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The Cell Cycle Control Element of Histone H4 Gene Transcription Is Maximally Responsive to Interferon Regulatory Factor Pairs IRF-1/IRF-3 and IRF-1/IRF-7

2001; Elsevier BV; Volume: 276; Issue: 21 Linguagem: Inglês

10.1074/jbc.m010391200

ISSN

1083-351X

Autores

Ronglin Xie, André J. van Wijnen, Caroline van der Meijden, Mai X. Luong, Janet L. Stein, Gary S. Stein,

Tópico(s)

Protein Tyrosine Phosphatases

Resumo

Interferon regulatory factors (IRFs) are transcriptional mediators of interferon-responsive signaling pathways that are involved in antiviral defense, immune response, and cell growth regulation. To investigate the role of IRF proteins in the regulation of histone H4 gene transcription, we compared the transcriptional contributions of IRF-1, IRF-2, IRF-3, and IRF-7 using transient transfection assays with H4 promoter/luciferase (Luc) reporter genes. These IRF proteins up-regulate reporter gene expression but IRF-1, IRF-3, and IRF-7 are more potent activators of the H4 promoter than IRF-2. Forced expression of different IRF combinations reveals that IRF-2 reduces IRF-1 or IRF-3 dependent activation, but does not affect IRF-7 function. Thus, IRF-2 may have a dual function in histone H4 gene transcription by acting as a weak activator at low dosage and a competitive inhibitor of other strongly activating IRFs at high levels. IRF-1/IRF-3 and IRF-1/IRF-7 pairs each mediate the highest levels of site II-dependent promoter activity and can up-regulate transcription by 120–150-fold. We also find that interferon γ up-regulates IRF-1 and site II-dependent promoter activity. This up-regulation is not observed when the IRF site is mutated or if cells are preloaded with IRF-1. Our results indicate that IRF-1, IRF-2, IRF-3, and IRF-7 can all regulate histone H4 gene expression. The pairwise utilization of distinct IRF factors provides a flexible transcriptional mechanism for integration of diverse growth-related signaling pathways. Interferon regulatory factors (IRFs) are transcriptional mediators of interferon-responsive signaling pathways that are involved in antiviral defense, immune response, and cell growth regulation. To investigate the role of IRF proteins in the regulation of histone H4 gene transcription, we compared the transcriptional contributions of IRF-1, IRF-2, IRF-3, and IRF-7 using transient transfection assays with H4 promoter/luciferase (Luc) reporter genes. These IRF proteins up-regulate reporter gene expression but IRF-1, IRF-3, and IRF-7 are more potent activators of the H4 promoter than IRF-2. Forced expression of different IRF combinations reveals that IRF-2 reduces IRF-1 or IRF-3 dependent activation, but does not affect IRF-7 function. Thus, IRF-2 may have a dual function in histone H4 gene transcription by acting as a weak activator at low dosage and a competitive inhibitor of other strongly activating IRFs at high levels. IRF-1/IRF-3 and IRF-1/IRF-7 pairs each mediate the highest levels of site II-dependent promoter activity and can up-regulate transcription by 120–150-fold. We also find that interferon γ up-regulates IRF-1 and site II-dependent promoter activity. This up-regulation is not observed when the IRF site is mutated or if cells are preloaded with IRF-1. Our results indicate that IRF-1, IRF-2, IRF-3, and IRF-7 can all regulate histone H4 gene expression. The pairwise utilization of distinct IRF factors provides a flexible transcriptional mechanism for integration of diverse growth-related signaling pathways. interferon regulatory