Intracellular Interleukin-1α Functionally Interacts with Histone Acetyltransferase Complexes
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m306342200
ISSN1083-351X
AutoresMiroslava Buryskova, Martin Pospíšek, Arnhild Grothey, Thomas Simmet, Ladislav Burýšek,
Tópico(s)Immune Response and Inflammation
ResumoInterleukin-1α (IL-1α) is an inflammatory cytokine acting extracellularly via membrane receptors. Interestingly, a significant portion of synthesized IL-1α is not secreted; instead, it is actively translocated into the cell nucleus. IL-1α was indeed shown to be involved in certain intracellular processes, such as control of proliferation, apoptosis, or migration, however, the mechanisms of such actions are not known. Here we show that intracellular IL-1α fused to the Gal4p DNA-binding domain (Gal4BD) possesses strong transactivation potential that can be boosted by overexpression of the transcriptional coactivator p300. We demonstrate that the IL-1α precursor interacts via its N-terminal peptide (IL-1NTP) with histone acetyltransferases p300, PCAF, Gcn5 and with the adaptor component Ada3, and that it integrates into the PCAF·p300 complex in a non-destructive manner. In analogy with known acidic coactivators, yeast strains expressing Gal4BD/IL-1NTP display a toxic phenotype that can be relieved by depletion of various components of the SAGA complex. Our data provide the first solid evidence for the nuclear target of the IL-1α precursor and suggest its novel function in transcriptional control. Interleukin-1α (IL-1α) is an inflammatory cytokine acting extracellularly via membrane receptors. Interestingly, a significant portion of synthesized IL-1α is not secreted; instead, it is actively translocated into the cell nucleus. IL-1α was indeed shown to be involved in certain intracellular processes, such as control of proliferation, apoptosis, or migration, however, the mechanisms of such actions are not known. Here we show that intracellular IL-1α fused to the Gal4p DNA-binding domain (Gal4BD) possesses strong transactivation potential that can be boosted by overexpression of the transcriptional coactivator p300. We demonstrate that the IL-1α precursor interacts via its N-terminal peptide (IL-1NTP) with histone acetyltransferases p300, PCAF, Gcn5 and with the adaptor component Ada3, and that it integrates into the PCAF·p300 complex in a non-destructive manner. In analogy with known acidic coactivators, yeast strains expressing Gal4BD/IL-1NTP display a toxic phenotype that can be relieved by depletion of various components of the SAGA complex. Our data provide the first solid evidence for the nuclear target of the IL-1α precursor and suggest its novel function in transcriptional control. The constantly growing interleukin-1 (IL-1) 1The abbreviations used are: IL-1interleukin-1IL-1NTPinterleukin-1α N-terminal peptideGSTglutathione S-transferaseHAThistone acetyltransferaseSAGASpt-Ada-Gcn5-acetyltransferaseNLSnuclear localization signalIL-1αMATmature interleukin-1αaaamino acid(s)HAhemagglutininIPimmunoprecipitatedTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineGal4BDGal4p DNA-binding domainGal4ADGal4p DNA activation domainIRFintracellular regulatory factor. family currently consists of eight different protein ligands, and as much as 10 surface or soluble receptors (1Smith D.E. Renshaw B.R. Ketchem R.R. Kubin M. Garka K.E. Sims J.E. J. Biol. Chem. 2000; 275: 1169-1175Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 2Debets R. Timans J.C. Homey B. Zurawski S. Sana T.R. Lo S. Wagner J. Edwards G. Clifford T. Menon S. Bazan J.F. Kastelein R.A. J. Immunol. 2001; 167: 1440-1446Crossref PubMed Scopus (226) Google Scholar). The IL-1 system represents an evolutionary conserved signaling mechanism homologous to the Drosophila Toll pathway, being a central mediator of the host innate defense mechanisms (3Takeda K. Akira S. Genes Cells. 