Selective DNA Binding and Association with the CREB Binding Protein Coactivator Contribute to Differential Activation of Alpha/Beta Interferon Genes by Interferon Regulatory Factors 3 and 7
Recent studies implicate the interferon (IFN) regulatory factors (IRF) IRF-3 and IRF-7 as key activators of the alpha/beta IFN (IFN-α/β) genes as well as the RANTES chemokine gene. Using coexpression analysis, the human IFNB, IFNA1, and RANTES promoters were stimulated by IRF-3 coexpression, whereas the IFNA4, IFNA7, and IFNA14 promoters were preferentially induced by IRF-7 only. Chimeric proteins containing combinations of different IRF-7 and IRF-3 domains were also tested, and the results provided evidence of distinct DNA binding properties of IRF-3 and IRF-7, as well as a preferential association of IRF-3 with the CREB binding protein (CBP) coactivator. Interestingly, some of these fusion proteins led to supraphysiological levels of IFN promoter activation. DNA binding site selection studies demonstrated that IRF-3 and IRF-7 bound to the 5′-GAAANNGAAANN-3′ consensus motif found in many virus-inducible genes; however, a single nucleotide substitution in either of the GAAA half-site motifs eliminated IRF-3 binding and transactivation activity but did not affect IRF-7 interaction or transactivation activity. These studies demonstrate that IRF-3 possesses a restricted DNA binding site specificity and interacts with CBP, whereas IRF-7 has a broader DNA binding specificity that contributes to its capacity to stimulate delayed-type IFN gene expression. These results provide an explanation for the differential regulation of IFN-α/β gene expression by IRF-3 and IRF-7 and suggest that these factors have complementary rather than redundant roles in the activation of the IFN-α/β genes.
Interferons (IFNs) are multifunctional secreted proteins involved in antiviral defense, cell growth regulation, and immune activation (44). Alpha/beta IFN (IFN-α/β) is produced by virus-infected host cells and constitutes the primary response against virus infection, while gamma IFN (IFN-γ), a TH1 cytokine produced by activated T cells and natural killer cells, is crucial in eliciting the proper immune response and pathogen clearance. Virus infection induces the transcription and synthesis of multiple IFN genes (16, 33, 44); newly synthesized IFN interacts with neighboring cells through cell surface receptors and the Janus-activated kinase (JAK)–STAT signaling pathway, resulting in the induction of over 30 new cellular proteins that mediate the diverse functions of the IFNs (6, 18, 21, 39). Among the many virus- and IFN-inducible proteins are members of the growing family of interferon regulatory factors (IRFs), which now consists of nine members, as well as several virus-encoded IRFs (4). The presence of IRF-like binding sites in the promoter regions of the IFNB and IFNA genes implicated the IRFs as direct regulators of IFN-α/β gene induction (11–14, 29). Within the IRF family, IRF-3 and IRF-7 have recently been identified as key regulators of the induction of IFNs (reviewed in reference 26).
IRF-3 is expressed constitutively in a variety of tissues and demonstrates a unique response to virus infection (1). Latent cytoplasmic IRF-3 is posttranslationally modified and activated through phosphorylation of specific serine residues located in its C-terminal end following virus infection or treatment with double-stranded RNA (24, 45–47). Overexpression of IRF-3 significantly enhances virus-mediated expression of IFN-α/β genes and results in the induction of an antiviral state (19). Other studies have demonstrated that transcription of the CC-chemokine RANTES is upregulated by virus infection, mediated through IRF-3 activation and binding to overlapping ISRE-like elements in the −100 region of the RANTES promoter (23).
