Interferon regulatory factor 1 induces the expression of the interferon‐stimulated genes
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The interferon regulatory factor 1 (IRF‐1) is a positive transcriptional regulatory protein which acts in the interferon signal transduction pathway to activate the transcription of the type I interferon genes by binding to the PRDI response element. The aim of this study was to explore the role of IRF‐1 in regulating the expression of other interferon‐stimulated genes in the interferon signal transduction pathway. A transient transfection assay was used to show that IRF‐1 induced the expression of interferon‐stimulated genes. The induction was a direct result of IRF‐1 binding to the promoters of the interferon‐stimulated response element (ISRE). The levels of endogenous mRNA of two interferon‐stimulated genes, 6‐16 and 9‐27, were increased in cells containing increased levels of IRF‐1. In addition, IRF‐1 activates the expression of IRF‐2, a negative regulator of the type I interferon genes themselves. Two sequences were found in the IRF‐2 promoter which were the binding sites for IRF‐1. Mutations in the oligonucleotide sequences of these sites could abolish the binding of the IRF‐1. These data suggested that IRF‐1 not only plays an important role in the induction of type I interferon genes, but also in the activation of interferon‐stimulated genes.Keywords:
IRF1
Response element
IRF8
Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells. In previous studies, we found that the interferon consensus sequence-binding protein (ICSBP; also referred to as interferon regulatory factor 8) activates transcription of the gene encoding Nf1 (the NF1 gene) in differentiating myeloid cells. We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8. In this study, we found that ICSBP/IRF8 cooperates with PU.1 and interferon regulatory factor 2 to activate a composite ets/IRF-cis element in the NF1 promoter. We found that PU.1 binds directly to the NF1-cis element, and DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element. This interaction requires cytokine-induced phosphorylation of specific serine residues in the PU.1 PEST domain and of a conserved tyrosine residue in the IRF domain of IRF2. We found that ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2. The conserved IRF domain tyrosine in ICSBP/IRF8 is required for interaction with the DNA-bound PU.1-IRF2 heterodimer. NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation. Therefore, these studies identify a target gene for the previously observed tumor-suppressor effect of PU.1. Additionally, these studies identify a tumor-suppressor function for the "oncogenic" transcription factor, IRF2. Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells. In previous studies, we found that the interferon consensus sequence-binding protein (ICSBP; also referred to as interferon regulatory factor 8) activates transcription of the gene encoding Nf1 (the NF1 gene) in differentiating myeloid cells. We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8. In this study, we found that ICSBP/IRF8 cooperates with PU.1 and interferon regulatory factor 2 to activate a composite ets/IRF-cis element in the NF1 promoter. We found that PU.1 binds directly to the NF1-cis element, and DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element. This interaction requires cytokine-induced phosphorylation of specific serine residues in the PU.1 PEST domain and of a conserved tyrosine residue in the IRF domain of IRF2. We found that ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2. The conserved IRF domain tyrosine in ICSBP/IRF8 is required for interaction with the DNA-bound PU.1-IRF2 heterodimer. NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation. Therefore, these studies identify a target gene for the previously observed tumor-suppressor effect of PU.1. Additionally, these studies identify a tumor-suppressor function for the "oncogenic" transcription factor, IRF2. Neurofibromin 1 (Nf1) is 2818-amino acid protein with Ras-GAP activity encoded by the NF1 gene (1Ballester R. Marchuk D. Boguski M. Saulino A. Lecher R. Wigler M. Collins F. Cell. 1990; 63: 851-859Abstract Full Text PDF PubMed Scopus (649) Google Scholar). In myeloid progenitor cells and differentiating phagocytes, Nf1-Ras-GAP activity antagonizes granulocyte-macrophage colony-stimulating factor, stem cell factor, or macrophage colony-stimulating factorinduced Ras activation (2Basu T.N. Gutmann D.H. Fletcher J.A. Glover T.W. Colling F.S. Downward J. Nature. 