factor luciferase interferon, EBNA-1, Epstein-Barr virus nuclear antigen-1 cyclin-dependent kinase cell cycle element chloramphenicol acetyltransferase polyacrylamide gel electrophoresis phosphate-buffered saline Interferon regulatory factors (IRFs)1 form a large family of transcription factors involved in antiviral defense, immune activation, and cell growth regulation. IRFs were initially identified as regulators of interferon genes in response to viral infection. However, it has subsequently been shown that there are at least nine cellular IRF proteins (IRF-1, IRF-2, IRF-3, IRF-4/Pip/ICSAT, IRF-5, IRF-6, IRF-7, ICSBP/IRF8, and ISGF3γ/p48/IRF9), as well as virally encoded forms, with broad biological functions (1Vaughan P.S. van Wijnen A.J. Stein J.L. Stein G.S. J. Mol. Med. 1997; 75: 348-359Crossref PubMed Scopus (44) Google Scholar, 2Mamane Y. Heylbroeck C. Genin P. Algarte M. Servant M.J. LePage C. DeLuca C. Kwon H. Lin R. Hiscott J. Gene. 1999; 237: 1-14Crossref PubMed Scopus (465) Google Scholar). All members of the IRF family share significant homology in the N-terminal 115 amino acids, which comprise the DNA-binding domain. For the IRF-3, IRF-4, IRF-5, IRF-8, and IRF-9 proteins, the homology extends into the C-terminal region with which these IRFs interact with other proteins or family members. Current data indicate that IRFs can function as transcriptional activators (e.g. IRF-1, IRF-3, and IRF-9), repressors (e.g. IRF-8), or both (e.g. IRF-2, IRF-4, and IRF-7). Studies with IRF-expressing cell lines and IRF knockout mice reveal that IRF family members have distinct roles in various biological processes, including cytokine signaling, responses to pathogens, cell growth regulation, and hematopoietic development (1Vaughan P.S. van Wijnen A.J. Stein J.L. Stein G.S. J. Mol. Med. 1997; 75: 348-359Crossref PubMed Scopus (44) Google Scholar, 2Mamane Y. Heylbroeck C. Genin P. Algarte M. Servant M.J. LePage C. DeLuca C. Kwon H. Lin R. Hiscott J. Gene. 1999; 237: 1-14Crossref PubMed Scopus (465) Google Scholar, 3Nguyen H. Hiscott J. Pitha P.M. Cytokine Growth Factor Rev. 1997; 8: 293-312Crossref PubMed Scopus (423) Google Scholar). IRF-1 and IRF-2 are transcription factors that interact with the same DNA sequence element (designated ISRE/IRF-E) in the promoters of type I interferon (IFN) and other cytokine inducible genes (4Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Abstract Full Text PDF PubMed Scopus (807) Google Scholar, 5Miyamoto M. Fujita T. Kimura Y. Maruyama M. Harada H. Sudo Y. Miyata T. Taniguchi T. Cell. 1988; 54: 903-913Abstract Full Text PDF PubMed Scopus (795) Google Scholar, 6Fujita T. Kimura Y. Miyamoto M. Barsoumian E.L. Taniguchi T. Nature. 1989; 337: 270-272Crossref PubMed Scopus (318) Google Scholar, 7Pine R. J. Virol. 1992; 66: 4470-4478Crossref PubMed Google Scholar, 8Hiscott J. Nguyen H. Lin R. Semin. Virology. 1995; 6: 161-173Crossref Scopus (48) Google Scholar). IRF-1 is up-regulated by type I interferons and the type II interferon, IFN-γ (2Mamane Y. Heylbroeck C. Genin P. Algarte M. Servant M.J. LePage C. DeLuca C. Kwon H. Lin R. Hiscott J. Gene. 1999; 237: 1-14Crossref PubMed Scopus (465) Google Scholar). IRF-2 is up-regulated by IRF-1 and antagonizes IRF-1 activation by competing with IRF-1 for its DNA-binding site (4Harada H. Fujita T. Miyamoto M. Kimura Y. Maruyama M. Furia A. Miyata T. Taniguchi T. Cell. 1989; 58: 729-739Abstract Full Text PDF PubMed Scopus (807) Google Scholar, 9Harada H. Willison K. Sakakibara J. Miyamoto M. Fujita T. Taniguchi T. Cell. 1990; 63: 303-312Abstract Full Text PDF PubMed Scopus (320) Google Scholar, 10Tanaka N. Kawakami T. Taniguchi T. Mol. Cell. Biol. 1993; 13: 4531-4538Crossref PubMed Scopus (388) Google Scholar, 11Taniguchi T. Harada H. Lamphier M. J. Cancer Res. Clin. Oncol. 