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Nramp1-mediated Innate Resistance to Intraphagosomal Pathogens Is Regulated by IRF-8, PU.1, and Miz-1

2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês

10.1074/jbc.m307954200

1083-351X

Michal Alter-Koltunoff, Sharon Ehrlich, Natalie Dror, Aviva Azriel, Martin Eilers, H. Häuser, H. J. M. Bowen, C. Howard Barton, Tomohiko Tamura, Keiko Ozato, Ben-Zion Levi,

MicroRNA in disease regulation

Natural resistance-associated macrophage protein 1 (Nramp1) is a proton/divalent cation antiporter exclusively expressed in monocyte/macrophage cells with a unique role in innate resistance to intraphagosomal pathogens. In humans, it is linked to several infectious diseases, including leprosy, pulmonary tuberculosis, visceral leishmaniasis, meningococcal meningitis, and human immunodeficiency virus as well as to autoimmune diseases such as rheumatoid arthritis and Crohn's disease. Here we demonstrate that the restricted expression of Nramp1 is mediated by the macrophage-specific transcription factor IRF-8. This factor exerts its activity via protein-protein interaction, which facilitates its binding to target DNA. Using yeast two-hybrid screen we identified Myc Interacting Zinc finger protein 1 (Miz-1) as new interacting partner. This interaction is restricted to immune cells and takes place on the promoter Nramp1 in association with PU.1, a transcription factor essential for myelopoiesis. Consistent with these data, IRF-8 knockout mice are sensitive to a repertoire of intracellular pathogens. Accordingly, IRF-8–/– mice express low levels of Nramp1 that can not be induced any further. Thus, our results explain in molecular terms the role of IRF-8 in conferring innate resistance to intracellular pathogens and point to its possible involvement in autoimmune diseases. Natural resistance-associated macrophage protein 1 (Nramp1) is a proton/divalent cation antiporter exclusively expressed in monocyte/macrophage cells with a unique role in innate resistance to intraphagosomal pathogens. In humans, it is linked to several infectious diseases, including leprosy, pulmonary tuberculosis, visceral leishmaniasis, meningococcal meningitis, and human immunodeficiency virus as well as to autoimmune diseases such as rheumatoid arthritis and Crohn's disease. Here we demonstrate that the restricted expression of Nramp1 is mediated by the macrophage-specific transcription factor IRF-8. This factor exerts its activity via protein-protein interaction, which facilitates its binding to target DNA. Using yeast two-hybrid screen we identified Myc Interacting Zinc finger protein 1 (Miz-1) as new interacting partner. This interaction is restricted to immune cells and takes place on the promoter Nramp1 in association with PU.1, a transcription factor essential for myelopoiesis. Consistent with these data, IRF-8 knockout mice are sensitive to a repertoire of intracellular pathogens. Accordingly, IRF-8–/– mice express low levels of Nramp1 that can not be induced any further. Thus, our results explain in molecular terms the role of IRF-8 in conferring innate resistance to intracellular pathogens and point to its possible involvement in autoimmune diseases. Macrophages are essential components of innate immunity but also provide important links between innate and adaptive immunity. Mature macrophages differentiate from a bipotential myeloid progenitor stem cell that can also differentiate to granulocyte in a regulated process in which many transcription factors are involved. Macrophages are an essential component of the host defense mechanisms against invading pathogens. These invading foreign bodies activate the macrophages, which in response engulf and subsequently entrap the pathogens in the phagosome/lysosome compartment. In this specialized compartment, the pathogens are subjected to massive attack by reactive oxygen and nitrogen intermediates (1Nathan C. Shiloh M.