Structure-function analysis has revealed that IRF-3 contains an N-terminal DNA binding domain (DBD); a strong but atypical transactivation domain, located between amino acids 134 and 394, a region that also contains a nuclear export sequence element; a proline-rich region; and an IRF association domain (IAD). Two autoinhibitory domains in IRF-3 form an intramolecular interaction that results in a closed conformation and masks the IAD and the DBD to prevent nuclear translocation and subsequent DNA binding (25). Following virus infection, inducible phosphorylation of IRF-3 at the carboxy terminus relieves the intramolecular association between the two autoinhibitory domains, unmasking the IAD and the DBD. The conformational change in IRF-3 results in the formation of homodimers through the IAD. IRF-3 dimerization leads to cytoplasmic to nuclear translocation, association with the CREB binding protein (CBP) coactivator, and stimulation of DNA binding and transcriptional activities (reviewed in references 17 and 26). IRF-3 phosphorylation ultimately results in its degradation via the ubiquitin-proteasome pathway (24, 34). These biological features implicate IRF-3 as an important component of the immediate-early response to virus infection (17, 26).
IRF-7 was first described to bind and repress the Qp promoter region of the Epstein-Barr virus (EBV) EBNA-1 gene, which contains an ISRE-like element (31, 48). Unlike IRF-3, IRF-7 is not expressed constitutively in cells; rather, expression is induced by IFN, lipopolysaccharide, and virus infection. As with IRF-3, virus infection appears to induce the phosphorylation of IRF-7 at its carboxy terminus, a region that is highly homologous to the IRF-3 C-terminal end (27, 37). IRF-7 also localizes to the cytoplasm in uninfected cells and translocates to the nucleus after phosphorylation (2, 37). Two groups have identified potential serine residues targeted for inducible phosphorylation by analogy to IRF-3. Marie et al. mutated Ser425 and Ser426 in murine IRF-7, based on homology to Ser385 and Ser386 in IRF-3. The mutant was not phosphorylated and did not activate IFN-α gene expression (27). Sato et al. generated a deletion mutant in which the region containing the potential sites of inducible phosphorylation between amino acids 411 to 453 was truncated. The mutant no longer translocated to the nucleus following virus infection, implicating inducible phosphorylation as a critical step for translocation (37).
Because of the common and distinct biological features of IRF-3 and IRF-7, we sought to identify the molecular basis for the differential activation of IFN-α/β genes by IRF-3 and IRF-7 in response to virus infection. Our results indicate that the distinct DNA binding specificities of IRF-3 and IRF-7—together with the different capacities of the IRF-3 and IRF-7 C-terminal domains to bind the CBP coactivator—provide an explanation for the differential regulation of IFN-α/β gene expression by these two transcription factors.
We thank Paula Pitha, Luwen Zhang, Joseph Pagano, Xiang-Jiao Yang, and Illka Julkunen for reagents used in this study and members of the Molecular Oncology Group, Lady Davis Institute for Medical Research, for helpful discussions.
This research was supported by grants from the Cancer Research Society Inc. and the Medical Research Council of Canada. R.L. was supported in part by a Fraser Monat McPherson fellowship from McGill University, P.G. was supported by an FRSQ postdoctoral fellowship, Y.M. was supported by an MRC studentship, and J.H. was supported by an MRC senior scientist award.
- 1. Au W-C, Moore P A, Lowther W, Juang Y-T, Pitha P MIdentification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc Natl Acad Sci USA. 1995;92:11657–11661.
- 2. Au W C, Moore P A, LaFleur D W, Tombal B, Pitha P MCharacterization of the interferon regulatory factor-7 and its potential role in the transcription activation of interferon A genes. J Biol Chem. 1998;273:29210–29217.
- 3. Braganca J, Civas AType I interferon gene expression: differential expression of IFN-A genes induced by viruses and double-stranded RNA. Biochimie. 1998;80:673–687.
- 4. Burysek L, Yeow W S, Pitha P MUnique properties of a second human herpesvirus 8-encoded interferon regulatory factor (vIRF-2) J Hum Virol. 1999;2:19–32.
- 5. Chen J, Attardi L, Verrijzer P, Yokomori K, Tjian RAssembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators. Cell. 1994;79:93–105.
- 6. Darnell J E, Jr, Kerr I M, Stark G RJak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science. 1994;264:1415–1421.
- 7. Dikstein R, Ruppert S, Tjian RTAFII250 is a bipartite protein kinase that phosphorylates the basal transcription factor RAP74. Cell. 1996;84:781–790.
- 8. Escalante C R, Yie J, Thanos D, Aggarwal A KStructure of IRF-1 with bound DNA reveals determinants of interferon regulation. Nature. 1998;391:103–106.