2001; 6: 733-742Crossref PubMed Scopus (217) Google Scholar, 4Kimbrell D.A. Beutler B. Nat. Rev. Genet. 2001; 2: 256-267Crossref PubMed Scopus (499) Google Scholar). IL-1 proteins are expressed by most of the cell types, and they act as the first alarming signals inducing release of the full set of proinflammatory molecules such as prostaglandins, tumor necrosis factor α, IL-6, chemokines, proteins of the acute phase, and IL-1 per se (5Dinarello C.A. Int. Rev. Immunol. 1998; 16: 457-499Crossref PubMed Scopus (679) Google Scholar). interleukin-1 interleukin-1α N-terminal peptide glutathione S-transferase histone acetyltransferase Spt-Ada-Gcn5-acetyltransferase nuclear localization signal mature interleukin-1α amino acid(s) hemagglutinin immunoprecipitated N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Gal4p DNA-binding domain Gal4p DNA activation domain intracellular regulatory factor. IL-1α is translated as a 33-kDa precursor, which is proteolytically processed into mature IL-1α (IL-1MAT) and the IL-1α N-terminal peptide (IL-1NTP) (6Lomedico P.T. Gubler U. Hellmann C.P. Dukovich M. Giri J.G. Pan Y.C. Collier K. Semionow R. Chua A.O. Mizel S.B. Nature. 1984; 312: 458-462Crossref PubMed Scopus (598) Google Scholar). For many years, the striking IL-1NTP sequence homology among different species fostered speculations about its putative biological function. The nuclear localization signal (NLS), present in the IL-1NTP molecule, was shown to be essential for the biological activity of intracellular IL-1α (7Wessendorf J.H. Garfinkel S. Zhan X. Brown S. Maciag T. J. Biol. Chem. 1993; 268: 22100-22104Abstract Full Text PDF PubMed Google Scholar, 8Burysek L. Houstek J. Cytokine. 1996; 8: 460-467Crossref PubMed Scopus (25) Google Scholar). Indeed, overexpressed intracellular IL-1α precursor (pre-IL-1α), but not IL-1MAT, was able to inhibit cell growth and to induce the expression of the plasminogen activator inhibitor-1 and collagenase genes (9Martel-Pelletier J. McCollum R. DiBattista J. Faure M.P. Chin J.A. Fournier S. Sarfati M. Pelletier J.P. Arthritis Rheum. 1992; 35: 530-540Crossref PubMed Scopus (152) Google Scholar, 10Seki T. Gelehrter T.D. J. Cell Physiol. 1996; 168: 648-656Crossref PubMed Google Scholar). Pre-IL-1α was shown to stimulate proliferation of smooth muscle cells (11Beasley D. Cooper A.L. Am. J. 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IL-1NTP was also found to be specifically associated with the novel protein HAX-1 that has been implicated in modulating apoptosis and cytoskeletal functions (15Yin H. Morioka H. Towle C.A. Vidal M. Watanabe T. Weissbach L. Cytokine. 2001; 15: 122-137Crossref PubMed Scopus (35) Google Scholar). Recently, a strong proapoptotic effect of ectopically expressed IL-1NTP in numerous human tumor cell lines has been reported (16Pollock A.S. Turck J. Lovett D.H. FASEB J. 2003; 17: 203-213Crossref PubMed Scopus (62) Google Scholar). Taken together, several lines of evidence point to a role of IL-1NTP in certain yet unknown nuclear processes. Initiation of transcription in eukaryotes is mediated by recruitment of different multiprotein complexes to the promoter sequence involving the RNA polymerase II complex, general transcription factors and transcriptional cofactors, histone acetytransferase (HAT) complexes, and sequence-specific transcription factors (17Nakatani Y. Genes Cells. 2001; 6: 79-86Crossref PubMed Scopus (50) Google Scholar, 18Young B.A. Gruber T.M. Gross C.A. Cell. 2002; 109: 417-420Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The sequence-specific transcription factors are often composed of separate DNA-binding and activation domains (19Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1179) Google Scholar, 20Triezenberg S.J. Curr. Opin. Genet. Dev. 1995; 5: 190-196Crossref PubMed Scopus (350) Google Scholar). Depending on the amino acid composition, three types of activation domains are recognized: glutamine-, proline-, and acidic-rich domains. The transcription factors containing the acidic-rich activation domain, such as yeast Gal4p, viral proteins VP16 and E1A, and human c-Jun and NF-κB RelA(p65), are evolutionary well conserved, and are able to activate transcription in Saccharomyces cerevisiae as well as in mammalian cells (21Kornuc M. Altman R. Harrich D. Garcia J. Chao J. Kayne P. Gaynor R. 