1992; 356: 713-715Crossref PubMed Scopus (571) Google Scholar, 3Bollan G. Clapp D.W. Shih S. Adler F. Zhang Y.Y. Thompson P. Lange B.J. Freedman M.H. McCormick F. Jacks T. Shannon K. Nat. 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Acquired Nf1 deficiency has been documented in myeloid cells from human subjects with acute myeloid leukemia (AML) 2The abbreviations used are: AML, acute myeloid leukemia; ICSBP, interferon consensus sequence-binding protein; IRF2, interferon regulatory factor 2; GST, glutathione S-transferase; MDS, myelodysplastic syndromes; IFN, interferon; EMSA, electrophoretic mobility shift assay; CREB, cAMP-response element-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPA, DNA affinity purification assay; shRNA, short hairpin RNA; ds, double stranded; ERK, extracellular signal-regulated kinase; CAT, chloramphenicol acetyltransferase.2The abbreviations used are: AML, acute myeloid leukemia; ICSBP, interferon consensus sequence-binding protein; IRF2, interferon regulatory factor 2; GST, glutathione S-transferase; MDS, myelodysplastic syndromes; IFN, interferon; EMSA, electrophoretic mobility shift assay; CREB, cAMP-response element-binding protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPA, DNA affinity purification assay; shRNA, short hairpin RNA; ds, double stranded; ERK, extracellular signal-regulated kinase; CAT, chloramphenicol acetyltransferase. and myelodysplastic syndromes (MDS) (7Lu D. Nounou R. Beran M. Estey E. Manshouri T. Kantarjian H. Keating M.J. Albitar M. Cancer. 2003; 97: 441-449Crossref PubMed Scopus (22) Google Scholar). Therefore, although Nf1 expression is not restricted to hematopoietic cells, Nf1 deficiency is implicated in the pathogenesis of malignant myeloid disorders. Consistent with this, Nf1 deficiency induces a myeloproliferative disorder in murine models (8Brannan C.I. Perkins A.S. Vogel K.S. Ratner N. Nordlund M.L. Reid S.W. Buchberg A.M. Jenkins N.A. Parada L.F. Copeland N.G. Genes Dev. 1994; 8: 1019-1029Crossref PubMed Scopus (524) Google Scholar, 9Zhang Y. Vik T.A. Ryder J.W. Srour E.F. Jacks T. Shannon K. Clapp D.W. J. Exp. Med. 1998; 187: 1893-1902Crossref PubMed Scopus (128) Google Scholar). Previously, we found that NF1 transcription and Nf1 expression increase during cytokine-induced differentiation of myeloid leukemia cell lines or murine myeloid progenitor cells (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We also found that cytokine-induced NF1 transcription requires the interferon consensus sequence-binding protein (ICSBP or IRF8) (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). ICSBP/IRF8 is expressed exclusively in myeloid and B-cells (10Nelson N. Marks M.S. Driggers P.H. Ozato K. Mol. Cell. Biol. 1993; 13: 588-599Crossref PubMed Scopus (174) Google Scholar), and acquired ICSBP/IRF8 deficiency is found in bone marrow cells from subjects with chronic myeloid leukemia, AML, and MDS (11Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 12Schmidt M. Nagel S. Proba J. Theide C. Ritter M. Waring J.F. Rosenbauer F. Huhn D. Wittig B. Horak I. Neubauer A. Blood. 1998; 91: 22-29Crossref PubMed Google Scholar). Interestingly, ICSBP/IRF8 deficiency induces myeloproliferation in mice, and myeloid cells from these mice are hypersensitive to the same cytokines as Nf1-deficient cells (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 13Holtchke 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 (542) Google Scholar, 14Scheller M. Foerster J. Heyworth C.M. Waring J.F. Lohler J. Gilmore G.L. Shadduck R.K. Dexter T.M. Horak I. Blood. 1999; 94: 3764-3771Crossref PubMed Google Scholar). Consistent with a role in NF1 transcription, proliferative abnormalities in ICSBP/IRF8-deficient myeloid progenitor cells can be rescued by expression of the Nf1GAP-related domain (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In previous studies, we also found that activation of NF1 transcription requires cytokine-induced phosphorylation of a specific ICSBP/IRF8 tyrosine residue (Tyr-95) (15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar). This residue is in the conserved IRF domain that is thought to be involved in DNA-binding or protein/protein interactions of IRF proteins. ICSBP/IRF8 is a substrate for SHP1 and SHP2 protein-tyrosine phosphatases in undifferentiated myeloid cells (15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar, 16Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar). However, a