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DEAF1 is a Pellino1-interacting protein required for interferon production by Sendai virus and double-stranded RNA.

Ordureau A, Enesa K, Nanda S, Le Francois B, Peggie M, Prescott A, Albert PR, Cohen P - J. Biol. Chem. (2013)

Bottom Line: The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1.DEAF1 is also needed for TLR3-dependent IFNβ production.Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

ABSTRACT
Double-stranded (ds) RNA of viral origin, a ligand for Melanoma Differentiation-associated gene 5 (MDA5) and Toll-Like Receptor 3 (TLR3), induces the TANK-Binding Kinase 1 (TBK1)-dependent phosphorylation and activation of Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1, which are required for interferon β (IFNβ) gene transcription. Here, we report that Pellino1 interacts with the transcription factor Deformed Epidermal Autoregulatory Factor 1 (DEAF1). The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1. We show that DEAF1 binds to the IFNβ promoter and to IRF3 and IRF7, that it is required for the transcription of the IFNβ gene and IFNβ secretion in MEFs infected with Sendai virus or transfected with poly(I:C). DEAF1 is also needed for TLR3-dependent IFNβ production. Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

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IFNβ promoters associated with IRF3 are reduced in MEFs from DEAF1−/− mice.A, MEFs from wild type mice (black bars) or DEAF1−/− mice (white bars) were infected with Sendai virus for the times indicated. The cells were lysed, and ChIP assays performed after immunoprecipitating IRF3 from the extracts with a specific anti-IRF3 antibody. The ordinate shows the fold enrichment of the IFNβ promoter in the anti-IRF3 immunoprecipitates compared with that measured after immunoprecipitation from uninfected cells (time = 0). The IFNβ promoter DNA was measured by qRT-PCR, as described in “Experimental Procedures.” The results are shown ± S.D. for triplicate determinations using MEFs from two different wild type and two different DEAF1−/− mice at each time point. Similar results were obtained in two independent experiments. B, experiment was carried out as in A, except that the ChIP assay was performed after immunoprecipitating DEAF1 from the extracts of MEFs from wild type mice (black bars) or from DEAF1−/− mice (white bars) with an anti-DEAF1 antibody.
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Figure 4: IFNβ promoters associated with IRF3 are reduced in MEFs from DEAF1−/− mice.A, MEFs from wild type mice (black bars) or DEAF1−/− mice (white bars) were infected with Sendai virus for the times indicated. The cells were lysed, and ChIP assays performed after immunoprecipitating IRF3 from the extracts with a specific anti-IRF3 antibody. The ordinate shows the fold enrichment of the IFNβ promoter in the anti-IRF3 immunoprecipitates compared with that measured after immunoprecipitation from uninfected cells (time = 0). The IFNβ promoter DNA was measured by qRT-PCR, as described in “Experimental Procedures.” The results are shown ± S.D. for triplicate determinations using MEFs from two different wild type and two different DEAF1−/− mice at each time point. Similar results were obtained in two independent experiments. B, experiment was carried out as in A, except that the ChIP assay was performed after immunoprecipitating DEAF1 from the extracts of MEFs from wild type mice (black bars) or from DEAF1−/− mice (white bars) with an anti-DEAF1 antibody.

Mentions: The results presented in the preceding section suggested that DEAF1 must be exerting its effects “upstream” of IFNβ secretion. Since DEAF1 is a transcription factor (13), we investigated whether it could bind to the IFNβ promoter and/or whether it affected the interaction of IRF3 with the IFNβ promoter. We showed that infection with Sendai virus enriched the association of IRF3 with IFNβ promoters in MEFs from wild type mice, which was maximal after 2 h, but this did not occur in MEFs from the DEAF1−/− mice (Fig. 4A). IFNβ promoters were not enriched if the anti-IRF3 was replaced by a control IgG (not shown). These ChIP experiments indicated that DEAF1 was required for the binding of IRF3 to the IFNβ promoter.


