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Interferon Regulatory Factor-8 Is Indispensable for the Expression of Promyelocytic Leukemia and the Formation of Nuclear Bodies in Myeloid Cells

2006; Elsevier BV; Volume: 282; Issue: 8 Linguagem: Inglês

10.1074/jbc.m607825200

ISSN

1083-351X

Autores

Natalie Dror, Naama Rave-Harel, Andreas Burchert, Aviva Azriel, Tomohiko Tamura, Prafullakumar Tailor, Andreas Neubauer, Keiko Ozato, Ben-Zion Levi,

Tópico(s)

interferon and immune responses

Resumo

Interferon (IFN) regulatory factor-8 (IRF-8), previously known as ICSBP, is a myeloid cell essential transcription factor. Mice with null mutation in IRF-8 are defective in the ability of myeloid progenitor cells to mature toward macrophage lineage. Accordingly, these mice develop chronic myelogenous leukemia (CML). We demonstrate here that IRF-8 is an obligatory regulator of the promyelocytic leukemia (PML) gene in activated macrophages, leading to the expression of the PML-I isoform. This regulation is most effective together with two other transcription factors, IRF-1 and PU.1. PML is a tumor suppressor gene that serves as a scaffold protein for nuclear bodies. IRF-8 is not only essential for the IFN-γ-induced expression of PML in activated macrophages but also for the formation of nuclear bodies. Reduced IRF-8 transcript levels were reported in CML patients, and a recovery to normal levels was observed in patients in remission following treatment with IFN-α. We demonstrate a significant correlation between the levels of IRF-8 and PML in these CML patients. Together, our results indicate that some of the myeloleukemia suppressor activities of IRF-8 are mediated through the regulation of PML. When IRF-8 levels are compromised, the reduced PML expression may lead to genome instability and eventually to the leukemic phenotype. Interferon (IFN) regulatory factor-8 (IRF-8), previously known as ICSBP, is a myeloid cell essential transcription factor. Mice with null mutation in IRF-8 are defective in the ability of myeloid progenitor cells to mature toward macrophage lineage. Accordingly, these mice develop chronic myelogenous leukemia (CML). We demonstrate here that IRF-8 is an obligatory regulator of the promyelocytic leukemia (PML) gene in activated macrophages, leading to the expression of the PML-I isoform. This regulation is most effective together with two other transcription factors, IRF-1 and PU.1. PML is a tumor suppressor gene that serves as a scaffold protein for nuclear bodies. IRF-8 is not only essential for the IFN-γ-induced expression of PML in activated macrophages but also for the formation of nuclear bodies. Reduced IRF-8 transcript levels were reported in CML patients, and a recovery to normal levels was observed in patients in remission following treatment with IFN-α. We demonstrate a significant correlation between the levels of IRF-8 and PML in these CML patients. Together, our results indicate that some of the myeloleukemia suppressor activities of IRF-8 are mediated through the regulation of PML. When IRF-8 levels are compromised, the reduced PML expression may lead to genome instability and eventually to the leukemic phenotype. Interferon (IFN) 2The abbreviations used are: IFN, interferon; IRF, IFN regulatory factor; CML, chronic myelogenous leukemia; NBs, nuclear bodies, AML, acute myelocytic leukemia; WT, wild type; KO, knock-out; RARα, retinoic acid receptor-α; APL, acute promyelocytic leukemia; PML, promyelocytic leukemia; RT, reverse transcription; LPS, lipopolysaccharide; ISRE, IFN-simulated response element; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RA, retinoic acid; DC, dendritic cell. regulatory factor-8 (IRF-8), previously known as ICSBP (1Driggers P.H. Ennist D.L. Gleason S.L. Mak W.H. Marks M.S. Levi B.Z. Flanagan J.R. Appella E. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3743-3747Crossref PubMed Scopus (314) Google Scholar), is a myeloid cell essential transcription factor that belongs to the IRF family (for review see Refs. 2Tamura T. Ozato K. J. Interferon Cytokine Res. 2002; 22: 145-152Crossref PubMed Scopus (166) Google Scholar and 3Levi B.Z. Hashmueli S. Gleit-Kielmanowicz M. Azriel A. Meraro D. J. Interferon Cytokine Res. 2002; 22: 153-160Crossref PubMed Scopus (65) Google Scholar). This factor is expressed exclusively in immune cells and accordingly plays a major role in the differentiation, development, and function of macrophages (4Tamura T. Thotakura P. Tanaka T.S. Ko M.S. Ozato K. Blood. 2005; 106: 1938-1947Crossref PubMed Scopus (105) Google Scholar, 5Tsujimura H. Nagamura-Inoue T. Tamura T. Ozato K. J. Immunol. 2002; 169: 1261-1269Crossref PubMed Scopus (76) Google Scholar, 6Tamura T. Nagamura-Inoue T. Shmeltzer Z. Kuwata T. Ozato K. Immunity. 2000; 13: 155-165Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), dendritic cells (7Tamura T. Tailor P. Yamaoka K. Kong H.J. Tsujimura H. O'Shea J.J. Singh H. Ozato K. J. Immunol. 2005; 174: 2573-2581Crossref PubMed Scopus (338) Google Scholar, 8Schiavoni G. Mattei F. Borghi P. Sestili P. Venditti M. Morse H.C. II I Belardelli F. Gabriele L. Blood. 2004; 103: 2221-2228Crossref PubMed Scopus (94) Google Scholar, 9Tsujimura H. Tamura T. Ozato K. J. Immunol. 2003; 170: 1131-1135Crossref PubMed Scopus (195) Google Scholar, 10Schiavoni G. Mattei F. Sestili P. Borghi P. Venditti M. Morse H.C. II I Belardelli F. Gabriele L. J. Exp. Med. 2002; 196: 1415-1425Crossref PubMed Scopus (348) Google Scholar), and B-cells (11Lu R. Medina K.L. Lancki D.W. Singh H. Genes Dev. 2003; 17: 1703-1708Crossref PubMed Scopus (207) Google Scholar). Mice with IRF-8 null mutation are defective in the ability of myeloid progenitor cells to mature toward macrophage lineage and eventually develop chronic myelogenous leukemia (CML)-like syndrome (6Tamura T. Nagamura-Inoue T. Shmeltzer Z. Kuwata T. Ozato K. Immunity. 2000; 13: 155-165Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 12Holtschke 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 H.C. Ozato K. Horak I. Cell. 1996; 87: 307-317Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). Furthermore, IRF-8 blocks the development of murine Bcr-Abl-induced CML (13Hao S.X. Ren R. Mol. Cell. Biol. 2000; 20: 1149-1161Crossref PubMed Scopus (127) Google Scholar) through the inhibition of Bcl-2 (14Burchert A. Cai D. Hofbauer L.C. Samuelsson M.K. Slater E.P. Duyster J. Ritter M. Hochhaus A. Muller R. Eilers M. Schmidt M. Neubauer A. Blood. 2004; 103: 3480-3489Crossref PubMed Scopus (89) Google Scholar) and induction of anti-leukemic immunity (15Deng M. Daley G.Q. Blood. 2001; 97: 3491-3497Crossref PubMed Scopus (49) Google Scholar). In humans, down-regulation of IRF-8 expression was reported in CML and acute myelocytic leukemia (AML) patients. Accordingly, a significant increase in IRF-8 expression was correlated with remission following treatment with IFN-α (14Burchert A. Cai D. Hofbauer L.C. Samuelsson M.K. Slater E.P. Duyster J. Ritter M. Hochhaus A. Muller R. Eilers M. Schmidt M. Neubauer A. Blood. 2004; 103: 3480-3489Crossref PubMed Scopus (89) Google Scholar, 16Schmidt M. Nagel S. Proba J. Thiede C. Ritter M. Waring J.F. Rosenbauer F. Huhn D. Wittig B. Horak I. Neubauer A. Blood. 1998; 91: 22-29Crossref PubMed Google Scholar, 17Schmidt M. Hochhaus A. Nitsche A. Hehlmann R. Neubauer A. Blood. 