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The eukaryotic chromatin structure with DNA wrapped around histone proteins forming a repeating array of nucleosomes leaves promoter DNA sequences inaccessible to the transcriptional machinery (28Paranjape S.M. Kamakaka R.T. Kadonaga J.T. Annu. Rev. Biochem. 1994; 63: 265-297Crossref PubMed Scopus (321) Google Scholar). Therefore, the chromatin structure must be remodeled prior to gene targeting (17Nakatani Y. Genes Cells. 2001; 6: 79-86Crossref PubMed Scopus (50) Google Scholar). Up to date, a number of different factors with chromatin remodeling activities have been identified (29Kingston R.E. Bunker C.A. Imbalzano A.N. Genes Dev. 1996; 10: 905-920Crossref PubMed Scopus (404) Google Scholar). The fact that histone hyperacetylation is associated with the transcriptionally active chromatin suggests that histone acetylation is one of the chromatin remodeling mechanisms important for transcription initiation (30Turner B.M. J. Cell Sci. 1991; 99: 13-20Crossref PubMed Google Scholar). 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Workman J.L. Mol. Cell. Biol. 1999; 19: 6621-6631Crossref PubMed Scopus (152) Google Scholar). Mass spectrometric analysis has revealed that the human PCAF complex involves, besides transcriptional coactivator CBP/p300 and others, human counterparts of yeast HAT cofactors Ada2, Ada3, and Spt3 (34Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). This mega-dalton complex is composed of more than 14 different subunits that seem to be organized in the three functionally divergent modules responsible: (i) for HAT activity, (ii) for interaction with the sequence-specific transcription factors, and (iii) for association with the preinitiation complex containing TATA-binding protein-associated factors and RNA polymerase II (35Sterner D.E. Grant P.A. Roberts S.M. Duggan L.J. Belotserkovskaya R. Pacella L.A. Winston F. Workman J.L. Berger S.L. Mol. Cell. Biol. 1999; 19: 86-98Crossref PubMed Scopus (291) Google Scholar). Therefore, the HAT complex represents a physical bridge between distant DNA-bound activators and the transcriptional machinery at the core promoter (36Marmorstein R. Roth S.Y. Curr. Opin. Genet. Dev. 2001; 11: 155-161Crossref PubMed Scopus (318) Google Scholar). In this study we have investigated functions of the intracellular forms of IL-1α. We have found that when fused to Gal4BD, IL-1NTP acts as an activator of transcription in both yeast and mammalian cells. The transcriptional activation requires an intact SAGA complex in yeast and the transcriptional coactivator p300 in mammalian cells. We also demonstrate that IL-1NTP physically interacts with mammalian histone acetyltransferases Gcn5, PCAF, and p300, and with adaptor protein Ada3 in vitro. In coimmunoprecipitation experiments we show in detail the specific interactions between intracellular forms of IL-1α and different histone acetyltransferases in vivo. Yeast Strains—The yeast strain AH109 (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ) was from the Matchmaker Two-hybrid System 3 (Clontech). The strains H-224 (MATa, ade2, his3, leu2, trp1, ura3, ahc1Δ::LEU2), YMH171 (MATα, ura3–52, leu2-3, 112, his3, trp1Δ) and its isogenic derivatives YMH511 (gcn5Δ::hisG), YMH 535 (ada2Δ::hisG), YMH537 (ada3Δ::hisG), and YMH567 (spt7Δ::LEU2) were kindly provided by M. Hampsey (37Chen B.S. Sun Z.W. Hampsey M. J. Biol. Chem. 2001; 276: 23881-23887Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). The expression level of Gal4BD fusion proteins was detected using rabbit polyclonal antibody against Gal4BD (sc-729; Santa Cruz Biotechnology). Plasmids—Mouse IL-1α/pLXSN construct was a gift from R. Apte (38Douvdevani A. Huleihel M. Zoller M. Segal S. Apte R.N. Int. J. Cancer. 