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8841-8848Crossref PubMed Scopus (1131) Google Scholar). This is achieved by two macrophage specific enzymatic pathways, phagocyte oxidase (phox) 1The abbreviations used are: phox, phagocyte oxidase; iNOS, inducible nitric-oxide synthase; IL, interleukin; ICSBP, interferon consensus sequence binding protein; DBD, DNA binding domain; IFN, interferon; IAD, IRF association domain; LPS, lipopolysaccharide.1The abbreviations used are: phox, phagocyte oxidase; iNOS, inducible nitric-oxide synthase; IL, interleukin; ICSBP, interferon consensus sequence binding protein; DBD, DNA binding domain; IFN, interferon; IAD, IRF association domain; LPS, lipopolysaccharide. (2Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and inducible nitric-oxide synthase (iNOS) (3Lyons C.R. Orloff G.J. Cunningham J.M. J. Biol. Chem. 1992; 267: 6370-6374Abstract Full Text PDF PubMed Google Scholar). In addition, macrophages mediate innate resistance to host infection by intracellular pathogens such as Mycobacterium, Salmonella, and Leishmania. This is controlled by a single dominant gene termed natural resistance-associated macrophage protein 1 (Nramp1) also known as solute carrier family 11 member a1 (Slc11a1) or Ity/Lsh/Bcg. Nramp1 demonstrates high similarity to Nramp2, which encodes a membrane-bound protein that functions as iron, and other divalent cations that are transporters into the cell cytoplasm (4Fleming M.D. Trenor III, C.C. Su M.A. Foernzler D. Beier D.R. Dietrich W.F. Andrews N.C. Nat. Genet. 1997; 16: 383-386Crossref PubMed Scopus (1013) Google Scholar). Based on the high identity between these two Nramp members, it was shown that Nramp1 is also a proton/divalent cation antiporter with a unique role in innate resistance to intraphagosomal pathogens and autoimmune disease (for review see Refs. 5Wyllie S. Seu P. Goss J.A. Microbes Infect. 2002; 4: 351-359Crossref PubMed Scopus (79) Google Scholar, 6Barton C.H. Biggs T.E. Baker S.T. Bowen H. Atkinson P.G. J. Leukoc. Biol. 1999; 66: 757-762Crossref PubMed Scopus (81) Google Scholar, 7Blackwell J.M. Searle S. Mohamed H. White J.K. Immunol. Lett. 2003; 85: 197-203Crossref PubMed Scopus (120) Google Scholar). The exact function of Nramp1 as iron and divalent cation transporter is still controversial. It either functions to increase transphagosomal Fe2+ catalyzing the Haber-Weiss/Fenton reaction to generate the highly toxic hydroxyl radical essential for macrophage bactericidal activity. On the other hand, it is thought to deprive the intraphagosomal bacterium of the availability of Fe2+ and other divalent cations, which are critical for the ability of the invading pathogens to survive the phagosomal damage (5Wyllie S. Seu P. Goss J.A. Microbes Infect. 2002; 4: 351-359Crossref PubMed Scopus (79) Google Scholar). Unlike the ubiquitous expression of Nramp2, Nramp1 is exclusively expressed in monocyte/macrophage cells (8Vidal S.M. Malo D. Vogan K. Skamene E. Gros P. Cell. 1993; 73: 469-485Abstract Full Text PDF PubMed Scopus (966) Google Scholar). In addition, in response to the invading pathogen, activated macrophages secrete proinflammatory cytokines such as IL-12 and IL-18 that recruit the cell-mediated adaptive immunity, e.g. Th1 cells (9Trinchieri G. Int. Rev. Immunol. 1998; 16: 365-396Crossref PubMed Scopus (267) Google Scholar, 10Dinarello C.A. Methods. 1999; 19: 121-132Crossref PubMed Scopus (392) Google Scholar, 11Akira S. Curr. Opin. Immunol. 2000; 12: 59-63Crossref PubMed Scopus (325) Google Scholar). It is, therefore, not surprising that, among the many transcription factors that participate in these well coordinated macrophage activities against intracellular pathogens, IRF-8 is alternatively termed as interferon consensus sequence binding protein (ICSBP). Mice with null mutation to IRF-8 are defective in the differentiation of bone marrow myeloid progenitor cells toward mature macrophages (12Tamura T. Nagamura-Inoue T. Shmeltzer Z. Kuwata T. Ozato K. Immunity. 