubMed Scopus (1188) Google Scholar).It is well known that NF-κB-dependent transcription requires the recruitment of multiple coactivator proteins. CBP and its homologue p300 interact with the p65 subunit of NF-κB to activate responsive gene promoters (31Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (709) Google Scholar, 32Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar), and this interaction is enhanced by inducible phosphorylation of p65 (33Zhong H. May M.J. Jimi E. Ghosh S. Mol. Cell. 2002; 9: 625-636Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar). The histone acetyltransferase activity of p/CAF is also required for the NF-κB-activated transcription (34Sheppard K.A. Rose D.W. Haque Z.K. Kurokawa R. McInerney E. Westin S. Thanos D. Rosenfeld M.G. Glass C.K. Collins T. Mol. Cell. Biol. 1999; 19: 6367-6378Crossref PubMed Google Scholar), whereas steroid receptor coactivator-1 interacts with the p50 subunit of NF-κB to potentiate the transcription (35Na S.Y. Lee S.K. Han S.J. Choi H.S. Im S.Y. Lee J.W. J. Biol. Chem. 1998; 273: 10831-10834Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Inversely, HDAC1/2 (36Ashburner B.P. Westerheide S.D. Baldwin Jr., A.S. Mol. Cell. Biol. 2001; 21: 7065-7077Crossref PubMed Scopus (621) Google Scholar), SNIP1 (37Kim R.H. Flanders K.C. Reffey S.B. Anderson L.A. J. Biol. Chem. 2001; 276: 46297-46304Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and PIAS3 (38Jang H.D. Yoon K. Shin Y.J. Kim J. Lee S.Y. J. Biol. Chem. 2004; 279: 24873-24880Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) interact with p65 to negatively regulate gene expression. More recently, the candidate tumor suppressor gene product ING4 has been reported to interact with p65 and suppress the NF-κB-dependent anti-apoptosis (39Garkavtsev I. Kozin S.V. Chernova O. Xu L. Winkler F. Brown E. Barnett G.H. Jain R.K. Nature. 2004; 428: 328-332Crossref PubMed Scopus (311) Google Scholar). Thus, the differential binding of p65 with coactivators or repressors determines the extent of activation or repression of NF-κB-dependent gene expression. However, our knowledge is still limited about what molecule(s) modulate NF-κB-dependent transcription in stress response.In this report, we explored the functional role of ATF3ΔZip2 by using TNF-α-stimulated cells. It was demonstrated that ATF3ΔZip2, but not full-length ATF3, sensitizes cells to apoptotic cell death, partly by suppressing the expression of NF-κB-dependent anti-apoptotic genes cIAP2 and XIAP. ATF3ΔZip2 is further shown to bind directly to the p65 subunit of NF-κB and down-regulate the CBP/p300 recruitment. Our study provides evidence that ATF3ΔZip2, which is generated through stress-activated alternative splicing, represses NF-κB activity and may play pro-apoptotic role in stress response.EXPERIMENTAL PROCEDURESPlasmids, Antibodies, and Reagents—A mammalian expression plasmid pMEFLAGATF3ΔZip2 and a bacterial expression plasmid encoding the GST fusion of ATF3ΔZip2 were as described (19Hashimoto Y. Zhang C. Kawauchi J. Imoto I. Adachi M.T. Inazawa J. Amagasa T. Hai T. Kitajima S. Nucleic Acids Res. 2002; 30: 2398-2406Crossref PubMed Scopus (68) Google Scholar). Mammalian expression vectors for the p65 and p50 subunits of NF-κB were generous gifts from Dr. Fujita at the Tokyo Metropolitan Institute, Tokyo, Japan. Plasmid vector for expressing p65 in Escherichia coli was constructed by subcloning into pET21a (Novagen). Expression plasmids, pCI-cIAP2 and pCI-XIAP, were prepared by subcloning cDNAs encoding human cIAP2 (GenBank™ accession number NM182962) and XIAP (GenBank™ accession number NM001167) into the pCIneo vector (Clontech), respectively. A bacterial expression plasmid pGST-CBP2N encoding a GST fusion of the N-terminal 117-738 amino acids of CBP and a mammalian expression vector pcDNA3/mCBP-HA for mouse CBP (40Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 41Araya N. Hirota K. Shimamoto Y. Miyagishi M. Yoshida E. Ishida J. Kaneko S. Kaneko M. Nakajima T. Fukamizu