The histone acetylase PCAF is a phorbol-ester-inducible coactivator of the IRF family that confers enhanced interferon responsiveness.
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Transcription factors of the interferon regulatory factor (IRF) family bind to the type I interferon (IFN)-responsive element (ISRE) and activate transcription from IFN-inducible genes. To identify cofactors that associate with IRF proteins, DNA affinity binding assays were performed with nuclear extracts prepared from tissue culture cells. The results demonstrated that the endogenous IRFs bound to the ISRE are complexed with the histone acetylases, PCAF, GCN5, and p300/CREB binding protein and that histone acetylase activities are accumulated on the IRF-ISRE complexes. By testing recombinant proteins, we show that PCAF directly binds to some but not all members of the IRF family through distinct domains of the two proteins. This interaction was functionally significant, since transfection of PCAF strongly enhanced IRF-1- and IRF-2-dependent promoter activities. Further studies showed that expression of PCAF and other histone acetylases was markedly induced in U937 cells upon phorbol ester treatment, which led to increased recruitment of PCAF to the IRF-ISRE complexes. Coinciding with the induction of histone acetylases, phorbol ester markedly enhanced IFN-alpha-stimulated gene expression in U937 cells. Supporting the role for PCAF in conferring IFN responsiveness, transfection of PCAF into U937 cells led to a large increase in IFN-alpha-inducible promoter activity. These results demonstrate that PCAF is a phorbol ester-inducible coactivator of the IRF proteins which contributes to the establishment of type I IFN responsiveness. (+info)
Interferon regulatory factor 2 represses the Epstein-Barr virus BamHI Q latency promoter in type III latency.
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Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is the essential protein for maintenance of the EBV episome and establishment of latency. The BamHI Q promoter (Qp) is used for the transcription of EBNA-1 mRNA in type I and type II latency, which are EBV infection states exemplified by Burkitt's lymphoma and nasopharyngeal carcinoma. However, Qp is inactive in type III latency, and other promoters (the BamHI C promoter and/or the BamHI W promoter) are used for EBNA-1. The involvement of interferon regulatory factors (IRFs) in the regulation of Qp is suggested by the presence of an essential interferon-stimulated response element (ISRE) in the promoter. In this work, expression of IRF-2 is shown to be inversely associated with Qp status, i.e., IRF-2 levels are high in type III latency (when Qp is inactive) and low in type I latency (when Qp is active). Also, IRF-2 is identified by electrophoretic mobility shift assay as the major protein binding to the Qp ISRE in type III latency. In transient transfection assays, IRF-2 represses the activity of Qp-reporter constructs. Overexpression of IRF-2 in a type I latency cell line did not activate the endogenous Qp but marginally reduced the EBNA-1 mRNA level. Switching from type III latency (Qp inactive) to type II latency (Qp active), as produced by cell fusion, is directly associated with greatly reduced expression of IRF-2. These data strongly suggest that IRF-2 is a negative regulator of Qp and may contribute to the silencing of Qp in type III latency. (+info)
Role of interferon and interferon regulatory factors in early protection against Venezuelan equine encephalitis virus infection.