1995; 121: 516-520Crossref PubMed Scopus (117) Google Scholar, 12Taniguchi T. Lamphier M.S. Tanaka N. Biochim. Biophys. Acta. 1997; 1333: M9-17Crossref PubMed Scopus (136) Google Scholar). IRF-2 also functions as a transcriptional activator (13Yamamoto H. Lamphier M.S. Fujita T. Taniguchi T. Harada H. Oncogene. 1994; 9: 1423-1428PubMed Google Scholar) and has been shown to activate the genes for histone H4 (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar, 15Vaughan P.S. van der Meijden C.M.J. Aziz F. Harada H. Taniguchi T. van Wijnen A.J. Stein J.L. Stein G.S. J. Biol. Chem. 1998; 273: 194-199Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), Epstein-Barr virus nuclear antigen-1 (EBNA-1) (16Schaefer B.C. Paulson E. Strominger J.L. Speck S.H. Mol. Cell. Biol. 1997; 17: 873-886Crossref PubMed Scopus (65) Google Scholar), and murine muscle vascular cell adhesion molecule-1 (17Jesse T.L. LaChance R. Iademarco M.F. Dean D.C. J. Cell Biol. 1998; 140: 1265-1276Crossref PubMed Scopus (129) Google Scholar). In addition, IRF-1 and IRF-2 can co-occupy the Class II transactivator type IV promoter element IRF-E and synergistically activate this promoter (18Xi H. Eason D.D. Ghosh D. Dovhey S. Wright K.L. Blanck G. Oncogene. 1999; 18: 5889-5903Crossref PubMed Scopus (45) Google Scholar). IRF-1 and IRF-2 are key regulators of cell growth, cell cycle, and apoptosis, and function as an anti-oncogene and oncogene, respectively (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar, 19Harada H. Kitagawa M. Tanaka N. Yamamoto H. Harada K. Ishihara M. Taniguchi T. Science. 1993; 259: 971-974Crossref PubMed Scopus (429) Google Scholar, 20Tanaka H. Samuel C.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7995-7999Crossref PubMed Scopus (113) Google Scholar, 21Tamura T. Ishihara M. Lamphier M.S. Tanaka N. Oishi I. Aizawa S. Matsuyama T. Mak T.W. Taki S. Taniguchi T. Nature. 1995; 376: 596-599Crossref PubMed Scopus (422) Google Scholar, 22Willman C.L. Sever C.E. Pallavicini M.G. Harada H. Tanaka N. Slovak M.L. Yamamoto H. Harada K. Meeker T.C. List A.F. Science. 1993; 259: 968-971Crossref PubMed Scopus (381) Google Scholar). Our laboratory has established that IRF-1 and IRF-2 can each functionally interact with and transcriptionally activate the H4 promoter (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar, 15Vaughan P.S. van der Meijden C.M.J. Aziz F. Harada H. Taniguchi T. van Wijnen A.J. Stein J.L. Stein G.S. J. Biol. Chem. 1998; 273: 194-199Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Furthermore, the gene for p21WAF1/cip1, a member of the family of cyclin-dependent kinase (CDK) inhibitors, which plays a primary role in cell cycle control, is regulated in response to DNA damage by both IRF-1 and p53 (23Tanaka N. Ishihara M. Lamphier M.S. Nozawa H. Matsuyama T. Mak T.W. Aizawa S. Tokino T. Oren M. Taniguchi T. Nature. 1996; 382: 816-818Crossref PubMed Scopus (303) Google Scholar, 24Sherr C.J. Roberts J.M. Genes Dev. 1995; 9: 1149-1163Crossref PubMed Scopus (3221) Google Scholar, 25Coccia E.M. Del Russo N. Stellacci E. Orsatti R. Benedetti E. Marziali G. Hiscott J. Battistini A. Oncogene. 