2001; 97: 3648-3650Crossref PubMed Scopus (79) Google Scholar). Together, these observations point toward a role for IRF-8 as a tumor suppressor gene. To characterize the IRF-8 regulatory network in activated macrophages, a differential expression profile of peritoneal macrophages extracted from wild type (WT) mice versus IRF-8-/- mice, before and following 4 h of stimulation with IFN-γ and LPS, was studied using DNA microarray assay (18Dror N. Alter-Koltunoff M. Azriel A. Amariglio N. Jacob-Hirsch J. Zeligson S. Morgenstern A. Tamura T. Hauser H. Rechavi G. Ozato K. Levi B.Z. Mol. Immunol. 2007; 44: 338-346Crossref PubMed Scopus (64) Google Scholar). Among the many putative IRF-8-target genes, the promyelocytic leukemia (PML) gene was identified. PML is a tumor suppressor gene that serves as a scaffold protein for nuclear bodies (NBs). PML-NBs associate with numerous proteins and therefore were implicated in a variety of cellular processes such as cell cycle regulation, apoptosis, proteolysis, tumor suppression, DNA repair, and transcription (for review see Refs. 19Salomoni P. Pandolfi P.P. Cell. 2002; 108: 165-170Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, 20Bernardi R. Pandolfi P.P. Oncogene. 2003; 22: 9048-9057Crossref PubMed Scopus (159) Google Scholar, 21Dellaire G. Bazett-Jones D.P. BioEssays. 2004; 26: 963-977Crossref PubMed Scopus (327) Google Scholar, 22Ching R.W. Dellaire G. Eskiw C.H. Bazett-Jones D.P. J. Cell Sci. 2005; 118: 847-854Crossref PubMed Scopus (112) Google Scholar). These NBs are disrupted during oncogenesis (23Mu Z.M. Le X.F. Glassman A.B. Chang K.S. Leuk. Lymphoma. 1996; 23: 277-285Crossref PubMed Scopus (19) Google Scholar) and viral infection (24Muller S. Dejean A. J. Virol. 1999; 73: 5137-5143Crossref PubMed Google Scholar). PML is involved in acute promyelocytic leukemia (APL) because of a reciprocal translocation between chromosome 15 (PML locus) and chromosome 17 (retinoic acid receptor-α (RARα) locus) leading to in-frame fusion peptides between these two loci (25de The H. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1202) Google Scholar, 26Goddard A.D. Borrow J. Freemont P.S. Solomon E. Science. 1991; 254: 1371-1374Crossref PubMed Scopus (440) Google Scholar, 27Kakizuka A. Miller Jr., W.H. Umesono K. Warrell Jr., R.P. Frankel S.R. Murty V.V. Dmitrovsky E. Evans R.M. Cell. 1991; 66: 663-674Abstract Full Text PDF PubMed Scopus (1297) Google Scholar). In this study we show that PML is regulated by IRF-8 in myeloid cells, and we provide evidence that the IRF-8-mediated CML tumor suppressor activity may be delegated via PML. Patient Samples—The cDNAs of peripheral blood from 13 healthy donors, 13 patients in chronic phase of CML at diagnosis, and 7 patients with CML in major or complete cytogenetic remission under an IFNα-based therapy were studied (14Burchert A. Cai D. Hofbauer L.C. Samuelsson M.K. Slater E.P. Duyster J. Ritter M. Hochhaus A. Muller R. Eilers M. Schmidt M. Neubauer A. Blood. 2004; 103: 3480-3489Crossref PubMed Scopus (89) Google Scholar). All patients and healthy donors gave written informed consent for the use of their samples. Animals—The mouse strains, C57BL/6J (Harlan Biotech, Israel), IRF-1-/- (kindly obtained from Dr. Rubinstein, The Weizmann Institute, Israel, originally from The Jackson Laboratory), and IRF-8-/- (12Holtschke 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 H.C. Ozato K. Horak I. Cell. 1996; 87: 307-317Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar), were maintained in microisolator cages in a viral pathogen-free facility. All animal work conformed to the guidelines of the animal care and use committee of the Technion. Cell Lines—U937 human leukemic monocyte lymphoma, HeLa cervical adenocarcinoma, RAW264.7 mouse monocyte macrophage, and NIH3T3 mouse fibroblast cell lines were obtained from the ATCC (Manassas, VA). CL-2 murine macrophage cell line and Tot2 myeloid progenitor cell line were derived from IRF-8-/- mice and maintained as described previously (6Tamura T. Nagamura-Inoue T. Shmeltzer Z. Kuwata T. Ozato K. Immunity. 