2000; 13: 155-165Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Therefore, these defective mice overproduce granulocytes, which eventually lead to lymphadenopathy and hepatosplenomegaly resembling chronic myelogenous leukemia syndrome in humans (13Holtschke T. Lohler J. Kanno Y. Fehr T. Giese N. Rosenbauer F. Lou J. Knobeloch K.P. Gabriele L. Waring J.F. Bachmann M.F. Zinkernagel R.M. Morse III, H.C. Ozato K. Horak I. Cell. 1996; 87: 307-317Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). Thus, IRF-8 is an essential factor for the differentiation of bipotential myeloid progenitor cells toward mature macrophages while inhibiting the differentiation pathway toward granulocytes (14Tamura T. Ozato K. J. Interferon Cytokine Res. 2002; 22: 145-152Crossref PubMed Scopus (162) Google Scholar). In addition, it was shown that IRF-8 is an essential factor for proper functioning of mature macrophages. For example, it is essential for the regulated expression of specific phagosomal components like phagocyte oxidase complex (gp91 phox and p67 phox (15Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105PubMed Google Scholar), iNOS (16Xiong H. Zhu C. Li H. Chen F. Mayer L. Ozato K. Unkeless J.C. Plevy S.E. J. Biol. Chem. 2003; 278: 2271-2277Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and Nramp1 (as shown here)). Furthermore, it is an essential regulatory element for the production of the proinflammatory cytokines IL-12 (17Wang I.M. Contursi C. Masumi A. Ma X. Trinchieri G. Ozato K. J. Immunol. 2000; 165: 271-279Crossref PubMed Scopus (158) Google Scholar), IL-18 (18Kim Y.M. Im J.Y. Han S.H. Kang H.S. Choi I. J. Immunol. 2000; 165: 3198-3205Crossref PubMed Scopus (71) Google Scholar), and IL-1β (19Marecki S. Riendeau C.J. Liang M.D. Fenton M.J. J. Immunol. 2001; 166: 6829-6838Crossref PubMed Scopus (96) Google Scholar). IRF-8 belongs to a family of nine cellular members all sharing significant similarity at the N-terminal 115 amino acids, which comprise the DNA binding domain (DBD). This DBD binds to specific DNA sequence motif termed IFN-stimulated response element, which mediates in part IFN type I signaling (20Levi B.Z. Hashmueli S. Gleit-Kielmanowicz M. Azriel A. Meraro D. J. Interferon Cytokine Res. 2002; 22: 153-160Crossref PubMed Scopus (65) Google Scholar). Unlike other IRF members, IRF-8 is capable of binding to target DNA sequence only following association with other IRF or non-IRF transcription factors. The interaction with non-IRF members, such as PU.1, leads to the binding of the heterocomplex to a DNA composite element of which half is an IRF binding site and half is the DNA binding site for the interacting partner. The domain essential for these protein-protein associations was mapped and found to be conserved among all other IRF members, excluding IRF-1 and IRF-2, which associate with IRF-8. This module was termed IRF association domain (IAD) and demonstrates structural similarity with the MH2 domain of the Smad family of transcription factors, which mediate transforming growth factor-β signaling also through protein-protein interactions (21Massague J. Wotton D. EMBO J. 2000; 19: 1745-1754Crossref PubMed Google Scholar). This region has several α helix structures of which the α helix structure surrounding leucine 331 of IRF-8 is highly conserved and is essential for protein-protein interaction. The association modules of IRF-1, IRF-2 and PU.1, which interact with IRF-8, were identified and found to be a PEST domain, which is enriched with proline, glutamic acid, serine, and threonine (for review see Ref. 20Levi B.Z. Hashmueli S. Gleit-Kielmanowicz M. Azriel A. Meraro D. J. Interferon Cytokine Res. 2002; 22: 153-160Crossref PubMed Scopus (65) Google Scholar). The interacting partner with IRF-8 dictates not only the DNA binding site but also the transcriptional activity, e.g. activation or repression. Interaction with non-IRF factors primarily leads to transcriptional synergy, whereas interaction with IRF members such as IRF-1 or IRF-2 primarily leads to transcriptional repression (20Levi B.Z. Hashmueli S. Gleit-Kielmanowicz M. Azriel A. Meraro D. J. Interferon Cytokine Res. 2002; 22: 153-160Crossref PubMed Scopus (65) Google Scholar). The stoichiometry between the interacting partners was not determined, and it is possible that several PEST domains can interact with an IAD as observed for the promoters of the macrophage-specific genes gp91 phox , p67 phox (15Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105PubMed Google Scholar), and IL-18, where heterocomplexes between IRF-1, PU.1, and IRF-8 were reported. Yeast two-hybrid screen with the IAD of IRF-8 as bait was used to search for new interacting partners for IRF-8. This search led to the identification of the association of IRF-8 with subunit 2 of the COP9/signalosome complex (22Cohen H. Azriel A. Cohen