DEAF1 is a Pellino1-interacting protein required for interferon production by Sendai virus and double-stranded RNA.

Ordureau A, Enesa K, Nanda S, Le Francois B, Peggie M, Prescott A, Albert PR, Cohen P - J. Biol. Chem. (2013)

IFNβ promoters associated with IRF3 are reduced in MEFs from DEAF1−/− mice.A, MEFs from wild type mice (black bars) or DEAF1−/− mice (white bars) were infected with Sendai virus for the times indicated. The cells were lysed, and ChIP assays performed after immunoprecipitating IRF3 from the extracts with a specific anti-IRF3 antibody. The ordinate shows the fold enrichment of the IFNβ promoter in the anti-IRF3 immunoprecipitates compared with that measured after immunoprecipitation from uninfected cells (time = 0). The IFNβ promoter DNA was measured by qRT-PCR, as described in “Experimental Procedures.” The results are shown ± S.D. for triplicate determinations using MEFs from two different wild type and two different DEAF1−/− mice at each time point. Similar results were obtained in two independent experiments. B, experiment was carried out as in A, except that the ChIP assay was performed after immunoprecipitating DEAF1 from the extracts of MEFs from wild type mice (black bars) or from DEAF1−/− mice (white bars) with an anti-DEAF1 antibody.
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Related In: Results  -  Collection

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Figure 4: IFNβ promoters associated with IRF3 are reduced in MEFs from DEAF1−/− mice.A, MEFs from wild type mice (black bars) or DEAF1−/− mice (white bars) were infected with Sendai virus for the times indicated. The cells were lysed, and ChIP assays performed after immunoprecipitating IRF3 from the extracts with a specific anti-IRF3 antibody. The ordinate shows the fold enrichment of the IFNβ promoter in the anti-IRF3 immunoprecipitates compared with that measured after immunoprecipitation from uninfected cells (time = 0). The IFNβ promoter DNA was measured by qRT-PCR, as described in “Experimental Procedures.” The results are shown ± S.D. for triplicate determinations using MEFs from two different wild type and two different DEAF1−/− mice at each time point. Similar results were obtained in two independent experiments. B, experiment was carried out as in A, except that the ChIP assay was performed after immunoprecipitating DEAF1 from the extracts of MEFs from wild type mice (black bars) or from DEAF1−/− mice (white bars) with an anti-DEAF1 antibody.
Mentions: The results presented in the preceding section suggested that DEAF1 must be exerting its effects “upstream” of IFNβ secretion. Since DEAF1 is a transcription factor (13), we investigated whether it could bind to the IFNβ promoter and/or whether it affected the interaction of IRF3 with the IFNβ promoter. We showed that infection with Sendai virus enriched the association of IRF3 with IFNβ promoters in MEFs from wild type mice, which was maximal after 2 h, but this did not occur in MEFs from the DEAF1−/− mice (Fig. 4A). IFNβ promoters were not enriched if the anti-IRF3 was replaced by a control IgG (not shown). These ChIP experiments indicated that DEAF1 was required for the binding of IRF3 to the IFNβ promoter.

Bottom Line: The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1.DEAF1 is also needed for TLR3-dependent IFNβ production.Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom.

ABSTRACT
Double-stranded (ds) RNA of viral origin, a ligand for Melanoma Differentiation-associated gene 5 (MDA5) and Toll-Like Receptor 3 (TLR3), induces the TANK-Binding Kinase 1 (TBK1)-dependent phosphorylation and activation of Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1, which are required for interferon β (IFNβ) gene transcription. Here, we report that Pellino1 interacts with the transcription factor Deformed Epidermal Autoregulatory Factor 1 (DEAF1). The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1. We show that DEAF1 binds to the IFNβ promoter and to IRF3 and IRF7, that it is required for the transcription of the IFNβ gene and IFNβ secretion in MEFs infected with Sendai virus or transfected with poly(I:C). DEAF1 is also needed for TLR3-dependent IFNβ production. Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

Show MeSH
Related in: MedlinePlus