A. J. Biol. Chem. 2003; 278: 5427-5432Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) were generous gifts from Dr. Nakajima, St. Marianna University School of Medicine, Tokyo, Japan. Rabbit anti-ATF3 (C-19), anti-ATF2 (N-96), anti-p65, anti-p50, anti-IκBα (FL), anti-cIAP2 (H-85), anti-CBP (C-20), anti-p300 (C-20), goat anti-HDAC1 (C-19), and monoclonal anti-GST (B-14) antibodies were purchased from Santa Cruz Biotechnology. Monoclonal anti-FLAG M2 and anti-β-tubulin antibodies were from Sigma. Anti-ATF3 antibody was also generated by immunizing rabbits with full-length ATF3 expressed in E. coli. Recombinant human TNF-α was purchased from Genzyme. Other chemicals were reagent grade.Cell Culture and Transient Expression—TGR-1 is a subclone of the immortalized rat embryonic Rat-1 cell line, and U2OS is a human osteosarcoma cell line, respectively. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a 5% CO2 atmosphere at 37 °C. For transient expression, plasmid DNA was vortex-mixed with SuperFect (Qiagen) and transfected into cells according to the manufacturer's instruction.Adenovirus Preparation and Gene Transfer—An adenovirus vector AdATF3 encoding the full-length ATF3 was as described (11Kawauchi J. Zhang C. Nobori K. Hashimoto Y. Adachi M.T. Noda A. Sunamori M. Kitajima S. J. Biol. Chem. 2002; 277: 39025-39034Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and a vector AdATF3ΔZip2 for FLAG-tagged ATF3ΔZip2 was constructed as in a protocol of an adenovirus expression vector kit from Takara (Otsu, Japan). Briefly, a blunt-ended cDNA fragment of ATF3ΔZip2 was subcloned into the Swa1 site of the E1-deleted region of a cassette cosmid vector pAxCAwt and was cotransfected into 293 cells with DNA-terminal protein complex (42Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (786) Google Scholar). Cells expressing recombinant viruses for FLAG-tagged ATF3ΔZip2 were screened by anti-FLAG immunoblot analysis and cloned by a limiting dilution. An adenovirus vector encoding β-galactosidase (AdLacZ) was a generous gift from Dr. Saito of Tokyo University, Japan. These adenoviruses were plaque-purified, and their titers were determined by titration in 293 cells. For adenovirus-mediated gene transfer, cells were exposed to adenoviral vectors at the indicated multiplicity of infection (m.o.i.) and were cultured for the specified times.Isolation of U2OS Cells Stably Expressing ATF3ΔZip2 siRNA—An oligonucleotide that targets the C-terminal amino acid regions unique for human ATF3ΔZip2 from 126 to 132, 5′-ACTTGCATGTGGGCTGTTGGC-3′, was subcloned into pMX-puroII-U6 (43Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar). The resulting vector was transfected into Plat E cells, and recombinant retrovirus was generated. U2OS cells stably expressing mouse ecotropic retrovirus receptor were infected with the recombinant retrovirus and were cultured in medium containing 20 μg/ml puromycin for 4 days.Cell Death Assay—TGR1 cells (6 × 104 cells) that were infected with AdATF3ΔZip2, AdATF3, or AdLacZ virus at 25 m.o.i. for 24 h or U2OS cells (1 × 105 cells) were treated with the indicated concentration of TNF-α for 36 h or 10 ng/ml TNF-α and the indicated amount of actinomycin D for 18 h, respectively. Both attached and floating cells were combined and stained with 0.2% trypan blue. Viable cells were counted and expressed as a proportion of total cells as described before (11Kawauchi J. Zhang C. Nobori K. Hashimoto Y. Adachi M.T. Noda A. Sunamori M. Kitajima S. J. Biol. Chem. 2002; 277: 39025-39034Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Alternatively, cells (6 × 104 cells) were transfected with indicated the amounts of pMEFLAGATF3ΔZip2, pCMVGFP, and effector plasmids. At 16 h post-transfection, cells were treated with 20 ng/ml TNF-α and incubated for another 36 h. Cells were then examined for GFP expression and nuclear fragmentation by DAPI staining under a fluorescence microscope (Nikon). Cell death was quantitated as a fraction of cells with fragmented