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• W2042263540 abstract "Infections of bacteria and viruses induce host defense reactions known as innate responses including the activation of interferon regulatory factor-3 (IRF-3), critical for the activation of type I interferon system. Upon immediate early signals triggered by the infection, IRF-3 is phosphorylated and a homodimer results. The homodimer complexes with the coactivator CREB-binding protein (CBP)/p300 in the nucleus; thus, holocomplex of IRF-3 competent in DNA binding is generated. We showed CBP/p300 to be indispensable for the DNA binding activity of the holocomplex and to aid the binding through direct interaction with the DNA. We demonstrated that p300 binds with the IRF-3 homodimer via a Q-rich domain and that an intact histone acetyltransferase (HAT) domain is indispensable for the DNA binding of the holocomplex along with a CH3 domain, which connects the HAT and Q-rich domains. These results highlight a novel function of CBP/p300: direct involvement in sequence-specific DNA binding. Furthermore, the critical function of these domains in virus-induced gene activation was demonstrated in vivo by using p300 mutants. Infections of bacteria and viruses induce host defense reactions known as innate responses including the activation of interferon regulatory factor-3 (IRF-3), critical for the activation of type I interferon system. Upon immediate early signals triggered by the infection, IRF-3 is phosphorylated and a homodimer results. The homodimer complexes with the coactivator CREB-binding protein (CBP)/p300 in the nucleus; thus, holocomplex of IRF-3 competent in DNA binding is generated. We showed CBP/p300 to be indispensable for the DNA binding activity of the holocomplex and to aid the binding through direct interaction with the DNA. We demonstrated that p300 binds with the IRF-3 homodimer via a Q-rich domain and that an intact histone acetyltransferase (HAT) domain is indispensable for the DNA binding of the holocomplex along with a CH3 domain, which connects the HAT and Q-rich domains. These results highlight a novel function of CBP/p300: direct involvement in sequence-specific DNA binding. Furthermore, the critical function of these domains in virus-induced gene activation was demonstrated in vivo by using p300 mutants. double-stranded RNA bromodeoxyuridine chloramphenicol acetyltransferase cAMP-responsive element-binding protein CREB-binding protein deoxycholate electrophoretic mobility shift assay glutathione S-transferase glutathione hemagglutinin histone acetyltransferase interferon interferon regulatory factor interferon-stimulated response element Newcastle disease virus nuclear export signal Toll like receptor Bacterial and viral infections induce a series of host responses known collectively as innate immunity, in which a set of genes encoding proteins crucial to the primary phase of host defense are activated. Infection by a virus or treatment with double-stranded RNA (dsRNA)1 induces the activation of an array of genes including the gene for type I interferon (IFN-α and -β) in various cell types (1DeMaeyer E. DeMaeyer-Guignard J. Interferons and Other Regulatory Cytokines. John Wiley & Sons, New York1988: 1-153Google Scholar, 2Sen G.C. Lengyel P. J. Biol. Chem. 1992; 267: 5017-5020Abstract Full Text PDF PubMed Google Scholar). In certain cells such as macrophages, however, bacterial endotoxin triggers a signal leading to the activation of type I IFN genes along with other cytokine genes. Type I IFN genes are directly activated by the immediate early response, and the secreted IFN expands the response by activating additional sets of genes involved in antiviral responses and the modulation of cellular functions. Therefore, the activation of type I IFN genes is crucial to the amplification of the signal triggering late responses. A very low basal expression and a rapid reversal to efficient expression after activation are characteristics of type I IFN genes. It has been shown that the versatile transcription factors NF-κB, ATF-2, and c-Jun are involved in the activation of the IFN-β gene (3Du W. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2150-2154Crossref PubMed Scopus (96) Google Scholar, 4Du W. Thanos D. Maniatis T. Cell. 1993; 74: 887-898Abstract Full Text PDF PubMed Scopus (396) Google Scholar, 5Fujita T. Miyamoto M. Kimura Y. Hammer J. Taniguchi T. Nucleic Acids Res. 1989; 17: 3335-3346Crossref PubMed Scopus (77) Google Scholar). However, when these factors were activated by nonviral/dsRNA stimuli such as tumor necrosis factor-α or interleukin-1, no significant activation of the IFN-β gene was observed, indicating that activation of these factors alone are not sufficient (6Fujita T. Reis L.F. Watanabe N. Kimura Y. Taniguchi T. Vilcek J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9936-9940Crossref PubMed Scopus (250) Google Scholar, 7Watanabe N. Sakakibara J. Hovanessian A.G. Taniguchi T. Fujita T. Nucleic Acids Res. 1991; 19: 4421-4428Crossref PubMed Scopus (177) Google Scholar). Promoter analysis of IFN-α and -β revealed the involvement of the interferon regulatory factor (IRF) family of proteins (8Yoneyama M. Suhara W. Fukuhara Y. Sato M. Ozato K. Fujita T. J. Biochem. (Tokyo). 1996; 120: 160-169Crossref PubMed Scopus (119) Google Scholar). IRF-1–9 contain a conserved DNA binding domain at their N termini and potentially bound to the IRF motif in the promoter (9Taniguchi T. Ogasawara K. Takaoka A. Tanaka N. Annu. Rev. Immunol. 2001; 19: 623-655Crossref PubMed Scopus (1258) Google Scholar). Although some of the IRFs specifically bound to the promoter of the IFN-β gene or a synthetic IRF element in vitro, the exogenous expression of these IRFs is not sufficient to activate the gene as efficiently as viral infection (10Fujita T. Kimura Y. Miyamoto M. Barsoumian E.L. Taniguchi T. Nature. 1989; 337: 270-272Crossref PubMed Scopus (315) Google Scholar). Studies from different laboratories using dominant negative mutants, gene disruption techniques, and specific ribozymes show that IRF-3 plays a critical role in the viral induction of type I IFN genes (11Sato M. Suemori H. Hata N. Asagiri M. Ogasawara K. Nakao K. Nakaya T. Katsuki M. Noguchi S. Tanaka N. Taniguchi T. Immunity. 2000; 13: 539-548Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 12Yeow W.S., Au, W.C. Lowther W.J. Pitha P.M. J. Virol. 2001; 75: 3021-3027Crossref PubMed Scopus (30) Google Scholar, 13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar). IRF-3 is expressed ubiquitously and accumulated in cytoplasm in an inactive form. Viral infection or treatment with dsRNA triggers a signal, which results in the specific phosphorylation of serine residues of IRF-3 (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar, 14Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar, 15Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (296) Google Scholar). The phosphorylated IRF-3 becomes a homodimer and then forms a complex with the coactivators CBP/p300 in the nucleus (16Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (265) Google Scholar, 17Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (65) Google Scholar). This holocomplex of IRF-3 is conferred DNA binding activity for the IRF motif and possibly the potential to initiate gene activation (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar, 16Lin R. Mamane Y. Hiscott J. Mol. Cell. Biol. 1999; 19: 2465-2474Crossref PubMed Scopus (265) Google Scholar, 18Sato M. Tanaka N. Hata N. Oda E. Taniguchi T. FEBS Lett. 1998; 425: 112-116Crossref PubMed Scopus (224) Google Scholar, 19Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar). CBP/p300 interacts with various DNA-binding transcription factors with its respective domains and forms a multimeric complex on promoter DNA (20Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1359) Google Scholar). Because CBP and p300 are histone acetyltransferases, their involvement in the activation of nucleosomal loci by histone acetylation is suggested (21Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6448) Google Scholar). In vitro, CBP/p300 does not significantly alter the DNA binding affinity or specificity of the DNA-binding transcription factors, except in some cases (for example p53 and c-Myb) where the DNA binding is modulated by acetylation (22Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2144) Google Scholar, 23Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (645) Google Scholar, 24Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1010) Google Scholar, 25Tomita A. Towatari M. Tsuzuki S. Hayakawa F. Kosugi H. Tamai K. Miyazaki T. Kinoshita T. Saito H. Oncogene. 