T. Meraro D. Hashmueli S. Bech-Otschir D. Kraft R. Dubiel W. Levi B.Z. J. Biol. Chem. 2000; 275: 39081-39089Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The interaction with COP9/signalosome leads to the phosphorylation of serine 260 within the IAD of IRF-8. This phosphorylated residue within the IAD is essential for efficient interaction between IRF-8 and IRF-1 or IRF-2. The data collected from such screen suggested that IRF-8 is engaged in numerous associations with additional transcription factors. In this communication, we report the cloning of Myc interacting zinc-finger protein 1 (Miz-1) as a new interacting partner with IRF-8 identified in yeast cells. This interaction could be demonstrated only in mammalian cells of hematopoietic origin. Furthermore, Nramp1 was identified as the target gene synergistically activated by Miz-1 and IRF-8. This synergistic activation is further enhanced by PU.1. Thus, our results lay the molecular basis for the regulated expression of Nramp1 in macrophages and explain in molecular terms the role of IRF-8 in conferring innate resistance to intracellular pathogens. Cell Culture—COS-7, NIH3T3, U937, HL-60, and J774A.1 cells were obtained from ATCC (Manassas, VA). CL2 cells were described previously (17Wang I.M. Contursi C. Masumi A. Ma X. Trinchieri G. Ozato K. J. Immunol. 2000; 165: 271-279Crossref PubMed Scopus (158) Google Scholar). NIH3T3 and COS-7 were maintained in Dulbecco's modified Eagle's medium, whereas U937, HL-60, J447A.1, and CL2 cells were maintained in RPMI 1640 supplemented with 40 μm β-mercaptoethanol and in an addition 5 ng/ml M-CSF and GM-CSF (R&D Systems, Minneapolis, MN) for CL2 cells. All media were supplemented with 10% fetal calf serum and antibiotics. Plasmids—Mammalian expression vectors encoding for IRF-8, IRF-8 L331P, IRF-8 S260A, GAL4-IRF-8, GAL4-IRF-8 L331P, GAL4-IRF-8 S260A, PU.1, pSG424 (encoding for GAL4 DBD), and the reporter plasmid pGAL4× 5-Luc were all described previously (23Hashmueli S. Gleit-Kielmanowicz M. Meraro D. Azriel A. Melamed D. Levi B.Z. Int. Immunol. 2003; 15: 807-815Crossref PubMed Scopus (7) Google Scholar). The expression vectors encoding for Miz-1 (pPK7, kindly obtained from Dr. Hänel), GAL4-Miz-1, and the reporter plasmid pGL2-p15 ink4b , a 2500-bp segment of the p15 ink4b promoter driving the expression of the luciferase gene, were also described (24Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nat. Cell Biol. 2001; 3: 392-399Crossref PubMed Scopus (449) Google Scholar). pGL3-Nramp1 is a 1600-bp fragment from the promoter region of Nramp1 that was subcloned from the plasmid pHB4 (25Bowen H. Biggs T.E. Phillips E. Baker S.T. Perry V.H. Mann D.A. Barton C.H. J. Biol. Chem. 2002; 277: 34997-35006Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) following restriction digest with XbaI and BamHI and subsequent cloning to the corresponding NheI and BglII sites in the luciferase promoterless reporter vector pGL3 (Promega). Yeast Two-hybrid Screens—A GAL4 AD-tagged human B cell library (kindly obtained from Dr. Elledge through Dr. Wallach (26Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar) was screened for interacting clones with ICSBP-IAD as bait, which was cloned in the yeast pGBT9 plasmid. Experiments were performed according to the MATCHMAKER Two-Hybrid System Protocol (Clontech) in the presence of 5 mm 3-aminotriazole. Each positive clone was also reacted with baits harboring mutation in the IAD L331P, which ablates protein-protein interaction, and with the corresponding IADs of IRF-4 and IRF-7. DNA Transfections, Reporter Gene Analyses, Immunoprecipitation, and Mammalian Two-hybrid Assays—NIH3T3 and COS-7 cells were transfected by the calcium phosphate-DNA coprecipitation method as described previously (27Chen C. Okayama H. Mol. Cell Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar, 28Meraro D. Hashmueli S. Koren B. Azriel A. Oumard A. Kirchhoff S. Hauser H. Nagulapalli S. Atchison M.L. Levi B.Z. J. Immunol. 