1992; 51: 822-830Crossref PubMed Scopus (76) Google Scholar). Yeast Gal4BD expression vectors pAS2-1 and pGBKT7, as well as pGBKT7-53 and pGADT7-T constructs were obtained from Clontech. Mammalian Gal4BD expression vector pSG424 was a gift from M. Ptashne (39Sadowski I. Ptashne M. Nucleic Acids Res. 1989; 17: 7539Crossref PubMed Scopus (472) Google Scholar). The GST expression plasmid pGEX4T was from Amersham Biosciences. The Gal4BD/VP16-(412–490) yeast expression vector pM1536, and the reporter plasmids pN927 (UASGAL1-TATAGAL1-lacZ (TA/G1Z)), and pN928 (UASGAL1-TGTAGAL1-lacZ (TG/G1Z)) were provided by M. Hampsey (37Chen B.S. Sun Z.W. Hampsey M. J. Biol. Chem. 2001; 276: 23881-23887Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). The cDNAs encoding the mouse IL-1α precursor (aa 1–270), IL-1NTP (aa 1–109), as well as its deletion mutants IL-1NTP-ATSSN (internal deletion aa 78–87), IL-1NTP-TSETS (internal deletion aa 57–87), IL-1NTP-DYSSA (terminal deletion aa 1–21), IL-1NTP-FTEDD (terminal deletion aa 97–109), and IL-1NTP-DY/FT (terminal deletions aa 1–21 and 97–109) were amplified from the IL-1α/pLXSN template by PCR and cloned into EcoRI and BamHI sites of pAS2-1, pGBKT7 vectors, or into BamHI and EcoRI sites of pGEX4T vector, or into BamHI and XbaI sites of the pSG424 vector. Human cDNA encoding the full-length IL-1α protein was amplified from peripheral blood monocytes by reverse transcriptase-PCR and cloned into NcoI and BamHI sites of pGBKT7. This construct was sequenced and used as the template in all the following PCR amplifications of human IL-1NTP, mature IL-1α, and IL-1α precursor proteins for subsequent cloning into EcoRI and BamHI sites of pGBKT7 vector, BamHI and XhoI sites of pGEX4T vector, or into BamHI and XbaI sites of pSG424 vector. All the mouse and human IL-1α protein forms were subcloned into BamHI and XhoI sites of modified pcDNA4/TO/MycHis (Invitrogen) vector with added FLAG tag upstream of the multiple cloning site. The original vector contains Myc and His tags downstream of the cloned cDNA, therefore, all the IL-1α proteins were expressed as double tagged chimeras with N-terminal FLAG and C-terminal Myc and His tags. Fragments encoding human deletion mutants IL-1NTP-VVATN (internal deletion aa 78–87), IL-1NTP-EDSSS (terminal deletion aa 1–19), and IL-1NTP-ITDDD (terminal deletion aa 96–111) were amplified and cloned into BamHI and XhoI sites of modified pcDNA4/TO/MycHis (see above) vector. The E1A and HA-tagged human p300 expression plasmids were from A. Hecht (40Hecht A. Vleminckx K. Stemmler M.P. van Roy F. Kemler R. EMBO J. 2000; 19: 1839-1850Crossref PubMed Google Scholar). Plasmids encoding HA-tagged human p300 mutants were kindly provided by T. Fujita (41Suhara W. Yoneyama M. Kitabayashi I. Fujita T. J. Biol. Chem. 2002; 277: 22304-22313Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Plasmids expressing N-terminal FLAG-tagged mouse PCAF and GCN5 were a gift from T. Honjo (42Kurooka H. Honjo T. J. Biol. Chem. 2000; 275: 17211-17220Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) and the expression vector encoding HA-tagged human PCAF was kindly supplied by M. Ott (43Dorr A. Kiermer V. Pedal A. Rackwitz H.R. Henklein P. Schubert U. Zhou M.M. Verdin E. Ott M. EMBO J. 2002; 21: 2715-2723Crossref PubMed Scopus (123) Google Scholar). R. Brachmann provided us with the vector expressing the N-terminal FLAG-tagged human Ada3 (44Wang T. Kobayashi T. Takimoto R. Denes A.E. Snyder E.L. el-Deiry W.S. Brachmann R.K. EMBO