2000; 19: 444-451Crossref PubMed Scopus (109) Google Scholar). It has been observedin vitro that the complex of IRF motif DNA and the activated IRF-3 invariably associates with CBP/p300, suggesting an unusually high affinity of CBP/p300 for the phosphorylated IRF-3 and/or a direct association of CBP/p300 with the DNA in the context of an IRF-3 holocomplex (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar, 15Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (296) Google Scholar, 18Sato M. Tanaka N. Hata N. Oda E. Taniguchi T. FEBS Lett. 1998; 425: 112-116Crossref PubMed Scopus (224) Google Scholar, 26Kumar K.P. McBride K.M. Weaver B.K. Dingwall C. Reich N.C. Mol. Cell. Biol. 2000; 20: 4159-4168Crossref PubMed Scopus (172) Google Scholar). This efficient recruitment of CBP/p300 appears to be unique to IRF-3 and may be of physiological significance. In the present study, we analyzed molecular mechanism behind the formation of the IRF-3 holocomplex. The biochemical reconstitution of the holocomplex from IRF-3 homodimers and CBP/p300 and the use of various p300 mutants allowed us to identify the critical domains of p300. The physiological importance of these domains in gene expression was shown in cells using the corresponding mutants of p300. L929, 293T, and HeLa cells were maintained in Eagle's minimum essential medium supplemented with 5% fetal bovine serum. DNA transfection of L929 cells and Newcastle disease virus (NDV) infection were performed as described previously (8Yoneyama M. Suhara W. Fukuhara Y. Sato M. Ozato K. Fujita T. J. Biochem. (Tokyo). 1996; 120: 160-169Crossref PubMed Scopus (119) Google Scholar). DNA transfection of 293T cells was performed by calcium phosphate methods as reported (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar). Cell extracts were prepared as described previously (27Iwamura T. Yoneyama M. Yamaguchi K. Suhara W. Mori W. Shiota K. Okabe Y. Namiki H. Fujita T. Genes Cells. 2001; 6: 375-388Crossref PubMed Scopus (235) Google Scholar). Expression constructs of pEFp50IRF-3, pEFHAp300 and pEFGSTIRF-3 were described previously (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar, 17Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (65) Google Scholar). Expression plasmids for p300 deletion mutants were obtained by deletion of SpeI (Δ143–957), BamHI (Δ599–1240),BspHI and SmaI (Δ194–1572), orPmaCI (1–1946) fragments from pEFHAp300. pEFHAp300 (1235–2412), pEFHAp300 (1235–2221), and pEFHAp300 (1235–2412ΔHAT) were obtained by PCR with oligonucleotides corresponding to the end point sequences. To generate pEFHAp300 (Δ143–957MutAT2),XbaI and NdeI fragment from pBluescript KS containing p300 (MutAT2) was inserted into pEFHAp300 (Δ143–957) (28Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (200) Google Scholar). To obtain an expression construct of pEFHAp300 (Δ143–957ΔZZ), a PCR fragment corresponding to amino acids 1302–1663 was inserted into XbaI/MunI-digested pEFHAp300 (Δ143–957). The expression vector of pEFHAp300 (Δ143–957ΔTAZ) was constructed by insertion of two appropriate PCR fragments into pEFHAp300 (Δ143–957) to delete amino acids 1725–1806, and a MluI site was introduced to join these fragments without mutation. To obtain constructs of pEFHAp300 (Δ143–957ΔZn1/2, Δ143–957ΔZn1α1, Δ143–957ΔZn2/3, and Δ143–957ΔZn1), appropriate fragments were inserted into theMluI site of pEFHAp300 (Δ143–957ΔTAZ). To generate pEFHAp300 (Δ143–957Δα1), the Gene Editor in vitrosite-directed mutagenesis system (Promega) was used. The vector pLNCX-FLAG-CBP was used for expression of human CBP (29Kitabayashi I. Aikawa Y. Nguyen L.A. Yokoyama A. Ohki M. EMBO J. 2001; 20: 7184-7196Crossref PubMed Scopus (174) Google Scholar). The reporter construct p-55C1B-CAT was described previously (7Watanabe N. Sakakibara J. Hovanessian A.G. Taniguchi T. Fujita T. Nucleic Acids Res. 1991; 19: 4421-4428Crossref PubMed Scopus (177) Google Scholar). Anti-p50 epitope monoclonal antibody was established by Dr. N. Hanai (Kyowa Hakko Kogyo Co., Ltd.). Anti-human IRF-3 and anti-human