1999; 163: 6468-6478PubMed Google Scholar). NIH3T3 cells were plated in a 6-well dish, whereas COS-7 cells were transfected in 10-cm plates with indicated amounts of reporter gene constructs and expression vectors and with 600 ng of pMDISRLuc (SV40 promoter driving the expression of Renilla luciferase (Luc) to monitor transfection efficiencies), and pUC19 serving as carrier DNA up to 3 μg for NIH3T3 cells and 50 μg for COS-7 cells. The cells were harvested 48 h later and lysed using the lysis buffer of the Dual Luciferase assay kit (Promega), and luciferase activities were determined according to the manufacturer's instructions using a TD-20/20 luminometer (Turner Design, Promega). Reporter gene activities were normalized for protein concentration and transfection efficiencies as described (29Sharf R. Meraro D. Azriel A. Thornton A.M. Ozato K. Petricoin E.F. Larner A.C. Schaper F. Hauser H. Levi B.Z. J. Biol. Chem. 1997; 272: 9785-9792Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Western blot analyses were performed with each transfected expression vector to ensure an expected level of ectopic protein expression (data not shown). Each set of transfection experiments was repeated at least three times, each generating similar results. U937 cells were diluted to 106 cells/ml 16–24 h prior to transfection. At the time of transfection, the cells were washed twice in phosphate-buffered saline and suspended at 2.5 × 107 cells/ml in RPMI medium lacking fetal calf serum and antibiotics. 0.4 ml of the cell suspension was placed into a 0.4-cm electroporation cuvette (Bio-Rad, Richmond, CA) with up to 30 μg of plasmid DNA, suspended in no more than 20 μl of distilled water. Cells and plasmid DNA were incubated for 5 min at room temperature prior to electroporation at 960 microfarads and 300 V (Bio-Rad Gene Pulser). Following the electric shock, the cells were left at room temperature for an additional 15 min and then diluted into 10 ml of RPMI containing 10% fetal calf serum. 24 h after electroporation the cells were harvested and analyzed as described above for NIH3T3 cells. Mammalian two-hybrid assays were performed in principle as described previously (30Schaper F. Kirchhoff S. Posern G. Koster M. Oumard A. Sharf R. Levi B.Z. Hauser H. Biochem. J. 1998; 335: 147-157Crossref PubMed Scopus (79) Google Scholar). NIH3T3 or U937 cells were transfected as described above with indicated amounts of GAL4-driven luciferase gene (pGAL4× 5-Luc), GAL4-Miz-1 fusion construct and full-length ICSBP fused to VP16 (ICSBP-VP16) (31Sharf R. Azriel A. Lejbkowicz F. Winograd S.S. Ehrlich R. Levi B.-Z. J. Biol. Chem. 1995; 270: 13063-13069Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) together with Renilla luciferase expression plasmid for transfection efficiency. The cells were harvested 24 h later, lysed using the lysis buffer of the Dual Luciferase assay kit (Promega), and luciferase activities were determined and normalized as described above. The -fold of synergism was calculated as the ratio between the -fold of activation elicited by the two transfected factors divided by the sum of the -fold of activation for each factor alone. For coimmunoprecipitation studies, COS-7 cells were seeded 24 h before transfection (1.5 × 106cells/10-cm dish) and transfected as indicted in the text with 10 μg of pcDNA-ICSBP/myc-his or pPK7 encoding for Miz-1 or both. 48 h later, the cells were washed twice with cold phosphate-buffered saline and harvested in 800 μl of ice-cold TEN buffer (40 mm Tris-HCl, pH 7.4, 1 mm EDTA, 150 mm NaCl) using a rubber policeman. Following a short spin the cells were lysed in 200 μl of lysis buffer (50 mm HEPES buffer, pH 7.4, 1 mm EDTA, 150 mm NaCl, and 1% Nonidet P-40). 600 μl of cells lysate were incubated with gentle rotation for 16 h at 4 °C with 50 μl of 50% Protein A-Sepharose beads (Sigma) to which 3 μg of anti-Miz-1 antibodies (sc-5987, Santa Cruz Biotechnology) were bound. The beads were washed three times with lysis buffer and taken in 20 μl of protein Sample buffer and separated on 10% SDS-PAGE. In some of the experiments, 100 μl of cell extract corresponding to 3 × 107 HL-60 was mixed with 600 μl of COS-7 cell lysate prior to immunoprecipitation for 20 min at 25 °C. The combined cell lysates were subjected to immunoprecipitation with anti-Miz-1 antibodies and subsequent separation on 10% SDS-PAGE as described above. The gels were electroblotted to polyvinylidene difluoride membranes that were subsequently blocked for 2 h with 5% nonfat dry milk (Carnation) in TBS-T (20 