J. 2001; 20: 6404-6413Crossref PubMed Scopus (74) Google Scholar), which was also used as a template for amplification and subcloning of Ada3 into pCMV-HA (Clontech) vector expressing HA-tagged human Ada3 protein. All amplifications were done using Pfu DNA polymerase (Stratagene) and products were verified by direct sequencing. Cell Culture and Luciferase Assay—HEK293 cells from the American Type Culture Collection were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Twenty-four hours before transfection, cells were plated in 24-well plates so that they were 80% confluent the next day. The cells were cotransfected with 0.6 μg of total amount of expression plasmid DNA mixture and pFRLuc Gal4 reporter construct (Stratagene) per well using the Superfect transfection reagent (Qiagen) according to the manufacturer's instructions. Empty vectors were used to adjust the amounts of DNA in each experiment. After 48 h cells were harvested and lysates were assayed for luciferase activity using the dual luciferase reporter assay (Promega) and the DLR 96-well plate luminometer (STRATEC Biotechnology Systems AG, Birkenfeld, Germany). Alternatively, the measured luciferase activities were normalized according to total protein concentrations. All experiments were performed in triplicates. Preparation of GST Fusion Proteins and Pull-down Assay—Purifications of recombinant proteins as well as pull-down assays were described previously (45Burysek L. Yeow W.S. Lubyova B. Kellum M. Schafer S.L. Huang Y.Q. Pitha P.M. J. Virol. 1999; 73: 7334-7342Crossref PubMed Google Scholar). Briefly, GST fusion proteins (0.5 μg) bound to glutathione-agarose beads were incubated with 300 μl of whole cell lysates (see next chapter) from HA-tagged p300, PCAF, and Ada3 or FLAG-Gcn5-transfected HEK293 cells in 700 μl of immunoprecipitated (IP) lysis buffer at 4 °C for 4 h. After three washes (10 min at 4 °C) with IP lysis buffer, the proteins bound to the beads were resolved by Tricine-SDS-PAGE (8% gel), transferred onto nitrocellulose membranes (Bio-Rad), and probed with anti-FLAG (M2; Sigma), anti-HA (sc-805), or anti-RelB (sc-226; Santa Cruz Biotechnology) antibodies. Transient Transfection and Immunoprecipitation—HEK293 cells were transfected in 10-cm Petri dishes using 10 μg of total plasmid DNA as described before. Forty-eight hours later the cells were washed with phosphate-buffered saline and lysed in IP lysis buffer (50 mm Tris-HCl, pH 7.6, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 0.5% Nonidet P-40, 10% glycerol, Protease and Phosphatase Inhibitor Cocktails; Sigma) for 30 min on ice. After centrifugation, the supernatants were stored and the sediments containing nuclei and cell debris were extracted with buffer B (20 mm HEPES, pH 7.9, 420 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, protease and phosphatase inhibitor mixtures; Sigma) for 15 min on ice. The supernatants from both extractions were mixed. The resulting whole cell extracts were precleared with 50 μl of protein G-agarose for 1 h at 4 °C. After centrifugation, the cleared lysates were incubated with 50 μl of protein G-agarose and 1 μg of anti-FLAG (Santa Cruz Biotechnology) antibody in 700 μl of IP lysis buffer overnight at 4 °C. The beads were then washed three times with IP lysis buffer and subjected to Western blot detection of bound material as described before. β-Galactosidase Assay—Yeast strains harboring UASGAL1-lacZ reporter constructs were grown to late logarithmic phase, harvested by centrifugation, and resuspended in 200 μl of breaking buffer (100 mm potassium phosphate, pH 7.8, 0.2% Triton X-100, 0.5 mm dithiothreitol, and protease inhibitor mixture; Sigma). Cell extracts were prepared by vortexing with 0.5-mm glass beads four times for 30 s. Activities were determined from at least three independent clones using the luminescent β-galactosidase detection