IRF-3 NES antiserum were described previously (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar, 17Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (65) Google Scholar). Anti-HA polyclonal (Y-11, Santa Cruz Biotechnology, Inc.), anti-HA mouse monoclonal (12CA5), anti-p300 (N-15, Santa Cruz), anti-GST (B-14, Santa Cruz), and anti-CBP (A-22, Santa Cruz) polyclonal antibodies were obtained commercially. Recombinant full-length histidine-tagged human p300 was a gift from Dr. T. Ito of Saitama Medical University. The protein was expressed by a baculovirus system and purified to homogeneity on a nickel column. L929 cells were transfected with pEFp50IRF-3 and infected with NDV for 12 h. The whole cell lysate prepared by a standard procedure (150 μl) was treated with 1% DOC on ice for 10 min, then separated on a 5-ml gradient (10–40% glycerol containing 1% DOC, 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, and 1 mm sodium orthovanadate) by centrifugation for 16 h at 50,000 rpm in a Hitachi p55ST2 rotor. After fractionation of the gradient into 20 fractions, Nonidet P-40 was added to give a final concentration of 2%. Each fraction was assayed for IRF-3 and p300 by immunoblotting. The IRF-3 fraction free of p300 was pooled. L929 cells were transiently transfected with pEFGSTIRF-3 and infected with NDV for 12 h. The cell extract and HA-tagged recombinant p300 proteins generated from 293T cells were mixed and rotated with glutathione (GT)-Sepharose 4B (AmershamBiosciences) at 4 °C for 30 min. After an extensive wash with lysis buffer, the bound proteins were eluted with SDS loading buffer, separated by SDS-PAGE, and immunoblotted. EMSA was performed as described previously (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar). To examine the DNA binding activity of homomeric IRF-3, L929 or HeLa cell extract was treated with DOC (final 1%) on ice for 30 min and then subjected to EMSA. To investigate the DNA binding activities of p300 deletion mutants, L929 cells were transiently transfected with pEFp50IRF-3 and infected with NDV for 12 h. The extract and HA-tagged recombinant p300 mutants derived from 293T cells were incubated for 10 min at room temperature, and then the binding mixture was added. EMSA was performed with the lysate of infected L929 cells expressing p50-tagged IRF-3 and a32P-labeled ISG15 probe in which thymines were substituted with bromodeoxyuridines (BrdUrd). The probe sequence was: 5′-GAGAGGGAAACCGAAACUGAATTAGCTTTCAGUUUCGGUUUCCCTCTC-3′ (positions of BrdUrd are indicated by U, and the interferon-stimulated response element (ISRE) is underlined). After electrophoresis, the gel was irradiated (302 nm for 20 min) to cross-link the complex to the probe. The band corresponding to the holocomplex was excised from the gel, and the protein-DNA complexes were eluted with SDS loading buffer. Complexes were precipitated by acetone (80%) and dissolved with radioimmune precipitation buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.1% DOC, and 0.1% SDS). The samples were analyzed directly or subjected to immunoprecipitation followed by SDS-PAGE. To detect the intrinsic HAT activity of p300 deletion mutants, whole cell lysates of 293T cells expressing HA-tagged p300 mutants were prepared. The lysates were subjected to immunoprecipitation by anti-HA polyclonal antibody and then dissolved with reaction buffer (50 mm Tris-HCl, pH 8.0, 10% glycerol, 10 mm sodium butyrate, 0.1 mm dithiothreitol, and 0.1 mmphenylmethylsulfonyl fluoride). [14C]Acetyl-CoA (500 Bq) and 1 μg of histone were added to the reaction buffer and reacted at 30 °C for 1 h. SDS loading buffer was added to stop the reaction. After SDS-PAGE, proteins were transferred to PVDF membrane, and acetylated proteins were detected by autoradiography. The same membrane was reacted with anti-HA monoclonal antibody. To investigate the acetylation of IRF-3, L929 cells were transiently transfected with pEFGSTIRF-3 and mock-treated or infected with NDV for 12 h. The cell extracts were precipitated using GT-Sepharose 4B and then dissolved with reaction buffer as described above. [14C]Acetyl-CoA (500 Bq) was added to the reaction mixture and incubated at 30 °C for 1 h. SDS-PAGE, detection of acetylated proteins, and