2000; 13: 155-165Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 28Wang I.M. Contursi C. Masumi A. Ma X. Trinchieri G. Ozato K. J. Immunol. 2000; 165: 271-279Crossref PubMed Scopus (160) Google Scholar). Plasmids and Transfections—Mammalian expression vectors encoding for IRF-8, IRF-1, and PU.1 were all described previously (29Meraro 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). The pGL3-PML containing the 1.44-kb promoter region conjugated to luciferase (30Stadler M. Chelbi-Alix M.K. Koken M.H. Venturini L. Lee C. Saib A. Quignon F. Pelicano L. Guillemin M.C. Schindler C. de The H. Oncogene. 1995; 11: 2565-2573PubMed Google Scholar) was a kind gift from Dr. de Thé; (Hopital St. Louis, Paris, France). The PGL3-mut-PML promoter harboring 2-bp mutations (AA bases at positions 1422 and 1423 were changed to CG) that destroyed its IFN-simulated response element (ISRE, the DNA binding motif for IRFs) is described hereafter. Site-directed Mutagenesis—Mutagenesis of the ISRE site within the human PML promoter was performed using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using the following primers: 5′-GAT CTA AAC CGA GAA TCG CGA CTA AGC TGG GGT CC-3′ and the complementary oligonucleotide (positions 1404-1435 in the GenBank™ accession number X91752). The AA bases (1422 and 1423) were changed to CG and created an NruI site. Isolation of Peritoneal Macrophages—Peritoneal macrophages were harvested as described previously (31Alter-Koltunoff M. Ehrlich S. Dror N. Azriel A. Eilers M. Hauser H. Bowen H. Barton C.H. Tamura T. Ozato K. Levi B.Z. J. Biol. Chem. 2003; 278: 44025-44032Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 2.5 × 107 cells were plated in tissue culture Petri dishes (100 mm) and kept at 37 °C and 5% CO2. After 4 h, nonadherent cells were removed, and 24 h later, adherent cells were either not treated or treated for 4 h with 100 units/ml IFN-γ (CytoLab, Rehovot, Israel) and 10 ng/ml LPS (Sigma). Isolation of Dendritic Cells (DCs)—The Flt3L method was used with minor modifications. Briefly, BM mononuclear cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, and recombinant human Flt3L (100 ng/ml, PeproTech, Rocky Hill, NJ) for 9 days. During the final 24 h of culture, cells were either not stimulated or stimulated with IFN-γ (100 units/ml) and LPS (10 ng/ml). Nonadherent DCs were harvested by gentle pipetting leaving adherent cells behind. RNA Extraction and Northern Blot Analysis—Total RNA from cells and tissues was extracted with Tri-Reagent (Sigma) according to the manufacturer's protocol, separated (15 μg) on a 1.6% agarose/formaldehyde gel, and blotted as described before (31Alter-Koltunoff M. Ehrlich S. Dror N. Azriel A. Eilers M. Hauser H. Bowen H. Barton C.H. Tamura T. Ozato K. Levi B.Z. J. Biol. Chem. 2003; 278: 44025-44032Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Radiolabeled 600-bp murine PML cDNA fragment, generated by RT-PCR from total RNA of RAW264.7 cells by using the oligonucleotide primers 5′-AACTGTGGCTCCGTCCATAC-3′ (sense) and 5′-CCACAGGGAACAACTGACCT-3′ (antisense), was used as a probe. Real Time RT-PCR Analysis—100 ng of total RNA was reverse-transcribed to cDNA using Reverse-iT™ 1st strand synthesis kit (ABgene, Surrey, UK) according to manufacturer's protocol. cDNA was amplified with two primers for each gene using ABsolute SYBER Green ROX Mix (ABgene, Surrey, UK) and Rotor-Gene 3000™ Real Time thermal cycler (Corbett Research, Australia) according