kit Galacto-Light (Tropix, Bedford, MA). The measured activities were normalized according to optical densities of the used yeast cultures. The Gal4BD/IL-1NTP Fusion Protein Transactivates the Gal4 Promoter and Confers Growth Inhibition in Yeast—Experiments with the yeast two-hybrid system aiming to identify proteins interacting with IL-1NTP surprisingly revealed that expression of either mouse or human Gal4BD/IL-1NTP fusion protein enabled the growth of the reporter yeast strain on selection drop-out media lacking adenine, histidine and tryptophan (SD-AHT). Fig. 1A shows that the yeast strains expressing the mouse or human Gal4BD/IL-1NTP fusion proteins alone grow by a rate similar to that of the positive control strain expressing the interacting fusion proteins Gal4BD/p53 and Gal4AD/SV40 T large antigen, and when incubated with 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-α-gal) substrate, they turn blue (data not shown) indicating the presence of β-galactosidase activity. In contrast, strains expressing only Gal4BD, or mouse or human Gal4BD/IL-1MAT were unable to grow on SD-AHT even after 5 days of cultivation at 30 °C, and even when inoculated at high densities. Thus, in accordance with their high degree of homology, both mouse and human Gal4BD/IL-1NTP chimeras effectively transactivate the GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, and MEL1UAS-MEL1TATA-lacZ reporters harbored by the AH109 strain. Studies in the eukaryote S. cerevisiae have shown that expression of acidic transactivation proteins fused to Gal4BD can inhibit yeast growth (24Moore P.A. Ruben S.M. Rosen C.A. Mol. Cell. Biol. 1993; 13: 1666-1674Crossref PubMed Google Scholar, 25Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (989) Google Scholar, 27Bhaumik S.R. Green M.R. Genes Dev. 2001; 15: 1935-1945Crossref PubMed Scopus (238) Google Scholar). To test the effect of IL-1NTP on yeast growth, and to further investigate the mechanism of IL-1NTP-mediated activation, strain YMH171 and the indicated isogenic derivatives were transformed with the mouse IL-1NTP/pAS2-1 vector expressing higher levels of Gal4BD/IL-1NTP. As shown in Fig. 1B, strain YMH171 (wt) expressing Gal4BD/IL-1NTP grows markedly slower than the strain expressing Gal4BD only (control). However, the Gal4BD/IL-1NTP-induced growth inhibition was strictly dependent on the presence of the intact SAGA transcriptional activation complex. The growth inhibitory effect of IL-1NTP was completely relieved in yeast strains lacking one of the SAGA complex components: Gcn5, Ada2, Ada3, and Spt7. Surprisingly, deletion of the AHC1 gene product, a unique structural subunit of the yeast ADA transcriptional activation complex, did not relieve the Gal4BD/IL-1NTP-mediated growth inhibition. These data suggested specific interference of Gal4BD/IL-1NTP with certain components of the yeast SAGA complex as it has been described for the acidic coactivators Gal4p, E1A, or VP16 (26Kulesza C.A. Van Buskirk H.A. Cole M.D. Reese J.C. Smith M.M. Engel D.A. Oncogene. 2002; 21: 1411-1422Crossref PubMed Scopus (26) Google Scholar, 27Bhaumik S.R. Green M.R. Genes Dev. 2001; 15: 1935-1945Crossref PubMed Scopus (238) Google Scholar, 46Vignali M. Steger D.J. Neely K.E. Workman J.L. EMBO J. 2000; 19: 2629-2640Crossref PubMed Scopus (105) Google Scholar). Similar results were obtained when identical yeast strains harboring the UASGAL1-TATAGAL1-lacZ reporter and expressing Gal4BD, mouse Gal4BD/IL-1NTP, or the strong Gal4BD/VP16 activator were assayed for their respective β-galactosidase activities (Fig. 2B). The IL-1NTP-mediated β-galactosidase activities reached about 25% of activities stimulated by Gal4BD/VP16 in wild type strain YMH171 (Fig. 2B, wt). The gcn5Δ, ada2Δ, and ada3Δ deletions severely reduced the activities of both IL-1NTP and VP16, an effect more pronounced for IL-1NTP than for VP16 (5- versus 