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% Tween 20) and subjected to Western blot analysis by incubating for 16 h in the same blocking buffer at 4 °C with polyclonal antibodies directed against ICSBP (29Sharf R. Meraro D. Azriel A. Thornton A.M. Ozato K. Petricoin E.F. Larner A.C. Schaper F. Hauser H. Levi B.Z. J. Biol. Chem. 1997; 272: 9785-9792Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and subject to ECL analysis using SuperSignal (Pierce). For direct Western blot analysis, cells (1.5 × 106 cells/10-cm dish) were transfected with 10 μg of pcDNA-ICSBP/myc-his or pPK7 encoding for Miz-1 or both, harvested and lysed as above. The lysates were separated over 10% SDS-PAGE, and the proteins were transferred to polyvinylidene difluoride membrane and subjected to immunoblot analysis with either polyclonal antibodies directed against ICSBP or Miz-1. The corresponding proteins were identified by ECL analysis as described above. Each set of transfection experiments was repeated at least three times generating similar results. Northern Blot Analysis—Total RNA was extracted from CL2 and J477A.1 cell lines using Tri-Reagent (Sigma) according to the manufacturer's instruction before and 5 h after exposure to 100 units/ml IFN-γ (PeproTech Inc., Rocky Hill, NJ) and 50 ng/ml LPS (Sigma). Northern blot analysis was performed as previously described (32Weisz A. Kirchhoff S. Levi B.Z. Int. Immunol. 1994; 6: 1125-1131Crossref PubMed Scopus (43) Google Scholar) with 32P-labeled probes corresponding to a 600-bp HindIII and BamHI fragment from the coding region of murine Nramp1 and a 262-bp PvuII-BglII fragment corresponding to the C-terminal half of human ICSBP (33Weisz A. Marx P. Sharf R. Appella E. Driggers P.H. Ozato K. Levi B.-Z. J. Biol. Chem. 1992; 267: 25589-25596Abstract Full Text PDF PubMed Google Scholar). Yeast Two-hybrid Screens Demonstrated a Specific Interaction between IRF-8 and Miz-1—IRF-8 interacts with different transcription factors such as IRF-1, IRF-2, PU.1, and E47 through the IAD (for review see Ref. 20Levi B.Z. Hashmueli S. Gleit-Kielmanowicz M. Azriel A. Meraro D. J. Interferon Cytokine Res. 2002; 22: 153-160Crossref PubMed Scopus (65) Google Scholar). To identify other possible interacting factors with IRF-8, yeast two-hybrid screens were employed. The full-length IRF-8 missing only the first 33 amino acids and the IAD of IRF-8 (amino acids 201–377) were fused to the DNA binding domain of the yeast transcription factor GAL4 and used as baits against a human B-cells cDNA library fused to the activation domain of GAL4 (for details see “Experimental Procedures”). Yeast HF7C cells were transformed with the bait constructs, and the resulting cells were transformed with the human B-cells library (26Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar). Four interacting clones were identified among which was a truncated form of Miz-1 (accession number Y09723) (34Peukert K. Staller P. Schneider A. Carmichael G. Hanel F. Eilers M. EMBO J. 1997; 16: 5672-5686Crossref PubMed Scopus (290) Google Scholar). This truncated Miz-1, missing its 357 N-terminal amino acids (Miz-1ΔN357), demonstrated strong association with the full-length IRF-8 and moderate association with just its IAD in the yeast cells. Support for the specificity of interaction between IRF-8 and Miz-1ΔN357 stems from the fact that a mutated IAD of IRF-8, in which leucine 331 was mutated to proline (L331P), was incapable of interacting in this assay. This mutation is at a conserved leucine in a predicted α-helix structure in the IAD, essential for the interaction with other factors (28Meraro D. Hashmueli S. Koren B. Azriel A. Oumard A. Kirchhoff S. Hauser H. Nagulapalli S. Atchison M.L. Levi B.Z. J. Immunol. 