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.
Journal: 2000/September - Molecular and Cellular Biology
ISSN: 0270-7306
PUBMED: 10938111
Abstract:
Recent studies implicate the interferon (IFN) regulatory factors (IRF) IRF-3 and IRF-7 as key activators of the alpha/beta IFN (IFN-alpha/beta) 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-alpha/beta 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-alpha/beta genes.
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Mol Cell Biol 20(17): 6342-6353

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

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, and Departments of Microbiology and Immunology and Medicine, McGill University, Montreal, Quebec, Canada H3T 1E2
Corresponding author. Mailing address: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T 1E2. Phone: (514) 340-8222, ext. 3169. Fax: (514) 340-7576. E-mail: ac.lligcm.acisum@ildm.
Received 2000 Jan 27; Revisions requested 2000 Mar 22; Accepted 2000 Jun 2.

Abstract

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.

Abstract

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 (1114, 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, 4547). 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.

ACKNOWLEDGMENTS

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.

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