to the manufacturer's instructions. The amplification conditions for all reactions were one time at 95 °C for 15 min followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 15 s. Quantitative results of real time PCR were assessed by determining the relative calculated concentration values. Using Primer3 software primers were designed as follows: mouse PML 5′-CAGGCCCTAGAGCTGTCTAAG-3′ (sense) and 5′-ATACACTGGTACAGGGTGTGC-3′ (antisense); mouse GAPDH 5′-AGGTCGGTGTGAACGGATTTG-3′ (sense) and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (antisense); human PML 5′-AGGATGTCTCCAATACAACG-3′ (sense) and 5′-CTCCTCAGACTCCATCTTGA-3′ (antisense); human IRF-8 5′-GAAGACGAGGGTTACGCTGTG-3′ (sense) and 5′-TCCTCAGGAACAATTCGGTAA-3′ (antisense); human GAPDH 5′-CCTGGTATGACAACGAATTT-3′ (sense), 5′-GTGAGGGTCTCTCTCTTCCT-3′ (antisense). The primers used for PML isoforms amplification were designed using PrimerExpress software (ABI) are: PML-I 5′-CAAGATTGACAATGAAACCCAGAA-3′ (sense), 5′-GGATGACCACGCGGAACTT-3′ (antisense); PML-II 5′-GGTGATCAGCAGCTCGGAAG-3′ (sense), 5′-GGGCTCCATGCACGAGTT-3′ (antisense); PML-III 5′-AGGAAGTGAGGTCTTCCTGC-3′ (sense) and 5′-CTCCCTACCTGCCTCCCCGGC-3′ (antisense); PML-IV 5′-ACCCCCAAGCAGAAGACAGA-3′ (sense) and 5′-CCCAGGAGAACCCACTTTCA-3′ (antisense); PML-V 5′-ACGCGTTGTGGTGATCAGC-3′ (sense) and 5′-TGGGCCACTCACCGATT-3′ (antisense); and PML-VI 5′-TTCCAGCCCTCAGTCTGAGGT-3′ (sense) and 5′-GGTCTCCATGGGCTCCATG-3′ (antisense). The estimated amount of transcripts was normalized to GAPDH mRNA expression to compensate for variations in quantity or quality of starting mRNA and for differences in reverse transcriptase efficiency. The gene of interest/GAPDH ratio was expressed in percentage. The data are presented as the fold of induction for the gene of interest, e.g. the ratio between the relative mRNA levels in treated versus untreated cells. Retroviral Transduction—Retroviral pMSCV-puro and pMSCV-ICSBP-puro vectors and preparation of retroviruses were described previously (2Tamura T. Ozato K. J. Interferon Cytokine Res. 2002; 22: 145-152Crossref PubMed Scopus (166) Google Scholar). Tot2 cells or DCs were transduced with retroviruses by spinoculation (2500 rpm for 1 h at 33 °C, twice) in the presence of 4 μg/ml Polybrene (Sigma) on days 1 and 2. On day 4, puromycin (2 μg/ml) was added to select for transduced cells. Transduced cells were analyzed on day 6 (Tot2) or day 9 (DCs). Electrophoretic Mobility Shift Assay (EMSA)—Nuclear extracts from RAW264.7 cells and EMSAs were performed as described previously (32Meraro D. Gleit-Kielmanowicz M. Hauser H. Levi B.Z. J. Immunol. 2002; 168: 6224-6231Crossref PubMed Scopus (95) Google Scholar) with 32P-labeled double-stranded DNA oligonucleotide corresponding to the human PML ISRE (sense 5′-TAAACCGAGAATCGAAACTAAGCTGGGGT-3′ and antisense 5′-ACCCCAGCTTAGTTTCGATTCTCGGTTTA-3′). Competitions were performed with a 100-200-fold excess of WT or mutated ISRE (sense 5′-TAAACCGAGCCTCGACCCTAAGCTGGGGT-3′ and antisense 5′-ACCCCAGCTTAGGGTCGAGGCTCGGTTTA-3′). Supershift assays were performed by adding 0.2 μg of rabbit polyclonal antibodies against IRF-1, IRF-8, PU.1, and IgG (Santa Cruz Biotechnology). Gels were run at 250 V for 2 h and were then subjected to autoradiography. FAST Chromatin Immunoprecipitation (ChIP) Assay—RAW264.7 cells were grown to a final concentration of 1 × 107 with or without treatment with IFN-γ (100 units/ml) and LPS (10 ng/ml) for 12 h, and ChIP assays were performed exactly as described (33Nelson J.D. Denisenko O. Sova P. Bomsztyk K. Nucleic Acids Res. 2006; 34: e2Crossref PubMed Scopus (182) Google Scholar). Protein-DNA complexes were incubated with 5 μg of anti-rabbit IRF-1, PU.1, and anti-goat IRF-8 antibodies (Santa