1999; 163: 6468-6478PubMed Google Scholar, 35Ortiz M.A. Light J. Maki R.A. Assa-Munt N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2740-2745Crossref PubMed Scopus (41) Google Scholar). No interaction with Miz-1ΔN357 was observed if either the IAD of IRF-8 or the full factor were swapped with the corresponding segments of IRF-4, the closest homologue of IRF-8, and IRF-7. These data further demonstrate the specificity of interaction between IRF-8 and Miz-1 in yeast cells. The Interaction between IRF-8 and Miz-1 Is Identified Only in a Cell Line of Hematopoietic Origin—To show that the interaction between IRF-8 and Miz-1 takes place in mammalian cells, coimmunoprecipitation assays were performed in COS-7 cells that allow overexpression of these two factors. Both full-length IRF-8 and Miz-1 were cloned under the cytomegalovirus promoter, and their expression in transfected cells was readily detected by Western blotting (Fig. 1, A and C, lanes 1 and 6, respectively). However, when Miz-1 was immunoprecipitated, no coprecipitation of IRF-8 could be detected (Fig. 1A, lane 2). In addition, lanes 3 and 4 served as controls for the specificity of the coimmunoprecipitations, and lane 5 demonstrates that coexpression of IRF-8 had no effect on ectopic expression of Miz-1. Expression of just the truncated form of Miz-1 (Miz-1ΔN357) did not lead to coprecipitation of IRF-8 as was expected from the yeast two-hybrid assays (data not shown). Therefore, we decided to detect possible interaction between these two factors using mammalian two-hybrid assay. For that purpose, the C terminus of IRF-8 was fused to the herpes simplex virus VP16 transactivation domain (IRF-8-VP16) (30Schaper F. Kirchhoff S. Posern G. Koster M. Oumard A. Sharf R. Levi B.Z. Hauser H. Biochem. J. 1998; 335: 147-157Crossref PubMed Scopus (79) Google Scholar), and the mammalian expression vector encoding to this chimeric factor was cotransfected with a luciferase reporter gene driven by five repeats of the GAL4 binding site (pGAL4× 5Luc) into the fibroblast cell line NIH3T3. As expected, this chimeric IRF-8 was not able to induce the reporter gene (Fig. 1B, column 2). In addition, the DBD of the yeast transcription factor GAL4 was fused to the N terminus of Miz-1 and was cotransfected with the same reporter plasmid. This lead to a 25-fold increase in the level of the reporter gene indicating that as expected this GAL4-Miz-1 chimeric construct acted as a transcriptional activator (Fig. 1B, column 1). When both GAL4-Miz-1 and IRF-8-VP16 were cotransfected together with the reporter gene, no further induction of the reporter activity was observed, strongly suggesting that the two factors do not interact in the transfected cells (Fig. 1B, column 3). Because IRF-8 is a myeloid-specific factor, we repeated these experiments in the promyelocytic cell line U937. In this cell line, the GAL4-Miz-1 chimeric construct induced the reporter gene expression by 19-fold, but together with IRF-8-VP16 the induction was almost 50-fold (Fig. 1D, columns 1 and 3, respectively). These results strongly suggest that Miz-1 and IRF-8 lead to a statistically significant synergistic effect in this cell line as calculated in Fig. 1E. Thus, interaction between IRF-8 and Miz-1 might occur only in cells of hematopoietic origin. This suggests that these factors either have undergone specific modifications that occur only in immune cells or interact with a third-party element present only in such cells. To test this, coimmunoprecipitation experiments were performed as described in Fig. 1A except that, prior to the precipitation step, the COS-7 cell extracts were incubated first with cell extract from the promyelocytic cell line HL-60, which does not express detectable levels of IRF-8. As seen in Fig. 1C, under these conditions a weak yet reproducible band of IRF-8 coprecipitating with Miz-1 is detected (Fig. 1C, lane 2). IRF-8 Represses Miz-1 Activity on a Synthetic Promoter— Because no target promoter for both IRF-8 and Miz-1 was previously reported, we decided to test the effect of these factors on a synthetic promoter composed of five GAL4 binding sites driving the expression of the firefly luciferase reporter (pGAL4× 5-Luc). The full-length Miz-1 and IRF-8 were fused to the DBD of GAL4 (see schematic in Fig. 2A), and their effect on the reporter gene activity was tested. It is clear that GAL4-Miz-1 strongly activated this synthetic promoter (Fig. 2, B and C, lanes 4 and 2, respectively), whereas increasing amounts of GAL4-IRF-8 (Fig. 2B, columns 5–9) or just GAL4 DBD alone (Fig. 2B, columns 2 and 3) had no significant effect. However, cotransfection of increasing amounts of GAL4-IRF-8 with GAL4-Miz-1 leads to repression of the reporter gene that was not observed when increasing amounts of just GAL4D