1999; 18: 2129-2137Crossref PubMed Scopus (59) Google Scholar). These observations suggest that the transcriptional properties of IRF-1 and IRF-2 are linked to their cell growth regulatory potential. Cell cycle control of histone gene transcription at the onset of S phase is required for the functional coupling of histone gene expression and DNA replication (26Stein G.S. Stein J.L. Marzluff W.F. Histone Genes. John Wiley & Sons, Inc., New York1984Google Scholar, 27Stein G.S. Stein J.L. van Wijnen A.J. Lian J.B. Cell Biol. Int. 1996; 20: 41-49Crossref PubMed Scopus (44) Google Scholar). Transcriptional control of the human histone H4 gene designated FO108 (28Sierra F. Stein G. Stein J. Nucleic Acids Res. 1983; 11: 7069-7086Crossref PubMed Scopus (78) Google Scholar) has been extensively studied. The H4 gene is regulated by two multipartite proximal promoter elements (sites I and II), which together with two distal auxiliary domains (sites III and IV) modulate histone H4 promoter activity (27Stein G.S. Stein J.L. van Wijnen A.J. Lian J.B. Cell Biol. Int. 1996; 20: 41-49Crossref PubMed Scopus (44) Google Scholar). Site II mediates cell cycle control of histone H4 transcription by interacting with three distinct factors, including IRF-2/HiNF-M, CDP-cut/HiNF-D, and HiNF-P (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar, 29Ramsey-Ewing A. van Wijnen A.J. Stein G.S. Stein J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4475-4479Crossref PubMed Scopus (64) Google Scholar, 30van Wijnen A.J. Ramsey-Ewing A.L. Bortell R. Owen T.A. Lian J.B. Stein J.L. Stein G.S. J. Cell. Biochem. 1991; 46: 174-189Crossref PubMed Scopus (51) Google Scholar, 31van Wijnen A.J. van den Ent F.M. Lian J.B. Stein J.L. Stein G.S. Mol. Cell. Biol. 1992; 12: 3273-3287Crossref PubMed Scopus (78) Google Scholar, 32van der Meijden C.M.J. Vaughan P.S. Staal A. Albig W. Doenecke D. Stein J.L. Stein G.S. van Wijnen A.J. Biochim. Biophys. Acta. 1998; 1442: 82-100Crossref PubMed Scopus (23) Google Scholar, 33Aziz F. van Wijnen A.J. Vaughan P.S. Wu S. Shakoori A.R. Lian J.B. Soprano K.J. Stein J.L. Stein G.S. Mol. Biol. Rep. 1998; 25: 1-12Crossref PubMed Scopus (29) Google Scholar). The cell cycle element (CCE), 5′-CTTTCGGTTTT-3′, which is located in the distal part of site II (34Pauli U. Chrysogelos S. Stein G. Stein J. Nick H. Science. 1987; 236: 1308-1311Crossref PubMed Scopus (104) Google Scholar) and controls transcription at the G1/S phase transition (29Ramsey-Ewing A. van Wijnen A.J. Stein G.S. Stein J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4475-4479Crossref PubMed Scopus (64) Google Scholar), is known to interact functionally with both IRF-1 and IRF-2 (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar,15Vaughan P.S. van der Meijden C.M.J. Aziz F. Harada H. Taniguchi T. van Wijnen A.J. Stein J.L. Stein G.S. J. Biol. Chem. 1998; 273: 194-199Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Recently, other IRF proteins (e.g. IRF-3 and IRF-7) have been shown to contribute to transcriptional control via IRF-binding sites. For example, the formation of distinct heterodimers between activated IRF-3 and IRF-7 may lead to differential regulation of the IFN-α and IFN-β genes (2Mamane Y. Heylbroeck C. Genin P. Algarte M. Servant M.J. LePage C. DeLuca C. Kwon H. Lin R. Hiscott J. Gene. 1999; 237: 1-14Crossref PubMed Scopus (465) Google Scholar, 35Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar) which were initially characterized as responsive to IRF-1 and IRF-2. Both IRF-3 and IRF-7 are constitutively present in several cell types and can be activated in response to different biological stimuli, including viral infection, type I interferons, and/or DNA damage (2Mamane Y. Heylbroeck C. Genin P. Algarte