constitutively activated SHP2 mutant, described in human subjects with MDS, AML, and juvenile myelomonocytic leukemia, dephosphorylates ICSBP/IRF8 in differentiated and undifferentiated myeloid cells (15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar, 17Bentires-Ali M. Paez J.G. David F.S. Keilhack H. Halmos B. Naoki K. Maris J.M. Richardson A. Bardelli A. Sugarbaker D.J. Richards W.G. Du J. Girard L. Minna J.D. Loh M.L. Fisher D.E. Velculescu V.E. Vogelstein B. Meyerson M. Sellers W.R. Neel B.G. Cancer Res. 2004; 64: 8816-8820Crossref PubMed Scopus (412) Google Scholar). Such activated SHP2 mutants also induce cytokine hypersensitivity in myeloid progenitors (15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar, 18Chan R.J. Leedy M.B. Munugalavadla V. Voorhorst C.S. Li Y. Yu M. Kapur R. Blood. 2005; 105: 3737-3742Crossref PubMed Scopus (131) Google Scholar). IRF proteins regulate target gene transcription by interacting with several different DNA-binding site consensus sequences. ICSBP/IRF8 represses cis elements with PRDI consensus sequences (5′-TCACTT-3′) by interacting directly with DNA. In contrast, tyrosine-phosphorylated ICSBP/IRF8 can activate PRDI-cis elements by interaction with DNA-bound IRF1 (19Sharf 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). ICSBP/IRF8 also binds to interferon-stimulated response elements (5′-GAAANNGAAA-3′). Interferon-stimulated response elements can be repressed by an interaction between ICSBP/IRF8, the ets protein Tel, and histone deacetylase 3 (20Kuwata T. Gongora C. Kanno Y. Sakaguchi K. Tamura T. Kanno T. Basrur V. Martinez R. Appella E. Golub T. Ozato K. Mol. Cell. Biol. 2002; 22: 7439-7448Crossref PubMed Scopus (53) Google Scholar). ICSBP/IRF8 activates gene transcription by interacting with composite ets/IRF-cis elements (also referred to as EICE sequences). Such ets/IRF sequences have a loose consensus (5′-GGAA(A/G)TGNNA-3′) and are found in a number of genes expressed in mature phagocytes and involved in the immune response. Examples include genes encoding the respiratory burst oxidase proteins gp91PHOX and p67PHOX and the gene encoding the Toll-like receptor 4 (the CYBB, NCF2, and TLR4 genes, respectively) (11Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 22Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105PubMed Google Scholar). DNA-bound PU.1 interacts with IRF1, and this DNA-bound heterodimer recruits ICSBP/IRF8 and the CREB-binding protein (11Rehli M. Poltorak A. Schwarzfischer L. Krause S.W. Andreesen R. Beutler B. J. Biol. Chem. 2000; 275: 9773-9781Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 22Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105PubMed Google Scholar). These interactions require post-translational modification of the proteins in differentiating myeloid cells. Specifically, interaction of ICSBP/IRF8 with the DNA-bound PU.1-IRF1 heterodimer requires phosphorylation of PU.1 Ser-148 and ICSBP/IRF8 Tyr-95 (16Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar, 21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The ICSBP/IRF8-binding cis element in the proximal NF1 promoter has homology to composite ets/IRF consensus sequences found in myeloid-specific genes. This suggests possible involvement of PU.1 in NF1 transcriptional regulation and perhaps another IRF protein. The goal of these investigations is to determine the mechanism of cytokine-induced NF1 transcription in differentiating myeloid cells. This will be approached by identifying the components of the NF1 transcriptional activation complex. Although composite ets/IRF consensus sequences have been identified in a number of genes involved in the inflammatory response, no such cis elements have previously been identified in target genes regulating proliferation. Identification of homologous cis elements that interact with common trans-factors in genes that regulate both differentiation and proliferation would suggest a common mechanism of cytokine activation of different types of genes. This could have implications for understanding the inter-relationship between differentiation-progression and proliferation-arrest during myelopoiesis. Protein Expression Vectors—The ICSBP/IRF8 cDNA was obtained from Dr. Ben Zion-Levi (Technion, Haifa, Israel), and the full-length cDNA was generated by PCR and subcloned into the mammalian expression vector pcDNAamp, as described (21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). The cDNA for IRF2 in the pcDNAamp