- 9. Falvo J V, Thanos D, Maniatis TReversal of intrinsic DNA bends in the IFNβ gene enhancer by transcription factors and the architectural protein HMG I(Y) Cell. 1995;83:1101–1111.
- 10. Fujii Y, Shimizu T, Kusumoto M, Kyogoku Y, Taniguchi T, Hakoshima TCrystal structure of an IRF-DNA complex reveals novel recognition of and cooperative binding to a tandem repeat of core sequences. EMBO J. 1999;18:5028–5041.
- 11. Fujita T, Kimura Y, Miyamoto M, Barsoumian E L, Taniguchi TInduction of endogenous IFN-α and IFN-β genes by a regulatory transcription factor IRF-1. Nature. 1989;337:270–272.
- 12. Fujita T, Sakakibara J, Sudo Y, Miyamoto M, Kimura Y, Taniguchi TEvidence for a nuclear factor(s), IRF-1, mediating induction and silencing properties to human IFN-β gene regulatory elements. EMBO J. 1988;7:3397–3405.
- 13. Harada H, Fujita T, Miyamoto M, Kimura Y, Maruyama M, Furia A, Miyata T, Taniguchi TStructurally similar but functionally distinct factors, IRF-1 and IRF-2, bind to the same regulatory elements of IFN and IFN-inducible genes. Cell. 1989;58:729–739.
- 14. Harada H, Willison K, Sakakibara J, Miyamoto M, Fujita T, Taniguchi TAbsence of type I IFN system in EC cells: transcriptional activator (IRF-1) and repressor (IRF-2) genes are developmentally regulated. Cell. 1990;63:903–913.
- 15. Hiscott J, Cantell K, Weissmann CDifferential expression of human interferon genes. Nucleic Acids Res. 1984;12:3727–3746.
- 16. Hiscott J, Nguyen H, Lin RMolecular mechanisms of interferon beta gene induction. Semin Virol. 1995;6:161–173.
- 17. Hiscott J, Pitha P, Génin P, Nguyen H, Heylbroeck C, Mamane Y, Algarté M, Lin RTriggering the interferon response: the role of IRF-3 transcription factor. J Interferon Cytokine Res. 1999;19:1–13.
- 18. Ihle J NSTATs: signal transducers and activators of transcription. Cell. 1996;84:331–334.
- 19. Juang Y T, Lowther W, Kellum M, Au W C, Lin R, Hiscott J, Pitha P MPrimary activation of interferon A and interferon B gene transcription by interferon regulatory factory-3. Proc Natl Acad Sci USA. 1998;95:9837–9842.
- 20. Kim T K, Maniatis TThe mechanism of transcriptional synergy of an in vitro assembled interferon-β enhanceosome. Mol Cell. 1998;1:119–129.
- 21. Levy D EInterferon induction of gene expression through the Jak-Stat pathway. Semin Virol. 1995;6:181–190.
- 22. Lin R, Beauparlant P, Makris C, Meloche S, Hiscott JPhosphorylation of IκBα in the C-terminal PEST domain by casein kinase II affects intrinsic protein stability. Mol Cell Biol. 1996;16:1401–1409.
- 23. Lin R, Heylbroeck C, Genin P, Pitha P, Hiscott JEssential role of IRF-3 in direct activation of RANTES gene transcription. Mol Cell Biol. 1999;19:959–966.
- 24. Lin R, Heylbroeck C, Pitha P M, Hiscott JVirus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol Cell Biol. 1998;18:2986–2996.
- 25. Lin R, Mamane Y, Hiscott JStructural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains. Mol Cell Biol. 1999;19:2465–2474.
- 26. Mamane Y, Heylbroeck C, Genin P, Algarte M, Servant M, Lepage C, DeLuca C, Kwon H, Lin R, Hiscott JInterferon regulatory factors: the next generation. Gene. 1999;237:1–14.
- 27. Marie I, Durbin J E, Levy D EDifferential viral induction of distinct interferon-α genes by positive feedback through interferon regulatory factor-7. EMBO J. 1998;17:6660–6669.