M. Servant M.J. LePage C. DeLuca C. Kwon H. Lin R. Hiscott J. Gene. 1999; 237: 1-14Crossref PubMed Scopus (465) Google Scholar). These recent findings necessitate evaluation of the extent to which distinct combinations of IRF proteins may regulate histone H4 gene expression. To investigate the role of multiple IRF members in histone H4 gene transcription, we performed transfection studies with H4 promoter-luciferase reporter genes and a panel of IRF expression vectors. Our results suggest that IRF-1, IRF-2, IRF-3, and IRF-7 can all actively regulate histone H4 gene expression and that specific IRF pairs (i.e. IRF-1/IRF-3 and IRF-1/IRF-7) are strong activators. The wild type H4 promoter/luciferase reporter gene construct wtH4/Luc was derived from pFO108 wt/CAT (30van Wijnen A.J. Ramsey-Ewing A.L. Bortell R. Owen T.A. Lian J.B. Stein J.L. Stein G.S. J. Cell. Biochem. 1991; 46: 174-189Crossref PubMed Scopus (51) Google Scholar, 31van Wijnen A.J. van den Ent F.M. Lian J.B. Stein J.L. Stein G.S. Mol. Cell. Biol. 1992; 12: 3273-3287Crossref PubMed Scopus (78) Google Scholar, 36Kroeger P. Stewart C. Schaap T. van Wijnen A. Hirshman J. Helms S. Stein G. Stein J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3982-3986Crossref PubMed Scopus (51) Google Scholar), which contains the proximal promoter region of the H4 gene (nucleotides −240 to −38 relative to the ATG start codon; mRNA cap site at nucleotide −30) and spans sites I and II. The CAT gene was removed byPstI and HindIII cleavage and replaced by a 1.65-kilobase PstI/HindIII fragment spanning the luciferase (Luc) gene. The Luc gene was amplified from pGL3 (Promega, Madison, WI) by polymerase chain reaction amplification with two primers: forward PstI primer, 5′-gactgcagGCATTCCGGTACTGTTG-3′; reverse HindIII primer, 5′-gcaagcttACACGGCGATCTTTCC-3′; lowercase nucleotides were added to create restriction sites. The H4/Luc construct in which the IRF-binding site is mutated (IRF mutH4/Luc) was prepared from pMSP16-CAT (33Aziz F. van Wijnen A.J. Vaughan P.S. Wu S. Shakoori A.R. Lian J.B. Soprano K.J. Stein J.L. Stein G.S. Mol. Biol. Rep. 1998; 25: 1-12Crossref PubMed Scopus (29) Google Scholar) and the CAT gene was exchanged for the Luc reporter as described above. The 4X IRF/H4-Site II/Luc plasmid was constructed by inserting an oligonucleotide cassette containing a tandemly repeated IRF-binding site (5′-gatccGCTTTCGGTTTTCAGCTTTCGGTTTTCAGATCCGCTTTCGGTTTTCAGCTTTCGGTTTTCa-3′; 5′-gatctGAAAACCGAAAGCTGAAAACCGAAAGCGGATCTGAAAACCGAAAGCTGAAAACCGAAAGCg-3′;BamHI/BglII overhangs) into theBamHI site of pFP201CAT (29Ramsey-Ewing A. van Wijnen A.J. Stein G.S. Stein J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4475-4479Crossref PubMed Scopus (64) Google Scholar, 37van Wijnen A.J. Owen T.A. Holthuis J. Lian J.B. Stein J.L. Stein G.S. J. Cell. Physiol. 1991; 148: 174-189Crossref PubMed Scopus (33) Google Scholar) and was converted to a luciferase reporter using the same polymerase chain reaction-derived fragment described above. The FP201 segment of the H4 promoter spans nucleotides −97 to −38. All oligonucleotides were synthesized using a Beckman 1000M DNA synthesizer and all inserts were sequenced (ABI 100 model 377) to verify correct orientation and absence of polymerase chain reaction or chemical synthesis-related mutations. The 3X H4 distal site II wild type promoter-luciferase reporter gene construct (3X H4 distal Site II/Luc) which contains three copies of an oligonucleotide spanning the distal segment of H4 site II (5′-CGCTTTCGGTTTTCAATCTGGTCCGATAC-3′) fused to the TATA