vector was obtained from Dr. Gary S. Stein (University of Massachusetts Medical School, Worcester, MA). IRF2 with mutation of a conserved tyrosine residue in the IRF domain (Tyr-109) to phenylalanine was generated by PCR using the Clontech "QuikChange" protocol, as described (16Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar). Mutant clones were sequenced on both strands to verify that only intended mutations had been introduced. Wild type PU.1 and PU.1 mutants with serine 41 and 45, 148, or 132 and 133 changed to alanine were obtained from Dr. Michael L. Atchison (University of Pennsylvania School of Veterinary Medicine, Philadelphia) and subcloned into the pSRα mammalian expression vector. These cDNAs were also subcloned into the pGEX1 vector (Amersham Biosciences) for expression in Escherichia coli as glutathione S-transferase (GST) fusion proteins. Reporter Constructs—An artificial promoter construct with four copies of the ICSBP/IRF8-binding cis element from the NF1 promoter (bp -320 to -336) linked to a minimal promoter and a CAT reporter (the p-TATACAT vector) was previously described (15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar). This construct is referred to as nf1TATACAT. Oligonucleotides were custom-synthesized by MWG Biotec (Piedmont, NC). Double-stranded oligonucleotides used in EMSA and DNA affinity co-immunoprecipitation assays represent the -320- to -336-bp NF1 promoter sequence (NF1-320 to -336; 5′-ggatcccacttccggtggc-3′). The underlined sequence has homology to composite ets/IRF-cis elements from other ICSBP target genes. A double-stranded oligonucleotide with mutation of the ICSBP/IRF8-binding site (previously described (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar)) was also used in these assays (mutNF1 -320 to -336; 5′-ggatcccacaaccggtggc-3′). For in vitro binding competition assays, an oligonucleotide with the composite ets/IRF sequence for the CYBB gene was used 5′-gttttcatttcctcattgg-3′) (21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Subcloning these oligonucleotides into a plasmid vector to generate probes has been described (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The human myelomonocytic cell line U937 (24Larrick J.W. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-14PubMed Google Scholar) was obtained from Andrew Kraft (Hollings Cancer Center, Medical University of South Carolina, Charleston, SC). Cells were maintained and differentiated as described (21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). For differentiation experiments, U937 cells were treated for 24 or 48 h with 500 units/ml human recombinant IFNγ (Roche Applied Science) (21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). U937 cells were cultured and transfected as described previously (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar, 21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 22Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105PubMed Google Scholar). Cells (32 × 106 per sample) were transfected with a vector to express the following: wild type ICSBP/IRF8, Y95F ICSBP/IRF8, or empty vector control; wild type IRF2, Y109F IRF2, or empty vector control; wild type PU.1, S148A PU.1, S132A/S133A PU.1, or empty vector control; the minimal promoter/reporter vector pTATACAT with four copies of the -320- to -336-bp NF1 sequence (nf1TATACAT) or empty vector control (pTATACAT); and p-CMVβgal (to control for transfection efficiency). Transfectants were harvested 48 h after transfection, with or without incubation with recombinant human IFNγ (500 units/ml). Lysates were analyzed for CAT and β-galactosidase activity, as described (21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Nuclear extract proteins were isolated from U937 cells by the method of Dignam et al. (25Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1479Crossref PubMed Scopus (9145) Google Scholar) (with the addition of protease inhibitors but not phosphatase inhibitors, as described). In some experiments, U937 cells were differentiated with 500 units/ml of IFNγ before nuclear protein isolation. Oligonucleotides probes were prepared, and EMSA and antibody supershift assays were performed, as described (21Eklund E.A. Jalava A. Kakar R. J. Biol. Chem. 1998; 273: 13957-13965Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Antibodies to phosphotyrosine, ICSBP/IRF8, IRF1, IRF2, PU.1, and irrelevant, control GST antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Western Blots of U937 Lysates Proteins—U937 cells were lysed by boiling in 2× SDS sample buffer. Lysate proteins (30 μg) were separated by SDS-PAGE (12% acrylamide) and transferred to nitrocellulose, according to standard techniques. Western blots were serially probed with antibodies to Nf1, ICSBP/IRF8, IRF2, PU.1, and GAPDH (to control for loading). In other studies, nuclear proteins were isolated from U937 cells (with or without IFNγ treatment). 