Cruz Biotechnology) or acetyl-specific histone 3 antibody (Upstate Biotechnology). Immunoprecipitated DNA was analyzed for the murine PML promoter sequence by real time PCR (ABI 7300, Absolute quantification program) using the following primers: 5′-AGAGTTACTTCCATAGCTCCCTAGTCTTT-3′ (sense) and 5′-GAGAGGAAGTGAGACAGG-3′ (antisense). Relative quantity was calculated as described (33Nelson J.D. Denisenko O. Sova P. Bomsztyk K. Nucleic Acids Res. 2006; 34: e2Crossref PubMed Scopus (182) Google Scholar). Western Blot Analysis—Protein extracts (12 μg of nuclear/cytoplasmic) were separated on a 10% SDS-PAGE and blotted to polyvinylidene difluoride membrane that was subjected to Ponceau S staining (Sigma). Subsequently, Western blot was performed with rabbit polyclonal anti-PML (Santa Cruz Biotechnology) as described previously (34Hashmueli S. Gleit-Kielmanowicz M. Meraro D. Azriel A. Melamed D. Levi B.Z. Int. Immunol. 2003; 15: 807-815Crossref PubMed Scopus (7) Google Scholar). Immunoreactive proteins were visualized by the SuperSignal West Pico chemiluminescent ECL detection kit (Pierce). Immunofluorescent Staining—Immunofluorescent staining was performed as described by Boulware and Weber (35Boulware S.L. Weber P.C. J. Gen. Virol. 2000; 81: 1773-1777Crossref PubMed Scopus (5) Google Scholar). Cells were incubated for 2 h at room temperature with rabbit polyclonal anti-PML antibody (Santa Cruz Biotechnology) and subsequently for 1 h with the secondary antibody (donkey anti-rabbit Rhodamine Red X-conjugate, Jackson ImmunoResearch). Cells were stained with Hoechst 33342 (0.3 μg/ml) in PBS, at room temperature for 5 min. Following mounting, fluorescence was visualized under Leica TCS-SP2 confocal laser scanning unit. Bioinformatics Analysis of Promoter Region—Analysis of the promoter sequence was carried out with GEMS Launcher (Genomatix Software GmbH, Munich, Germany) using the FrameWorker tool which is based on the algorithms described. IRF-8 Is Essential for the Induced Expression of PML in Macrophage Cells and for the Constitutive Expression in Hematopoietic Organs—To identify genes that are regulated by IRF-8 and IRF-1 in macrophages, DNA microarray analysis was employed (18Dror N. Alter-Koltunoff M. Azriel A. Amariglio N. Jacob-Hirsch J. Zeligson S. Morgenstern A. Tamura T. Hauser H. Rechavi G. Ozato K. Levi B.Z. Mol. Immunol. 2007; 44: 338-346Crossref PubMed Scopus (64) Google Scholar). For this purpose, we compared the expression profile of genes in peritoneal macrophages from WT and from IRF-8-/- mice, before and following 4 h of stimulation with IFN-γ and LPS. This analysis led to the identification of PML as a putative gene regulated by IRF-8 in activated macrophages (18Dror N. Alter-Koltunoff M. Azriel A. Amariglio N. Jacob-Hirsch J. Zeligson S. Morgenstern A. Tamura T. Hauser H. Rechavi G. Ozato K. Levi B.Z. Mol. Immunol. 2007; 44: 338-346Crossref PubMed Scopus (64) Google Scholar). To further validate these results, Northern blot analyses were performed using nonstimulated as well as stimulated peritoneal macrophages and the macrophage cell lines RAW264.7 (expressing IRF-8) and CL-2 (originating from IRF-8 KO mice (28Wang I.M. Contursi C. Masumi A. Ma X. Trinchieri G. Ozato K. J. Immunol. 