box of the H2-L gene was a kind gift from Dr. Keiko Ozato (38Masumi A. Wang I.M. Lefebvre B. Yang X.J. Nakatani Y. Ozato K. Mol. Cell. Biol. 1999; 19: 1810-1820Crossref PubMed Scopus (94) Google Scholar). The companion construct with mutated IRF-binding sites (3X H4 distal site II IRF-mutant/Luc) was constructed by digesting the wild type plasmid withXhoI and BglII to remove the multimerized site II and then inserting the mutant multimer oligonucleotide (5′-CGCTTCAGGTTTTCAATCTGGTCCGATAC-3′). IRF expression constructs (pcDNA/IRF-1, pcDNA/IRF-2, and 6X-His-tagged human IRF-3 and IRF-7) were kindly provided by Dr. T. Maniatis (35Wathelet M.G. Lin C.H. Parekh B.S. Ronco L.V. Howley P.M. Maniatis T. Mol. Cell. 1998; 1: 507-518Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar, 39Palombella V.J. Maniatis T. Mol. Cell. Biol. 1992; 12: 3325-3336Crossref PubMed Scopus (67) Google Scholar). The CDK2/Luc construct, containing the 2.4-kilobase human cdk2 promoter inserted into the pGL2-basic plasmid, was a kind gift of Dr. Dov Shiffman (40Shiffman D. Brooks E.E. Brooks A.R. Chan C.S. Milner P.G. J. Biol. Chem. 1996; 271: 12199-12204Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The expression constructs pcDNA IRF-1, pcDNA IRF-3, and pcDNA IRF-7 were subjected to coupled in vitrotranscription and translation with unlabeled methionine or [35S]methionine in a rabbit reticulocyte lysate system according to the manufacturer's instructions (Promega). Aliquots of35S-labeled IRF-1 (5 μl), IRF-3 (20 μl), and IRF-7 (20 μl) were separated by SDS-PAGE in a 10% gel, which was subsequently dried and exposed for autoradiography. The bands for IRF-1, IRF-3, and IRF-7 were removed and incorporation of [35S]methionine for each protein was measured using an LS 6500 multipurpose scintillation counter (Beckman, Fullerton, CA). The measured radioactivity (in cpm) was used to calculate the molar ratios of IRF-1, IRF-3, and IRF-7. Electrophoretic mobility shift assays were performed as described previously (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar) with unlabeled in vitrotranslated IRF proteins. Each reaction contained 10 fmol of32P-labeled double-stranded CCE oligomer, IRF protein, 2 μg of poly(dG-dC)·(dG-dC), 1 μg of poly (dI-dC)·(dI-dC), and 1 pmol of unlabeled competitor oligomers where indicated. CCE-wt is 5′-GATCCCGGCGCGCTTTCGGTTTTCA and CCE-mut is 5′-GATCCCGGCGCGCTTTCAGGTTTTCA. The binding reactions were separated in a 4% polyacrylamide gel. Actively proliferating cultures of NIH3T3 cells were maintained at subconfluency in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin (Sigma), and 0.2 μml-glutamine, at 37 °C in humidified air containing 5% CO2. Cells were seeded in 6-well culture plates at a density of 1.5 × 105cells/well, and were transiently transfected 24 h later at ∼70% confluency by the Superfect transfection method (Qiagen, Valencia, CA). We co-transfected 0.8 μg of each H4/Luc reporter gene construct with different amounts of each IRF expression vector. The amount of DNA in each well was kept constant by supplementing the transfection mixture with the empty expression vector. Cells were also transfected with 50 ng/well of the pRL-CMV construct (Promega), which contains a cytomegalovirus promoter upstream of the Renilla luciferase gene, as an internal control for transfection efficiency (41Behre G. Smith L.T. Tenen D.G. BioTechniques. 