30 μg of protein were separated by SDS-PAGE (20% acrylamide gel) and transferred to nitrocellulose. Western blots were serially probed with antibodies to PU.1, IRF2, pERK2 (as a control for IFNγ treatment), total ERK2 (as a loading control), or GAPDH (as a loading control). Immunoprecipitation and Western Blots—Nuclear proteins were isolated from U937 cells with or without IFNγ treatment and immunoprecipitated under denaturing conditions with antibody to PU.1, IRF2, phosphotyrosine (Tyr(P)), or irrelevant control antibody (anti-GST), as described previously (14Scheller M. Foerster J. Heyworth C.M. Waring J.F. Lohler J. Gilmore G.L. Shadduck R.K. Dexter T.M. Horak I. Blood. 1999; 94: 3764-3771Crossref PubMed Google Scholar, 15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar). Precipitated proteins were collected with staphylococcus protein A-Sepharose, separated by SDS-PAGE, and transferred to nitrocellulose, as above. Western blots were serially probed with an anti-phosphotyrosine antibody (clone 4G10, Upstate) and antibodies to IRF2 or PU.1. DNA Affinity Co-immunoprecipitation—Nuclear proteins were isolated from U937 cells with or without IFNγ treatment. Proteins (300 μg) were incubated with a biotin-labeled double-stranded oligonucleotide probe representing the -320- to -336-bp NF1 promoter sequence or a specific mutant, non-ICSBP/IRF8-binding sequence. Probes were immunoprecipitated with anti-biotin antibody or irrelevant control antibody under nondenaturing conditions and recovered with staphylococcus protein A-Sepharose beads. Immunoprecipitates were separated by SDS-PAGE (10% acrylamide) and proteins transferred to nitrocellulose. Western blots were serially probed with antibodies to ICSBP/IRF8, IRF2, and PU.1. In other experiments, 35S-labeled, in vitro translated proteins were generated using rabbit reticulocyte lysate from in vitro transcribed RNA, as described previously (16Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar). These proteins were also used in DNA affinity co-immunoprecipitation experiments with biotin-labeled probes, as described above. For these experiments, DNA/protein interaction was identified by autoradiography of SDS-PAGE. Metabolic Labeling and Immunoprecipitation—U937 cells treated with IFN-γ for 0, 24, and 48 h were incubated for 4 h at 37 °C with [32P]orthophosphate. Cells were lysed under denaturing conditions, and lysate proteins (100 μg) were immunoprecipitated with antibody to PU.1 or irrelevant control antibody. Immunoprecipitates were collected with staphylococcus protein A-Sepharose beads and separated by SDS-PAGE (20% acrylamide). Phosphorylated PU.1 protein was identified by autoradiography of the fixed and dried gel. In vitro transcribed IRF2, Y109F IRF2, ICSBP/IRF8, Y95F ICSBP/IRF8, PU.1, S148A PU.1, or S132A/S133A PU.1 mRNAs were generated from linearized template DNA using the riboprobe system, according to manufacturer's instructions (Promega). In vitro translated proteins were generated in rabbit reticulocyte lysate, according to the manufacturer's instructions (Promega). Control (unprogrammed) lysates were generated in similar reactions in the absence of input RNA, and proteins were radiolabeled by including [35S]methionine in the translation reaction. JM109 E. coli transformed with PU.1, IRF2, or ICSBP/IRF8 in the pGEX vector (or empty control vector) were grown to log phase, supplemented to 0.1 mm isopropyl 1-thio-β-d-galactopyranoside, and incubated for 3 h at 37 °C with shaking. The cells were harvested and resuspended in HN buffer (20 mm HEPES (pH 7.4), 0.1 m NaCl, 2 mm MgCl2, 0.1 mm EDTA, 0.5% Nonidet P-40, 0.1% Triton X-100, 2 mm phenylmethylsulfonyl fluoride, 5 mm NaF) and sonicated on ice (22Eklund E.A. Kakar R. J. Immunol. 