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To investigate the role of type I interferon (IFN) and its regulatory transacting proteins, interferon regulatory factors (IRF-1 and IRF-2), in early protection against infection with virulent Venezuelan equine encephalitis virus (VEE), we utilized mice with targeted mutations in the IFN-alpha/beta receptor, IRF-1, or IRF-2 genes. IFN-alpha/beta-receptor knockout mice are highly susceptible to peripheral infection with virulent or attenuated VEE, resulting in their death within 24 and 48 h, respectively. Treatment of normal macrophages with anti-IFN-alpha/beta antibody prior to and during infection with molecularly cloned virulent VEE resulted in increased VEE replication. However, treatment with high doses of IFN or IFN-inducing agents failed to alter percentage mortality or average survival times in mice challenged with a low dose of virulent VEE. In IRF-1 and IRF-2 knockout mice (IRF-1(-/-) and IRF-2(-/-)), the 100% protection against virulent VEE that is conferred by attenuated VEE within 24 h in control C57BL/6 mice was completely absent in IRF-2(-/-) mice, whereas 50% of IRF-1(-/-) mice were protected. IRF-2(-/-) mice were deficient in clearing VEE virus from the spleen and the brain compared to the heterozygous IRF-2(+/-) knockout or C57BL/6 (+/+) mice. Furthermore, a distinct pattern of histopathological changes was observed in brains of IRF-2(-/-) mice after VEE exposure. Taken together, these findings imply that the altered immune response in IRF-1 and IRF-2 knockout mice results in altered virus dissemination, altered virus clearance, and altered virus-induced pathology. Thus, type I interferon, as well as IRF-1 and IRF-2, appears to play an important and necessary role in the pathogenesis of, and protection against, VEE infection. (+info)
Activation and repression of the 2-5A synthetase and p21 gene promoters by IRF-1 and IRF-2.
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The Interferon Regulatory Factors-1 and -2 (IRF-1 and IRF-2) were originally identified as transcriptional regulators of the interferon (IFN) and IFN-stimulated genes. These factors also modulate immune response and play a role in cell growth regulation. In this study we analysed the effect of the ectopic expression of IRF-1 and IRF-2 on the regulation of two potential IRF target genes involved in cell growth regulation, 2-5A synthetase and p21 (WAF/CP1), both of which contain consensus binding sites for IRF family members within their promoters. Following ectopic expression, IRF-1 transactivated 2-5A synthetase and p21 genes, an effect that was counterbalanced by concomitant ectopic expression of IRF-2. These effects were mediated by direct binding of IRF to the gene promoters. A construct expressing an IRF-2 antisense (FRI-2) was able to revert the inhibitory effect of IRF-2 on the IRF-1 transactivation. IRF-1 also induced expression of its homologous repressor IRF-2 as indicated by EMSA analysis using an IRF-E probe from the IRF-2 promoter; and by cotransfection of IRF-1 together with an IRF-2 promoter CAT construct. Therefore, the induction of IRF-1 by IFNs or other stimuli acts as a transactivator of genes involved in cell growth regulation, as well as of its own repressor IRF-2, thus providing autoinhibitory regulation of IRF-1 activated genes. (+info)
Defective binding of IRFs to the initiator element of interleukin-1beta-converting enzyme (ICE) promoter in an interferon-resistant Daudi subline.
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To investigate mechanisms of interferon (IFN) resistance, we have established an IFN-resistant Daudi subline (Daudi(res)), which is 1 X 10(4) times more resistant to IFN-alpha than parental cells. Among the IFN-inducible genes examined, only ICE mRNA expression was deficient in Daudi(res) cells. We then analyzed the regulatory mechanisms of ICE transcription, and found that IFN-induced activation of the ICE promoter was dependent on the binding of IRFs to its initiator (Inr) element. Inr binding of IRFs was markedly diminished in Daudi(res) cells, and forced expression of IRF-1 was able to activate the ICE promoter to the level of parental cells. These results suggest that IRFs and their target genes, as represented by ICE in this study, are involved in IFN resistance. (+info)
Interferon regulatory transcription factors are constitutively expressed and spatially regulated in the mouse lens.
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Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulation of cell growth and immunological responses. Nine IRFs have been described and they are expressed in a variety of cells, except for ICSBP and LSIRF/Pip, which are thought to be expressed exclusively in immune cells. Here, we show that IRF-1, IRF-2, ICSBP, and LSIRF/Pip are constitutively expressed in the mouse lens. These IRFs are present in both the cytoplasm and the nuclei of lens cells. However, the nuclear and cytoplasmic proteins exhibit distinct mobilities on SDS/PAGE. We further show that in the developing mouse lens, IRF-1 and IRF-2 are expressed at high levels in differentiated lens fiber cells with very low and barely detectable levels in undifferentiated lens epithelial cells. Although the level of ICSBP expression is very low in the normal mouse lens, in transgenic mice with constitutive expression of interferon gamma in the lens, its level is markedly elevated and ICSBP expression is detected exclusively in the nuclei of undifferentiated lens cells. Taken together, our data suggest that expression of IRF transcription factors is spatially regulated in the lens and that distinct IRFs may contribute to differential gene regulation in the epithelial and fiber compartments of the vertebrate lens. (+info)
IL-12 is dysregulated in macrophages from IRF-1 and IRF-2 knockout mice.