1999; 26 (, 28): 24-26Crossref PubMed Scopus (69) Google Scholar). Cell lysates were prepared for luciferase assay or for Western blot analysis 24 h after transfection. To monitor the effect of IFN-γ, cells transfected with reporter gene constructs at 0.8 μg/well were incubated with 0–1.0 ng/ml IFN-γ at 24 h post-transfection, and analyzed 12 h after treatment. Each transfection was performed in triplicate and repeated at least three times. Cells were washed twice with 1 × PBS buffer 24 h after transfection and lysed with 1 × lysis buffer (Promega). Luciferase assays were carried out according to the manufacturer's specifications using a dual-luciferase reporter assay system (Promega) and a Monolight 2010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA). The activity of Renilla luciferase was used to normalize for variation in transfection efficiency by calculating the ratio of firefly and Renilla luciferase activities. Cell lysates were centrifuged at 14,000 × g (4 °C for 30 min), and protein concentrations were determined using the Coomassie protein assay reagent (Pierce Chemical Co., Rockford, IL) according to the manufacturer's instructions. Equal amounts of total cellular protein were mixed with loading buffer (62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 2% β-mercaptoethanol, and bromphenol blue), boiled for 5 min, and subjected to 10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp., Bedford, MA). The membranes were saturated with phosphate-buffered saline containing 0.05% Tween 20 (1 × PBS-T buffer) and 5% fat-free dry milk (42Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1987Google Scholar) for 1 h at room temperature and incubated overnight with primary IRF antibodies at 1:1,000 dilution (α-IRF-1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); 1:3,000 dilution (α-IRF-2 (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar)), or α-His antibodies at 1:1,500 dilution (for detection of IRF-3 or IRF-7; Qiagen) in 1% fat-free dry milk in 1 × PBS-T buffer. After washing with 1 × PBS-T buffer containing 1% milk, blots were further incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) diluted 1:10,000 in milk/PBS-T buffer. Blots were then washed five times with the same buffer before visualization of immunoreactive protein bands by enhanced chemiluminescence detection (ECL kit; Amersham Pharmacia Biotech Inc., Piscataway, NJ). Densitometry was performed by using the Alpha Imager 2000 densitometer (Alpha Innotech Corp., San Leandro, CA) according to the manufacturer's instructions. The protein level of each IRF member in untransfected cells was set as control and the relative protein level of each IRF member in transfected cells was determined by dividing the densitometry measurements of IRF transfected cells by the densitometry measurements of the control. The CCE within site II of the histone H4 promoter has previously been shown to interact with IRF-1 and IRF-2 (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar) and has high similarity to the IRF-E and ISRE consensus elements. To determine whether more recently identified IRF family members (e.g.IRF-3 and IRF-7) are also capable of binding to the histone H4 promoter, we performed protein-DNA interaction studies with IRF proteins produced by coupled in vitro transcription and translation. The IRF-1, IRF-3, and IRF-7 proteins were analyzed by SDS-PAGE in a 10% gel for radiometric quantitation (Fig.1, A and B). Approximately equimolar amounts of these IRF proteins were evaluated by electrophoretic mobility shift assay for binding to an oligonucleotide spanning the CCE in the distal segment of histone H4 site II (Fig.1 C). All three proteins (Fig. 1), as well as IRF-2 (Ref. 