1999; 163: 6095-6105PubMed Google Scholar). Debris was removed by centrifugation, and the lysate was incubated with glutathione-agarose beads (Sigma) and washed extensively. The beads were preincubated with control rabbit reticulocyte lysate to induce serine/threonine phosphorylation of PU.1/GST and tyrosine phosphorylation of ICSBP/GST or IRF2/GST as described (16Kautz B. Kakar R. David E. Eklund E.A. J. Biol. Chem. 2001; 276: 37868-37878Abstract Full Text Full Text PDF PubMed Google Scholar, 26Pongubala J.M.R. Nagulapalli S. Klemsz M.J. McKercher S.R. Maki R.A. Atchison M.L. Mol. Cell. Biol. 1991; 12: 368-378Crossref Scopus (311) Google Scholar). GST proteins were then incubated with [35S]methionine-labeled in vitro translated protein. Proteins were eluted with SDS-PAGE sample buffer, separated on 15% SDS-PAGE, and identified by autoradiography of the fixed and dried gel. The Positive NF1-cis Element Binds a Multiprotein Complex—IFNγ treatment of U937 cells increases NF1 promoter activity and ICSBP/IRF8 binding to a positive cis element in the NF1 promoter (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar). To identify other proteins that interact with this cis element, we used in vitro DNA binding assays. EMSAs were performed with a radiolabeled, double-stranded oligonucleotide representing the NF1-cis element and nuclear proteins from U937 cells. We found that IFNγ differentiation of U937 cells increases in vitro protein binding to this probe, consistent with our previous results (Fig. 1A) (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 15Huang W. Zhu C. Saberwal G. Horvath E. Lindsey S. Eklund E.A. Mol. Cell. Biol. 2006; 26: 6311-6332Crossref PubMed Scopus (37) Google Scholar). We noted that the NF1-cis element has homology with the ets/IRF consensus sequences from the CYBB or NCF2 promoters. Therefore, we used double-stranded oligonucleotide competitors representing these cis element to investigate binding specificity of the NF1-protein complex. These competitors were compared with homologous oligonucleotide (ds NF1) or an oligonucleotide with mutation which abolishes protein binding to the NF1-cis element (ds mutNF1) (described (4Zhu C. Saberwal G. Ly Y.F. Platanias L.C. Eklund E.A. J. Biol. Chem. 2004; 279: 50874-50885Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar)). In EMSA with the NF1 probe and nuclear proteins from IFNγ-differentiated U937 cells, we found that the CYBB and NCF2 oligonucleotides compete for protein complex binding (Fig. 1B). This result suggests that the NF1-cis element interacts with PU.1 and one or more IRF proteins. Therefore, we performed EMSA with the NF1 probe, nuclear proteins from IFNγ-treated U937 cells, and antibodies to various transcription factors (Fig. 1C). In these studies, we found that the shifted complex is cross-immunoreactive with PU.1, ICSBP/IRF8, and IRF2 but not IRF1. Because the NF1-cis element appears to bind a multiprotein complex, we tested the ability of various combinations of PU.1, IRF2, and ICSBP/IRF8 antibodies to disrupt the complex (Fig. 1C). We found that the complex is completely disrupted by combining all three antibodies. We also used DAPA to determine the impact of differentiation on assembly of the multiprotein complex on the NF1-cis element. To determine specificity of binding, wild type NF1-cis element probe (ds NF1) was compared with the binding-mutant probe (ds mutNF1). These oligonucleotides were biotin-labeled and incubated with U937 nuclear proteins. Probes were precipitated with anti-biotin antibody, and co-precipitating proteins were separated by SDS-PAGE and identified by Western blot (Fig. 1D). In this assay, we also find that IFNγ treatment increases interaction of PU.1, IRF2, and ICSBP/IRF8 with the NF1-cis element probe. In contrast, we found previously that PU.1 and IRF1 bind the CYBB- and NCF2-cis elements in undifferentiated myeloid cells. For those cis elements, differentiation increases ICSBP/IRF8 interaction with these proteins but does not increase DNA binding of PU.1 and IRF1. Our current results suggest differences in the activation of various ets/IRF-cis elements, even in the same lineage. Identification of Protein/Protein/DNA Interactions Required for Assembly of the NF1-cis Element-binding Complex—Previous studies suggest that PU.1 binding to ets/IRF consensus sequences is required to p
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Interferon regulatory factor 1(IRF-1)is a important multifunctional transcription factor for regulating of expression of interferon(IFN)and having cantivirus action,antineoplastic activity.Indeed,IRF-1selectively modulates different sets of genes,depending on the cell type in order to evoke appropriate responses in each.The author summarizes the structure,the transcriptional regulation and the multiple biological effects.