immunoblotting with anti-p300 or anti-GST antibodies were performed as described above. For the digestion of GST-IRF-3 into GST and IRF-3, virus-activated GST-IRF-3 was precipitated as described above. After the in vitroacetylation assay was performed, the resin was dissolved with reaction buffer (20 mm Tris, pH 8.2, 150 mm NaCl, 2.5 mm CaCl2, and 5% glycerol) and incubated at 16 °C for 2 h with recombinant thrombin (Roche Molecular Biochemicals). SDS loading buffer was added to stop the reaction. After transfer to PVDF, acetylated proteins were detected by autoradiography and then reacted with anti-IRF-3 or anti-GST antibody. Immunoblotting and immunoprecipitation were performed as described previously (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar). The CAT assay was performed as described previously (7Watanabe N. Sakakibara J. Hovanessian A.G. Taniguchi T. Fujita T. Nucleic Acids Res. 1991; 19: 4421-4428Crossref PubMed Scopus (177) Google Scholar). It has been shown that viral infection or treatment with dsRNA results in the formation of a holocomplex of IRF-3, exhibiting activity to bind the ISRE or IRF motif (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar, 15Weaver B.K. Kumar K.P. Reich N.C. Mol. Cell. Biol. 1998; 18: 1359-1368Crossref PubMed Scopus (296) Google Scholar, 18Sato M. Tanaka N. Hata N. Oda E. Taniguchi T. FEBS Lett. 1998; 425: 112-116Crossref PubMed Scopus (224) Google Scholar, 26Kumar K.P. McBride K.M. Weaver B.K. Dingwall C. Reich N.C. Mol. Cell. Biol. 2000; 20: 4159-4168Crossref PubMed Scopus (172) Google Scholar). The holocomplex is composed of a homodimer of IRF-3 and the coactivators CBP/p300 (17Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (65) Google Scholar). A typical result of electrophoresis mobility shift assay (EMSA) with extract of mouse L929 cells expressing human IRF-3 and infected with NDV (Fig.1 A, lanes 1 and 2) or HeLa cells that had been treated with poly(I·C) for 60 min (Fig. 1 A, lanes 5 and 6) are shown. Supershift experiments with specific antibodies showed that the IRF-3 holocomplex contained endogenous IRF-3 and CBP/p300, as demonstrated previously (13Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (679) Google Scholar), but not IRF-7 (data not shown). The generation of the IRF-3 holocomplex was dependent on induction by virus or poly(I·C) (lanes 2 and 6) and independent of overexpression of IRF-3 (lanes 5 and 6, untransfected HeLa cells), demonstrating that the inducible interaction between IRF-3 and CBP/p300 is not because of an artifact of transient overexpression. Furthermore, co-precipitation experiment with untransfected cells demonstrated that the interaction is physiologically relevant (Fig.1 B). It was shown that treatment of extract with 1% DOC results in a dissociation of CBP/p300 from IRF-3 homodimers (17Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (65) Google Scholar, 19Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar). Under these conditions, the activity to bind ISRE of the holocomplex induced either by NDV or poly(I·C) disappeared (Fig. 1 A,lanes 4 and 8). It has been shown that bacterially expressed recombinant IRF-3 could bind to IRF motif (30Schafer S.L. Lin R. Moore P.A. Hiscott J. Pitha P.M. J. Biol. Chem. 1998; 273: 2714-2720Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar); however, we did not detect the faster migrating band corresponding to IRF-3 under these conditions (see below and “Discussion”). The bands indicated by asterisks were demonstrated as nonspecific bands by supershift experiments (data not shown). Because DOC treatment reversibly dissociates the tight association between the transcription factor NF-κB and its inhibitory subunit IκB (31Baeuerle P.A. Baltimore D. Genes Dev. 1989; 3: 1689-1698Crossref PubMed Scopus (365) Google Scholar), the result prompted us to dissociate the IRF-3 holocomplex into components and reconstitute them in vitro. To isolate IRF-3 homodimers, an extract of L929 cells expressing human IRF-3 and infected with NDV was treated with 1% DOC and subjected to glycerol density gradient fractionation. The fractionation allowed us to isolate human IRF-3 homodimers essentially free of p300 (Fig. 1 C). The isolated homodimers exhibited