14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholarand data not shown), form complexes with the CCE and these complexes are competed specifically by the unlabeled wild type but not the mutant CCE oligonucleotides. The relative intensities of the signals of protein-DNA complexes suggest that IRF proteins have different affinities for the same site (IRF-1 = IRF-2 > IRF-3 = IRF-7) (Fig. 1 and data not shown). Our results indicate that in addition to IRF-2 (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar), IRF-1, IRF-3, and IRF-7 are also capable of binding to the CCE in the histone H4 promoter. We have previously shown that the transcription factor IRF-2 can activate histone H4 gene expression (14Vaughan P.S. Aziz F. van Wijnen A.J. Wu S. Harada H. Taniguchi T. Soprano K.J. Stein J.L. Stein G.S. Nature. 1995; 377: 362-365Crossref PubMed Scopus (173) Google Scholar) and is involved in cell cycle regulation of histone H4 gene transcription (15Vaughan P.S. van der Meijden C.M.J. Aziz F. Harada H. Taniguchi T. van Wijnen A.J. Stein J.L. Stein G.S. J. Biol. Chem. 1998; 273: 194-199Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To determine whether IRF-1, IRF-3, and IRF-7 function as activators or repressors of histone H4 gene expression, we performed co-transfection assays with IRF expression vectors and histone H4 gene promoter/luciferase reporter gene constructs (Figs. 2 and3). We tested IRF-dependent activation in the context of the wild type histone H4 promoter spanning sites I and II, as well as with a mutant H4 promoter in which the IRF binding element in site II was altered by a two-nucleotide substitution that prevents IRF binding (33Aziz F. van Wijnen A.J. Vaughan P.S. Wu S. Shakoori A.R. Lian J.B. Soprano K.J. Stein J.L. Stein G.S. Mol. Biol. Rep. 1998; 25: 1-12Crossref PubMed Scopus (29) Google Scholar). The results show that IRF-3 and IRF-7 are each capable of activating transcription by 5–6-fold (Fig.3 A). For comparison, IRF-1 and IRF-2 increase transcription by ∼11- and 2-fold, respectively. When the IRF-binding site was mutated, activation of the histone H4 promoter by IRF factors was completely abrogated (Fig. 3 B). These results show that IRF-3 and IRF-7 can activate the histone H4 promoter via the IRF recognition motif in site II.Figure 3The effects of four IRFs on histone H4 transcription assayed with different promoter constructs. NIH3T3 cells were transfected with 0.8 μg/well of different luciferase promoter constructs: A, wtH4/Luc; B, IRF mutH4/Luc; C, 4X IRF/H4-site II/Luc; D, 3X H4 distal site II/Luc. The cells were co-transfected with 0.4 μg/well of pcDNA/IRF-1, pcDNA/IRF-2, pcDNA/IRF-3, pcDNA/IRF-7, and pcDNA as control. Renilla luciferase construct (50 ng/well) was used as an internal control for each sample. Samples were analyzed by the dual-luciferase assay 24 h after transfection as described under "Materials and Methods." The graphswere based on at least three independent experiments with triplicate samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess further the role of H4 site II promoter elements in mediating activation by IRF factors, we prepared two promoter constructs in which either the IRF element or the entire distal site II segment was tandemly repeated upstream of distinct minimal promoters,i.e. human histone H4 or the mouse MHC class I H2-L TATA box regions, respectively (Fig. 3, C and D). Our results indicate that IRF-1 can synergistically activate H4-related transcription in the presence of multimerized IRF e

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