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Interferon regulatory factor-1 (IRF-1), a transcriptional activator, and IRF-2, its antagonistic repressor, have been identified as regulators of type I interferon and interferon-inducible genes. The IRF-1 gene is itself interferon-inducible and hence may be one of the target genes critical for interferon action. When the IRF-2 gene was overexpressed in NIH 3T3 cells, the cells became transformed and displayed enhanced tumorigenicity in nude mice. This transformed phenotype was reversed by concomitant overexpression of the IRF-1 gene. Thus, restrained cell growth depends on a balance between these two mutually antagonistic transcription factors.
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Limb development
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Differential expression of the human interferon A (IFN-A) gene cluster is modulated following paramyxovirus infection by the relative amounts of active interferon regulatory factor 3 (IRF-3) and IRF-7. IRF-3 expression activates predominantly IFN-A1 and IFN-B, while IRF-7 expression induces multiple IFN-A genes. IFN-A1 gene expression is dependent on three promoter proximal IRF elements (B, C, and D modules, located at positions -98 to -45 relative to the mRNA start site). IRF-3 binds the C module of IFN-A1, while other IFN-A gene promoters are responsive to the binding of IRF-7 to the B and D modules. Maximal expression of IFN-A1 is observed with complete occupancy of the three modules in the presence of IRF-7. Nucleotide substitutions in the C modules of other IFN-A genes disrupt IRF-3-mediated transcription, whereas a G/A substitution in the D modules enhances IRF7-mediated expression. IRF-3 exerts dual effects on IFN-A gene expression, as follows: a synergistic effect with IRF-7 on IFN-A1 expression and an inhibitory effect on other IFN-A gene promoters. Chromatin immunoprecipitation experiments reveal that transient binding of both IRF-3 and IRF-7, accompanied by CBP/p300 recruitment to the endogenous IFN-A gene promoters, is associated with transcriptional activation, whereas a biphasic recruitment of IRF-3 and CBP/p300 represses IFN-A gene expression. This regulatory mechanism contributes to differential expression of IFN-A genes and may be critical for alpha interferon production in different cell types by RIG-I-dependent signals, leading to innate antiviral immune responses.
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Expression of Interferon Regulatory Factor Family and its Prognostic value in Acute Myeloid Leukemia
Aim: To elucidate the clinicopathological and prognostic values of interferon regulatory factor (IRF) family genes in acute myeloid leukemia (AML). Patients & methods: Differential expression analysis and survival analysis from several reliable databases were conducted and further validated using patients with AML. Results: The expression level of IRF1/2/4/5/7/8/9 in patients with AML was upregulated, while IRF3/6 expression was downregulated. High IRF1/7/9 expression indicated a worse overall survival rate. Conclusion: Overexpression of IRF1/7/9 may be associated with poor survival in patients with AML, suggesting that the IRF family may be a promising therapeutic target.
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The interferon regulatory factor 1 (IRF‐1) is a positive transcriptional regulatory protein which acts in the interferon signal transduction pathway to activate the transcription of the type I interferon genes by binding to the PRDI response element. The aim of this study was to explore the role of IRF‐1 in regulating the expression of other interferon‐stimulated genes in the interferon signal transduction pathway. A transient transfection assay was used to show that IRF‐1 induced the expression of interferon‐stimulated genes. The induction was a direct result of IRF‐1 binding to the promoters of the interferon‐stimulated response element (ISRE). The levels of endogenous mRNA of two interferon‐stimulated genes, 6‐16 and 9‐27, were increased in cells containing increased levels of IRF‐1. In addition, IRF‐1 activates the expression of IRF‐2, a negative regulator of the type I interferon genes themselves. Two sequences were found in the IRF‐2 promoter which were the binding sites for IRF‐1. Mutations in the oligonucleotide sequences of these sites could abolish the binding of the IRF‐1. These data suggested that IRF‐1 not only plays an important role in the induction of type I interferon genes, but also in the activation of interferon‐stimulated genes.
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General transcription factor
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Interferon regulatory factors (IRFs) are some of important transcriptional factors for regulating the expression of interferon (IFN) and interferon stimulated genes (ISG) as well as the genes related to IFN signal transduc-tion.The multiple biological effects of the members of interferon regulatory factor family have been shown to be mediated by the transcriptional activation for IFN, ISG and the related genes.
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