no significant DNA binding on EMSA (Fig.1 D, lane 1). The faster mobility bands indicated by asterisks were shown as a nonspecific bands by supershift experiment (data not shown). However, the addition of recombinant p300, which was produced in insect cells and exhibited no significant ISRE DNA binding on its own (Fig. 1 D, lane 4), generated DNA binding activity similar to the IRF-3 holocomplex (Fig.1 D, lanes 2 and 3). When the isolated IRF-3 homodimers were mixed with extracts of 293T cells, which had been transiently transfected with expression vectors for CBP or p300 (Fig. 1 E, lanes 2 and3) but not with vector alone (lane 1), DNA binding activity similar to that of the IRF-3 holocomplex was generated. A similar result was obtained using the IRF motif of IFN-β gene as a probe (data not shown). Thus, unlike most of the DNA-binding transcription factors, which utilize CBP/p300, IRF-3 absolutely requires the coactivators for DNA binding. The results prompted us to investigate whether p300 physically interacts with DNA. To test the contribution of p300 to the binding of IRF-3 to DNA, the ISG15 probe with thymines changed to BrdUrd was used. A complex containing32P-labeled probe and the holocomplex of IRF-3 was resolved by EMSA (Fig. 2 A) showing that the probe detects IRF-3 holocomplex and ISGF3 as the standard probe (Fig. 1 A, lane 2). The EMSA in Fig. 2 Awas performed on a large scale. The gel was UV-cross-linked in situ, and the band of IRF-3 holocomplex (NDV+) or corresponding area of the gel with uninfected extract (NDV−) was excised and extracted with SDS loading buffer. The recovered complex was analyzed by SDS-PAGE (Fig. 2 B). To our surprise, the size of the major complex was >200 kDa, much larger than the expected molecular size of the IRF-3/probe complex (lane 4). This band was not evident in the corresponding area of the gel in which extract from uninfected cells was run (lane 3) nor when UV irradiation was omitted (lanes 1 and2). To identify the peptide moiety of the recovered complex, the gel eluate was precipitated using specific antibodies. The >200-kDa complex was precipitated with anti-CBP and anti-p300 (lane 10) but not with the control antibody (lane 12), indicating that CBP/p300 is in direct contact with DNA in the context of the holocomplex bound to the target DNA. Anti-IRF-3 precipitated a small but significant amount of 66-kDa complex (lane 11), which likely corresponds to IRF-3 monomer cross-linked to the probe. The other >200 kDa complex, likely corresponding to IRF-3 and CBP/p300 simultaneously cross-linked to the probe. The results strongly suggest that p300 aids the binding of IRF-3 by directly interacting with the DNA. Next we examined the region of p300 required to induce the DNA binding activity of the IRF-3 holocomplex. A series of deletion mutants of p300 were expressed in 293T cells (Fig.3 A). The ability of these mutants to associate with phosphorylated IRF-3 was tested in vitro (Fig. 3 B, top panel,left). The extracts of 293T cells overexpressing HA-tagged p300 mutants were reacted in vitro with those of L929 cells that had been transfected with GST-IRF-3 and infected with NDV to phosphorylate specific serine residues of IRF-3. The bound p300 mutants were, respectively, co-precipitated with GT-Sepharose and detected by immunoblotting using anti-HA (Fig. 3 B, GST pull-down/α HA). In any case, GST-IRF-3 from uninfected cells did not form a complex with p300, indicating that phosphorylation is essential (data not shown). Because each of the mutants Δ143–957, Δ599–1240, Δ194–1572, 1235–2412, and 1235–2221 did form a complex with GST-IRF-3, the N-terminal region up to 1572 amino acids is dispensable for the association with IRF-3. Whereas 1–1946 did not bind to GST-IRF-3, indicating that the binding domain resides in the C-terminal region (1947–2221). This is consistent with our previous finding that GSTΔp300 (1752–2221) bound to IRF-3 in a phosphorylation-dependent manner (17Suhara W. Yoneyama M. Iwamura T. Yoshimura S. Tamura K. Namiki H. Aimoto S. Fujita T. J. Biochem. (Tokyo). 