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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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DEAF1 interacts with IRF3 and IRF7. HEK293FT cells in 10-cm dishes were transfected with DNA (0.6 μg/ml) encoding GFP-DEAF1 and either HA-IRF3 or HA-IRF7. The cells were extracted in lysis buffer containing 50 mm iodoacetamide as in Fig. 1 and incubated for 2 h at 4 °C in the absence or presence of the DNase benzonase (50 units/ml). A, aliquots of the cell extract (3 μg protein) were subjected to SDS-PAGE and immunoblotted as in Fig. 1. B, IRF3 or IRF7 were immunoprecipitated from 0.1 mg of cell lysate protein with an HA antibody and the immunoprecipitates washed, denatured in SDS, and subjected to SDS-PAGE and immunoblotting as in A. C, as in B except that DEAF1 was immunoprecipitated using anti-GFP. D–F, as in A–C except that the cells were co-transfected with DNA encoding GFP-DEAF1 and HA-CREB.
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Figure 6: DEAF1 interacts with IRF3 and IRF7. HEK293FT cells in 10-cm dishes were transfected with DNA (0.6 μg/ml) encoding GFP-DEAF1 and either HA-IRF3 or HA-IRF7. The cells were extracted in lysis buffer containing 50 mm iodoacetamide as in Fig. 1 and incubated for 2 h at 4 °C in the absence or presence of the DNase benzonase (50 units/ml). A, aliquots of the cell extract (3 μg protein) were subjected to SDS-PAGE and immunoblotted as in Fig. 1. B, IRF3 or IRF7 were immunoprecipitated from 0.1 mg of cell lysate protein with an HA antibody and the immunoprecipitates washed, denatured in SDS, and subjected to SDS-PAGE and immunoblotting as in A. C, as in B except that DEAF1 was immunoprecipitated using anti-GFP. D–F, as in A–C except that the cells were co-transfected with DNA encoding GFP-DEAF1 and HA-CREB.

Mentions: Since DEAF1 and IRF3 appeared to bind to the same region of the IFNβ promoter, and DEAF1 is required for IRF3 to associate with the IFNβ promoter and to enhance IFNβ gene transcription, we wondered whether DEAF1 might interact with IRF3 directly. We therefore co-transfected DNA encoding GFP-tagged DEAF1 and HA-tagged IRF3 into 293FT cells (Fig. 6A). We found that GFP-DEAF1 was immunoprecipitated from the cell extracts with anti-HA (Fig. 6B) while, conversely, HA-IRF3 was immunoprecipitated with anti-GFP (Fig. 6C). Similar overexpression experiments revealed that DEAF1 could also interact with IRF7 (Fig. 6, B and C), which also binds to the IFNβ promoter where it may function alone or as a heterodimer with IRF3 (see Introduction). The interaction between DEAF1 and IRF3 or IRF7 was not affected by DNase treatment prior to immunoprecipitation (Fig. 6, A–C), indicating that the interactions were specific and not mediated indirectly by the binding of DNA to these transcription factors. CREB (cyclic AMP response element binding protein), another transcription factor, did not interact with DEAF1 (Fig. 6, D–F), again indicating that the interactions with IRF3/7 were specific.


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)

DEAF1 interacts with IRF3 and IRF7. HEK293FT cells in 10-cm dishes were transfected with DNA (0.6 μg/ml) encoding GFP-DEAF1 and either HA-IRF3 or HA-IRF7. The cells were extracted in lysis buffer containing 50 mm iodoacetamide as in Fig. 1 and incubated for 2 h at 4 °C in the absence or presence of the DNase benzonase (50 units/ml). A, aliquots of the cell extract (3 μg protein) were subjected to SDS-PAGE and immunoblotted as in Fig. 1. B, IRF3 or IRF7 were immunoprecipitated from 0.1 mg of cell lysate protein with an HA antibody and the immunoprecipitates washed, denatured in SDS, and subjected to SDS-PAGE and immunoblotting as in A. C, as in B except that DEAF1 was immunoprecipitated using anti-GFP. D–F, as in A–C except that the cells were co-transfected with DNA encoding GFP-DEAF1 and HA-CREB.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: DEAF1 interacts with IRF3 and IRF7. HEK293FT cells in 10-cm dishes were transfected with DNA (0.6 μg/ml) encoding GFP-DEAF1 and either HA-IRF3 or HA-IRF7. The cells were extracted in lysis buffer containing 50 mm iodoacetamide as in Fig. 1 and incubated for 2 h at 4 °C in the absence or presence of the DNase benzonase (50 units/ml). A, aliquots of the cell extract (3 μg protein) were subjected to SDS-PAGE and immunoblotted as in Fig. 1. B, IRF3 or IRF7 were immunoprecipitated from 0.1 mg of cell lysate protein with an HA antibody and the immunoprecipitates washed, denatured in SDS, and subjected to SDS-PAGE and immunoblotting as in A. C, as in B except that DEAF1 was immunoprecipitated using anti-GFP. D–F, as in A–C except that the cells were co-transfected with DNA encoding GFP-DEAF1 and HA-CREB.
Mentions: Since DEAF1 and IRF3 appeared to bind to the same region of the IFNβ promoter, and DEAF1 is required for IRF3 to associate with the IFNβ promoter and to enhance IFNβ gene transcription, we wondered whether DEAF1 might interact with IRF3 directly. We therefore co-transfected DNA encoding GFP-tagged DEAF1 and HA-tagged IRF3 into 293FT cells (Fig. 6A). We found that GFP-DEAF1 was immunoprecipitated from the cell extracts with anti-HA (Fig. 6B) while, conversely, HA-IRF3 was immunoprecipitated with anti-GFP (Fig. 6C). Similar overexpression experiments revealed that DEAF1 could also interact with IRF7 (Fig. 6, B and C), which also binds to the IFNβ promoter where it may function alone or as a heterodimer with IRF3 (see Introduction). The interaction between DEAF1 and IRF3 or IRF7 was not affected by DNase treatment prior to immunoprecipitation (Fig. 6, A–C), indicating that the interactions were specific and not mediated indirectly by the binding of DNA to these transcription factors. CREB (cyclic AMP response element binding protein), another transcription factor, did not interact with DEAF1 (Fig. 6, D–F), again indicating that the interactions with IRF3/7 were specific.

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