2000; 165: 271-279Crossref PubMed Scopus (160) Google Scholar)). As seen in Fig. 1A, a strong induction of PML mRNA was observed following 4 h of treatment with IFN-γ and LPS in peritoneal macrophages and in RAW264.7 cells (Fig. 1A, lanes 2 and 6, respectively). This PML up-regulation was not observed in peritoneal macrophages and CL-2 cells originating from IRF-8 KO mice (Fig. 1A, lanes 4 and 8, respectively). By using real time PCR, it was clear that the mRNA level of PML was induced by 5-fold in peritoneal macrophages from WT mouse strain and by over 7-fold in the macrophage cell line RAW264.7 but not in cells from IRF-8 and IRF-1 null mice (Fig. 1B). RAW264.7 cells and peritoneal macrophages were also treated with either IFN-γ or LPS alone in 8-h intervals up to 24 h (data not shown). PML transcript levels were not affected by LPS at all times. However, IFN-γ induced both IRF-8 and PML mRNA to similar levels shown in Fig. 1B for the combined treatment (IFN-γ and LPS, for 4 h), and therefore the latter was used in subsequent experiments. Taken together, PML induction was compromised only in cells from the IRF-8-/- and IRF-1-/- mouse strains. We next examined the expression level of PML in hematopoietic tissues (thymus, liver, spleen, and lung), and in a non-hematopoietic tissue (heart). mRNA was extracted from these tissues from the WT as well as from the IRF-8-/- mouse strains and subjected to Northern blot analysis. A strong mRNA signal corresponding to PML was observed in samples from the WT tissues except for the heart (Fig. 1C, compare lanes 1, 3, 5, and 7 to lane 9). We attribute this strong signal to the hematopoietic nature of these tissues, including the lung, which is rich in alveolar macrophages. Interestingly, PML mRNA levels in all samples extracted from the KO mice were significantly lower than in the WT counterparts. In fact, they were similar to the level of PML mRNA observed in the heart of the WT mice (Fig. 1C, lanes 2, 4, 6, 8, and 10). Taken together, the data indicated that IRF-8 is essential for the expression of PML in hematopoietic tissues. To test the effect of IRF-8 on PML protein expression, cytoplasmic and nuclear extracts were prepared from RAW264.7 and CL-2 cells before and following activation with IFN-γ and LPS. Western blot analysis was performed using antibody against PML, and as seen in Fig. 1D, induced expression of PML is observed only in nuclear extract from the WT activated cells (e.g. RAW264.7 cells) in perfect alignment with the mRNA data. IRF-8 Is Essential for the Expression of Specific PML Isoform(s)—In humans, a large number of alternatively spliced PML transcripts generate a variety of PML isoforms that differ in their C-terminal sequences (see illustration in Fig. 2A). Recent studies assign specific activities to specific isoforms (36Fogal V. Gostissa M. Sandy P. Zacchi P. Sternsdorf T. Jensen K. Pandolfi P.P. Will H. Schneider C. Del Sal G. EMBO J. 2000; 19: 6185-6195Crossref PubMed Scopus (321) Google Scholar, 37Nguyen L.A. Pandolfi P.P. Aikawa Y. Tagata Y. Ohki M. Kitabayashi I. Blood. 2005; 105: 292-300Crossref PubMed Scopus (36) Google Scholar, 38Lin H.K. Bergmann S. Pandolfi P.P. Nature. 2004; 431: 205-211Crossref PubMed Scopus (265) Google Scholar, 39Xu Z.X. Zou W.X. Lin P. Chang K.S. Mol. Cell. 2005; 17: 721-732Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Although the murine PML genomic locus exhibit similar architecture, the murine orthologues were not described yet. To characterize the hematopoietic-specific PML isoform(s), we used a human promyelocytic cell line, U937, that was either activated or not with IFN-γ and LPS for 4 h. The relative mRNA levels for PML isoforms (I-VI) as well as for IRF-8 was determined. We found that in addition to the expected induction in IRF-8 mRNA level (∼18-fold; Fig. 2B), the level of PML-I was significantly induced (10-fold; Fig. 2B). The levels of the other isoforms were marginally elevated as compared with that of IRF-8 and PML-I. This induction of PML-I was not observed in HeLa cells, and the expression levels were rather low before and following activation (data not shown). These results suggest that PML-I isoform is the major splice variant that functions in IFN-γ and LPS-activated U937 cells. The IRF-8-regulated Expression of PML Is Mediated by ISRE Element(s) Located in the PML Promoter—PML expression is induced by IFNs through identified ISRE and GAS elements in its promoter (30Stadler M. Chelbi-Alix M.K. Koken M.H. Venturini L. Lee C. Saib A. Quignon F. Pelicano L. Guillemin M.C. Schindler C. de The H. Oncogene. 