nuclei per GFP-positive cells. For detection of DNA ladder formation, both attached and floating cells were collected 36 h after TNF-α treatment. Their chromosomal DNA was extracted using a kit from Wako (Osaka, Japan), separated on a 1.8% agarose gel, and visualized by staining with ethidium bromide.Cell Extract Preparation and Western Blot Analysis—TGR1 or U2OS cells treated as indicated were harvested, washed in PBS, and resuspended in lysis buffer (50 mm Hepes-KOH, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA, 0.1 mm PMSF, 10 μg/ml each leupeptin and aprotinin, 200 μm sodium vanadate, 100 mm NaF, and 10% glycerol). After incubation on ice for 10 min, the cells were centrifuged at 10,000 rpm for 10 min, and the supernatants were taken as whole cell extract. The amounts of protein were measured by the Lowry method using bovine serum albumin as standard (44Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Cell extracts (20 μg of protein) were separated on an SDS-PAGE, transferred onto a nitrocellulose membrane, and subjected to Western blot using the protocol of ECL kit (Amersham Biosciences).Nuclear Translocation of NF-κB—For nuclear translocation of NF-κB, U2OS cells (1 × 106 cells) were infected with AdATF3ΔZip2 or AdLacZ at 25 m.o.i. for 24 h and then treated with 20 ng/ml TNF-α. At the time points indicated after TNF-α treatment, cells were harvested and suspended in buffer of 10 mm Hepes-KOH, pH 7.8, 10 mm KCl, 0.1 mm EDTA, 1 mm dithiothreitol, 1 mm PMSF, and 0.1% Nonidet P-40. Cytoplasmic fraction was obtained by centrifugation of cells at 10,000 rpm for 10 min. The resultant pellet containing nuclei was washed by the above buffer, dissolved in sample buffer for SDS-PAGE, and subjected to Western blot analysis.Electrophoretic Mobility Shift Assay—U2OS cells (1.5 × 107 cells) were infected with 25 m.o.i. of AdATF3ΔZip2 or AdLacZ for 24 h and then treated with 20 ng/ml TNF-α for 3 h. Nuclear extracts were then prepared according to the method of Dignam et al. (45Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9142) Google Scholar). Nuclear extracts (2 μg of protein) were incubated in 20 μl of binding buffer (10 mm Hepes-KOH, pH 7.9, 60 mm KCl, 0.5 mm EDTA, 5 mm MgCl2, 0.1 mm PMSF, 5 mm β-mercaptoethanol) containing 0.5 mg of poly(dI-dC) and 0.5 ng of radiolabeled DNA probe at room temperature for 30 min. For supershift assays, anti-p65 or anti-p50 antibody (0.1 μg each) was added and incubated for another 30 min. An oligonucleotide DNA probe for consensus NF-κB motif (sc-2505) 5′-AGTTGAGGGGACTTTCCCAGGC-3′ was obtained from Santa Cruz Biotechnology and radiolabeled with 25 μCi of [γ-32P]ATP (6000 Ci/mmol) and polynucleotide kinase. Mutant oligonucleotide (sc-2511) used for the competition experiment was also product of Santa Cruz Biotechnology. Binding mixture was applied onto a 5% nondenatured polyacrylamide slab gel in Tris borate-EDTA buffer. After electrophoresis, the gel was dried on a 3MM Whatman paper and visualized by Fuji Bas 2500 image analyzer.RNA Isolation, Microarray Analysis, and Reverse Transcription (RT)-PCR—TGR1 cells (1.5 × 106 cells) were infected with AdATF3ΔZip2 or AdLacZ at 25 m.o.i. for 24 h and treated with 20 ng/ml TNF-α for 3 h. Total RNA was then isolated by acid-guanidinium method using a kit from Qiagen. After amplification and labeling of cRNA with Cy3 or Cy5, microarray analysis was performed using custom-made in situ synthesized 60-mer oligonucleotide microarrays containing 22,500 features, including controls (Agilent Technologies). Semi-quantitative measurements of mRNAs for cIAP2, XIAP, TRAF2, FLIP, or GAPDH were performed by RT-PCR of 1 μg of total RNA using a kit from Takara as described in Ref. 19Hashimoto Y. Zhang C. Kawauchi J. Imoto I. Adachi M.T. Inazawa J. Amagasa T. Hai T. Kitajima S. Nucleic Acids Res. 2002; 30: 2398-2406Crossref PubMed Scopus (68) Google Scholar. Primers used were as follows: cIAP2, 5′-CTCAGTATGCAGACACACTCTG-3′ and 5′-TGAGGTGTCTGAAGTGGACAAC-3′; XIAP, 5′-TCAGAGCACAGGAGACACTTTC-3′ and 5′-CTGGATACCACTTAGCATGCTG-3′; TRAF2, 5′-GTTACAGCGGTGCCAGATTTTG-3′ and 5′-GAAGTCGGAGATCTTCCAGATG-3′; FLIP, 5′-TGCCTGAAGAGCATCCACAGA-3′ and 5′-TCCACAGTAGTCATGCCTAGGT-3′; and GAPDH, 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and 5′-CATGTGGGCCATGAGGTCCACCAC-3′. Reaction products were separated on 2% agarose gel and stained with ethidium bromide.Luciferase Assay—Reporter plasmid, pLuc-cIAP2, was constructed by subcloning a 0.6-kb fragment of the human cIAP2 gene promoter (GenBank™ accession number AF233684) from -538 to +55 into an XhoI site of pGL3 vector (Promega). NF-κB-dependent reporter, pNFκB-Luc, which contains four tandem copies of the NF-κB consensus motif fused to a TATA-like promoter from the herpes simplex virus thymidine kinase promoter, was obtained from Clontech. U2OS cells (5 × 104 cells) were transfected with 0.5 μg of pLuc-cIAP2 or pNFκB-Luc and the indicated amounts of effector plasmids. At 16 h post-transfection, cells were stimulated with 10 ng/ml TNF-α for 24 h. Cells were then harvested, and their extracts were assayed for luciferase activity as described (19Hashimoto Y. Zhang C. Kawauchi J. Imoto I. Adachi M.T. Inazawa J. Amagasa T. Hai T. Kitajima S. Nucleic Acids Res. 2002; 30: 2398-2406Crossref PubMed Scopus (68) Google Scholar). Both firefly and seapansy luciferase activities were measured using a dual luciferase reporter assay system according to the manufacturer's protocol (Promega). pRL-TK (Toyo Ink, Tokyo, Japan) containing the seapansy luciferase gene was used as an internal control of transfection and expression.Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed as described (13Tamura K. Hua B. Adachi S. Guney I. Kawauchi J. Morioka M. Adachi T.M. Tanaka Y. Nakabeppu Y. Sunamori M. Sedivy J.M. Kitajima S. EMBO J. 2005; 24: 2590-2601Crossref PubMed Scopus (90) Google Scholar) according to the protocol supplied by Upstate (Charlottesville, VA). U2OS cells (4 × 107 cells) were infected with AdATF3ΔZip2 at 25 m.o.i. for 24 h and treated with 20 ng/ml TNF-α. At 8 h post-stimulation, cells were cross-linked with 1% formaldehyde for 10 min at room temperature. After washing twice with PBS, the cells were collected and lysed with 2 ml of SDS lysis buffer (50 mm Tris-HCl, pH 8.1, 10 mm EDTA, 1% SDS) containing a protease inhibitor mixture (Roche Applied Science), and sonicated to DNA lengths of ∼250 bp. After centrifugation at 13,000 rpm for 10 min, supernatants were diluted 10-fold in ChIP dilution buffer (16.7 mm Tris-HCl, pH 8.1, 167 mm NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA). Immunoprecipitations were then performed with the indicated antibodies. ChIP DNA was detected using standard PCR with the following primer pairs for the different regions of the cIAP2 gene promoter: the proximal promoter region at -260 to +55 5′-GTAAATGCCGCGAAGATATGCCAC-3′ and 5′-GCATGCACCAGCAAGGACAAG-3′ and the distal promoter region at -538 to -260 5′-CCTTTCACCTCTTACTTTCTTG-3′ and 5′-GTGGCATATCTTCGCGGCATTTAC-3′.Binding Assay of Zip2 Complex—For in vivo binding, U2OS cells (1 × 107 cells) were infected with AdATF3ΔZip2 at 25 m.o.i. for 24 h and then treated with 20 ng/ml TNF-α for 3 h. Alternatively, U2OS cells stably expressing ATF3ΔZip2 siRNA or control GFP siRNA were treated with 10 ng/ml TNF-α and 0.05 μg/ml actinomycin D for 3 h. Whole cell extracts were prepared and incubated with 0.5 μg each of anti-p65 or anti-FLAG antibody at 4 °C for 3 h, followed by incubation with 30 μl of protein G-Sepharose (Amersham Biosciences) for 2 h. The resulting immunocomplex was washed and subjected to Western blot analysis. For in vitro binding, full-length ATF3 and ATF3ΔZip2 were expressed as GST fusion as described (19Hashimoto Y. Zhang C. Kawauchi J. Imoto I. Adachi M.T. Inazawa J. Amagasa T. Hai T. Kitajima S. Nucleic Acids Res. 2002; 30: 2398-2406Crossref PubMed Scopus (68) Google Scholar). For deletion mutants of ATF3, cDNA fragments encoding 1-40-, 1-88-, and 1-147-a
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