4-fold reduction, respectively). The most severe reduction (20-fold for both IL-1NTP and VP16) was observed in the strain defective in expression of Spt7, the critical structural subunit of the SAGA complex. In agreement with previous results, ahc1Δ deletion had no impact on activities mediated by both VP16 (37Chen B.S. Sun Z.W. Hampsey M. J. Biol. Chem. 2001; 276: 23881-23887Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar) and IL-1NTP, suggesting that IL-1NTP acts via interaction with the SAGA, but not with the ADA transcriptional complex. Furthermore, the point mutation in the TATA core promoter element (UASGAL1-TGTAGAL1-lacZ) abolished the transactivation potentials of both IL-1NTP and VP16 proteins (Fig. 2A), indicating that the IL-1NTP-mediated activation occurs specifically through a common RNA polymerase II activation mechanism. Characterization of Domains in the IL-1NTP Molecule Responsible for the Transactivation Potential—Besides the well characterized NLS domain (aa 79–86) (7Wessendorf J.H. Garfinkel S. Zhan X. Brown S. Maciag T. J. Biol. Chem. 1993; 268: 22100-22104Abstract Full Text PDF PubMed Google Scholar), our structure-prediction analyses identified two additional highly conserved acidic α-helixes located at the N and C termini of the IL-1NTP molecule (aa 7–19 and 98–108, respectively). In a search for the structural motifs mediating the IL-1NTP transactivation potential, a set of deletion mutants fused to Gal4BD were prepared (Fig. 3A) and tested for their activities in the yeast reporter strain. The yeast strain AH109 was transformed with plasmid constructs encoding different Gal4BD/IL-1NTP deletion mutants. As shown in Fig. 3B, deletion of the NLS domain did not affect the transactivation potential of the Gal4BD/IL-1NTP, whereas deletions of either N- or C-terminal acidic helixes markedly slowed the growth rate. Moreover, the combination of both N- and C-terminal deletions had deteriorating impact on the growth potential on selection media. Analogously, the β-galactosidase activities in the Gal4BD/IL-1NTP expressing AH109 yeast strain were comparable with those in the positive control strain (expressing Gal4BD/p53 and Gal4AD/T-Ag together), and deletions of either one or both terminal helixes in the IL-1NTP molecule dramatically reduced β-galactosidase activities in the respective strains. Interestingly, the internal deletion mutants lacking the NLS domain (ATSSN and TSETS) showed enhanced activities that were as much as 2-fold higher than activities observed in the positive control strain (Fig. 3C). The expression levels of the corresponding Gal4BD fusion proteins in strains tested for growth on drop-out medium (Fig. 3B) and for β-galactosidase activities (Fig. 3C) are shown in Fig. 3D. Lower expression levels of Gal4BD/ATSSN and Gal4BD/TSETS reflect their higher transcriptional activities (Fig. 3C) and higher toxicities (data not shown), and further support the idea about the functional importance of both the IL-1NTP terminal helixes. These data indicate that the NLS domain is not required for the transactivation potential of Gal4BD/IL-1NTP, and that its deletion results even in enhanced transcriptional activity of the chimera. Gal4BD/IL-1NTP Transactivates the Gal4 Promoter in Mammalian Cells via Functional Cooperation and Physical Association with the Transcriptional Coactivator p300 —Our data from the yeast model are consistent with those of other acidic transcriptional coactivators and support our hypothesis about a novel nuclear function of IL-1α. To determine the physiological role of the intracellular IL-1α, we further tested its transactivation potential as well as its ability to cooperate with different histone acetyltransferases in mammalian cells. HEK293 cells cotransfected with a reporter plasmid and plasmid constructs encodin
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