1995; 11: 2565-2573PubMed Google Scholar). However, the transcription factors involved in this induced expression were not determined. To determine the role of IRF-8 in the regulated expression of PML, reporter gene assays were performed. The characterized human PML promoter was transfected into RAW264.7 and CL-2 cells. Because IRF-8 is commonly engaged in heterocomplexes with PU.1 (3Levi 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 role of the latter in this regulated expression was studied as well. PU.1 is a transcription factor obligatory for hematopoiesis (40Dahl R. Simon M.C. Blood Cells Mol. Dis. 2003; 31: 229-233Crossref PubMed Scopus (70) Google Scholar). In addition, because PML was selected in our DNA microarray analysis of macrophages from both IRF-8-/- and IRF-1-/- mice, we hypothesized that IRF-1 may also be an important regulator of PML, and therefore its effect on PML expression was investigated. Treatment of RAW264.7 cells with IFN-γ and LPS for 18 h led to the induced expression of the reporter gene (data not shown) that was equivalent to that observed following co-transfection of IRF-8-expressing vector (Fig. 3A, black columns). In addition, transfection of either IRF-1 or PU.1 expression vectors led to a significant increase in the reporter gene levels similar to that of IRF-8. Combined transfection of IRF-8 and IRF-1 led to a further increase in reporter activity. A strong additive activation of the reporter gene was observed when all three factors were cotransfected. Similar results were obtained with the CL-2 cells, lacking IRF-8. Here the reporter gene induced by IRF-8 was more profound (4-fold higher) than that of IRF-1 or PU.1 alone (Fig. 3A, white columns). This finding highlights the major role of IRF-8 in the expression of PML. To test the importance of the characterized ISRE (41Chelbi-Alix M.K. Quignon F. Pelicano L. Koken M.H. de The H. J. Virol. 1998; 72: 1043-1051Crossref PubMed Google Scholar), the binding site for IRFs in the PML promoter, 2-bp substitutions at positions 1422 and 1423 (AA to GC) were introduced to the PML reporter construct (mut ISRE). As seen in Fig. 3A (gray bars), only PU.1 could still affect this mutated reporter construct in RAW264.7 cells. The effects of IRF-8 and IRF-1 were reduced by more than 50%. This demonstrates that transcriptional activation of the PML promoter by both IRF-8 and IRF-1 is indeed mediated by this ISRE. To further characterize the binding activities of these three transcription factors, EMSA was performed using nuclear extracts from resting RAW264.7 cells. A DNA segment from the human PML promoter, encompassing the previously characterized ISRE element, was used as a probe (30Stadler M. Chelbi-Alix M.K. Koken M.H. Venturini L. Lee C. Saib A. Quignon F. Pelicano L. Guillemin M.C. Schindler C. de The H. Oncogene. 1995; 11: 2565-2573PubMed Google Scholar). A major slow migrating band was observed (Fig. 3B, indicated by arrowhead) that was competed effectively by an excess of unlabeled PML ISRE and not by an excess of mutated ISRE oligonucleotide (Fig. 3B, lanes 2 and 3, respectively). This indicates that the competed band results from specific binding to the ISRE. To identify the proteins engaged in this binding, specific antibodies directed against

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