- 28. Merika M, Williams A J, Chen G, Collins T, Thanos DRecruitment of CBP/p300 by the IFNβ enhanceosome is required for synergistic activation of transcription. Mol Cell. 1998;1:277–287.
- 29. Miyamoto M, Fujita T, Kimura Y, Maruyama M, Harada H, Sudo Y, Miyata T, Taniguchi TRegulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to the IFN-β gene regulatory elements. Cell. 1988;54:903–913.
- 30. Mizzen C, Yang X, Kokubo T, Brownell J, Bannister A, Owen-Hughes T, Workman J, Wang L, Berger S, Kouzarides T, Nakatani Y, Allis C DThe TAFII250 subunit of TFIID has histone acetyltransferase activity. Cell. 1996;87:1261–1270.
- 31. Nonkwello C, Ruf I K, Sample JInterferon-independent and -induced regulation of Epstein-Barr virus EBNA-1 gene transcription in Burkitt lymphoma. J Virol. 1997;71:6887–6897.
- 32. Parekh B S, Maniatis TVirus infection leads to localized hyperacetylation of histones H3 and H4 at the IFN-β promoter. Mol Cell. 1999;3:125–129.
- 33. Pitha P M, Au W-CInduction of interferon alpha gene expression. Semin Virol. 1995;6:151–159.
- 34. Ronco L, Karpova A, Vidal M, Howley PHuman papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes Dev. 1998;12:2061–2072.
- 35. Ruppert S, Tjian RHuman TAFII250 interacts with RAP74: implications for RNA polymerase II initiation. Genes Dev. 1995;9:2747–2755.
- 36. Ruppert S, Wang E, Tjian RCloning and expression of human TAFII250: a TBP-associated factor implicated in cell cycle regulation. Nature. 1993;362:175–179.
- 37. Sato M, Hata N, Asagiri M, Nakaya T, Taniguchi T, Tanaka NPositive feedback regulation of type I IFN genes by the IFN-inducible transcription factor IRF-7. FEBS Lett. 1998;441:106–110.
- 38. Schafer S L, Lin R, Moore P A, Hiscott J, Pitha P MRegulation of type 1 interferon gene expression by interferon regulatory factor 3. J Biol Chem. 1998;273:2714–2720.
- 39. Schindler C, Darnell J E., Jr Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. Annu Rev Biochem. 1995;64:621–651.
- 40. Struhl KChromatin structure and RNA polymerase II connection: implications for transcription. Cell. 1996;84:179–182.
- 41. Tanaka N, Kawakami T, Taniguchi TRecognition DNA sequences of interferon regulatory factor 1 (IRF-1) and IRF-2, regulators of cell growth and the interferon system. Mol Cell Biol. 1993;13:4531–4538.
- 42. Thanos D, Maniatis TVirus induction of human IFNβ gene expression requires the assembly of an enhanceosome. Cell. 1995;83:1091–1100.
- 43. Verrijzer C, Tjian RTAFs mediate transcriptional activation and promoter selectivity. Trends Biochem Sci. 1996;21:338–342.
- 44. Vilcek J, Sen G. Interferons and other cytokines. In: Fields B, Knipe D M, Howley P M, editors. Virology. Philadelphia, Pa: Lippincott-Raven; 1996. pp. 375–399.
- 45. Wathelet M G, Lin C H, Parakh B S, Ronco L V, Howley P M, Maniatis TVirus infection induces the assembly of coordinately activated transcription factors on the IFN-β enhancer in vivo. Mol Cell. 1998;1:507–518.
- 46. Weaver B K, Kumar K P, Reich N CInterferon regulatory factor 3 and CREB-binding protein/p300 are subunits of double-stranded RNA-activated transcription factor DRAF1. Mol Cell Biol. 1998;18:1359–1368.
- 47. Yoneyama M, Suhara W, Fukuhara Y, Fukada M, Nishida E, Fujita TDirect triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. EMBO J. 1998;17:1087–1095.
- 48. Zhang L, Pagano J SIRF-7, a new interferon regulatory factor associated with Epstein-Barr virus latency. Mol Cell Biol. 1997;17:5748–5757.