2000; 128: 301-307Crossref PubMed Scopus (65) Google Scholar), as well as with results from other laboratories (CBP, 1992–2441; Ref. 14Lin R. Heylbroeck C. Pitha P.M. Hiscott J. Mol. Cell. Biol. 1998; 18: 2986-2996Crossref PubMed Scopus (742) Google Scholar). Thus, the Q-rich region is defined as the interface of the interaction with IRF-3 homodimer. Next we tested whether the complex of IRF-3 homodimers and p300 mutants binds to DNA (Fig. 3 B, top panel,right). 293T cell lysate containing HA-p300 or the respective mutants was reacted with the lysate of L929 cells containing activated p50 tag-IRF-3. The mixture was subjected to EMSA using the ISG15 probe (EMSA). Under these conditions, the L929 extract exhibited DNA binding activity corresponding to the holocomplex composed of p50-IRF-3 and endogenous mouse p300 (EMSA, lane 1). However, the addition of excess p300 increased the intensity of the DNA-bound complex (lane 2), indicating that p300 is a limiting factor in the extract. Interestingly, the addition of p300 mutants Δ143–957, Δ599–1240, 1235–2412, and 1235–2221, which are smaller than the intact p300, generated a DNA-bound complex that migrated faster than the IRF-3 holocomplex (EMSA, arrows,lanes 3, 4, 7, and8). Supershift experiments showed that the complexes binding DNA contained both p300 mutants and IRF-3 (data not shown). Δ194–1572, which exhibited strong binding with the phosphorylated IRF-3 (Fig. 3 B, GST pull-down/α HA,lane 5), failed to promote DNA binding (Fig. 3 B,EMSA, lane 5). Comparison of the structures of Δ143–957, Δ194–1572, 1235–2412, and 1235–2221 suggests the presence of a region between 1241 and 1573 critical for DNA binding activity. Because this region corresponds to the HAT domain, several additional mutants were generated. Two sets of mutants of the HAT domain were generated (Fig.4 A). The internal region of 50 amino acids was deleted from the mutant 1235–2412 to generate 1235–2412ΔHAT. This deletion has been shown to remove the catalytic activity of HAT (32Boyes J. Byfield P. Nakatani Y. Ogryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (628) Google Scholar). Additionally, the substitution of 6 amino acids in a distinct region of HAT was shown to inactivate the enzyme (28Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (200) Google Scholar). This substitution was introduced into the mutant Δ143–957 to generate Δ143–957MutAT2. These mutants were overexpressed in 293T cells, and cell extracts were subjected to a HAT a" @default.
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• W2042263540 title "Direct Involvement of CREB-binding Protein/p300 in Sequence-specific DNA Binding of Virus-activated Interferon Regulatory Factor-3 Holocomplex" @default.
• W2042263540 cites W1558828789 @default.
• W2042263540 cites W1626483287 @default.
• W2042263540 cites W1966567943 @default.
• W2042263540 cites W1971966311 @default.
• W2042263540 cites W1976749826 @default.
• W2042263540 cites W1980710496 @default.
• W2042263540 cites W1988808478 @default.
• W2042263540 cites W2002301799 @default.
• W2042263540 cites W2002698073 @default.
• W2042263540 cites W2003247108 @default.
• W2042263540 cites W2011044131 @default.
• W2042263540 cites W2011529553 @default.
• W2042263540 cites W2024278937 @default.
• W2042263540 cites W2035524082 @default.
• W2042263540 cites W2037772620 @default.
• W2042263540 cites W2046263781 @default.
• W2042263540 cites W2049433808 @default.
• W2042263540 cites W2053472341 @default.
• W2042263540 cites W2061932605 @default.
• W2042263540 cites W2073784653 @default.
• W2042263540 cites W2075165827 @default.
• W2042263540 cites W2076361525 @default.
• W2042263540 cites W2076414137 @default.
• W2042263540 cites W2078480746 @default.
• W2042263540 cites W2081499148 @default.
• W2042263540 cites W2089077517 @default.
• W2042263540 cites W2092722802 @default.
• W2042263540 cites W2095194341 @default.
• W2042263540 cites W2096501714 @default.
• W2042263540 cites W2098901296 @default.
• W2042263540 cites W2107555074 @default.
• W2042263540 cites W2108399976 @default.
• W2042263540 cites W2110953702 @default.
• W2042263540 cites W2111447146 @default.
• W2042263540 cites W2130576390 @default.
• W2042263540 cites W2143471382 @default.
• W2042263540 cites W2151064612 @default.
• W2042263540 cites W2154161526 @default.
• W2042263540 cites W2156947910 @default.
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