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Macrophages derived from IFN-regulatory factor-1 (IRF-1) and IRF-2 knockout (-/-) and wild-type (+/+) mice were utilized to examine the role of these transcription factors in the regulation of IL-12 mRNA and protein expression. Induction of IL-12 p40 mRNA by LPS was markedly diminished in both IRF-1(-/-) and IRF-2(-/-) macrophages. In contrast, IRF-1(-/-), but not IRF-2(-/-), macrophages exhibited impaired LPS-induced IL-12 p35 mRNA expression. The ability of IFN-gamma to augment LPS-induced IL-12 p40 mRNA further when both stimuli were present simultaneously was significantly diminished in both IRF-1(-/-) and IRF-2(-/-) macrophages, with the most profound impairment observed for IRF-1(-/-) macrophages. Reductions in IL-12 mRNA expression after stimulation with LPS or LPS plus IFN-gamma were accompanied by substantial reductions in IL-12 p40 and IL-12 p70 protein in both IRF-1(-/-) and IRF-2(-/-) macrophages. Priming IRF-1(-/-) and IRF-2(-/-) macrophages with IFN-gamma for 24 h before LPS treatment partially restored impaired IL-12 mRNA and protein production in both IRF-1(-/-) and IRF-2(-/-) macrophages. Depressed IL-12 levels were paralleled by significant reductions in IFN-gamma mRNA expression in IRF-1(-/-) and IRF-2(-/-) macrophages. These results indicate that both IRF-1 and IRF-2 are critical transcription factors in the regulation of macrophage IL-12 and consequently IFN-gamma production. (+info)
Functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300.
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Human herpesvirus 8/Kaposi sarcoma-associated virus (HHV-8/KSHV) contains, in addition to genes required for viral replication, a unique set of nonstructural genes which may be part of viral mimicry and contribute to viral replication and pathogenesis in vivo. Among these, HHV-8 encodes four open reading frames (ORFs) that showed homology to the transcription factors of the interferon regulatory factor (IRF) family. The ORF K9, viral IRF 1 (vIRF-1), has been cloned, and it was shown that, when overexpressed, it down modulates the interferon-mediated transcriptional activation of the interferon-stimulated gene 15 (ISG 15) promoter, and the role of vIRF-1 in viral mimicry was implied. However, the molecular mechanism of this effect has not been clarified. Here, we extend this observation and show that vIRF-1 also downregulates the transcriptional activity of IFNA gene promoter in infected cells by interfering with the transactivating activity of cellular IRFs, including IRF-1 and IRF-3. We further show that ectopic expression of vIRF-1 in NIH 3T3 cells confers resistance to tumor necrosis factor alpha-induced apoptosis. While vIRF-1 is unable to bind DNA with the same specificity as cellular IRFs, we demonstrate by in vitro binding assay that it can associate with the family of cellular IRFs, such as IRF-1 and the interferon consensus sequence binding protein. vIRF-1 interaction domain was localized between amino acids (aa) 152 and 243. While no binding between the full-size IRF-3 and vIRF-1 could be detected by the same assay, we show that vIRF-1 also targets the carboxy-terminal region (aa 1623 to 2414) of the transcriptional coactivator p300 which could also bind IRF-3 and IRF-1. These results demonstrate that vIRF-1 can modulate the transcription of the IFNA genes by direct heterodimerization with members of the IRF family, as well as by competitive binding with cellular transcription factors to the carboxy-terminal region of p300. (+info)