Journal: The Journal of Experimental Medicine

Article Title: The methyltransferase NSD3 promotes antiviral innate immunity via direct lysine methylation of IRF3

doi: 10.1084/jem.20170856

Figure Lengend Snippet: K366 monomethylation of IRF3 in VSV-infected macrophages. (A) Immunoblot analysis of mono-/dimethylation of IRF3 from TAP-IRF3 RAW264.7 cells infected with HSV (10 multiplicity of infection [MOI]) or VSV (1 MOI) or stimulated with LPS (100 ng/ml) for the indicated times. Cell lysates were immunoprecipitated with streptavidin-binding protein antibody–conjugated magnetic beads and then subjected to immunoblot analysis with antimono-/dimethylated antibody against TAP-tag IRF3. (B) Endogenous IRF3 immunoprecipitated with IRF3 antibody from PMs infected with VSV (1 MOI) for the indicated times was immunoblotted with the indicated antibodies. (C) Illustration of methylated lysine residues of IRF3 identified by MS assay. DBD, DNA-binding domain; IAD, interaction domain of IRF3. (D) Ifnb activation in HEK293T cells transfected with WT IRF3 or IRF3 mutants was analyzed by luciferase reporter assay. (E) The tryptic peptide IRF3 (360–366, VVPTCLK), consistent with a 14-D mass modification of K366, was identified in VSV-infected TAP-IRF3 RAW264.7 cells by MS analysis. Images are representative of two independent experiments. (F) IRF3 immunoprecipitated from virus-infected or TLR-stimulated TAP-IRF3 RAW264.7 cells was immunoblotted with the indicated antibodies. (G) PMs from IRF3-deficient mice were transfected with WT IRF3 or IRF3 mutants by nuclear transfection for 24 h and then infected with VSV (1 MOI) for 8 h. The level of mRNA of Ifnb was measured by Q-PCR. (H) MEF cells from IRF3-deficient mice were transfected with WT IRF3 or IRF3 mutants by FuGENE HD transfection reagent for 24 h and then infected with VSV (1 MOI) for 8 h. The production of IFN-β was measured by ELISA. Mock, empty control vector. (A, B, and F) Immunoblots are representative of three independent experiments. (D) Data are mean ± SEM and representative of three independent experiments (one-way ANOVA; n = 4–6 per group). (G and H) Data are mean ± SEM and representative of three independent experiments (unpaired, two-tailed Student’s t test). *, P < 0.05; **, P < 0.01; ****, P < 0.0001. WCL, whole cell lysate.

Article Snippet: The polyclonal antibody against IRF3 K366 monomethylation (IRF3 K366me1) was custom produced by Abmart.

Techniques: Infection, Western Blot, Immunoprecipitation, Binding Assay, Magnetic Beads, Methylation, Activation Assay, Transfection, Luciferase, Reporter Assay, Modification, Virus, Enzyme-linked Immunosorbent Assay, Control, Plasmid Preparation, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: The methyltransferase NSD3 promotes antiviral innate immunity via direct lysine methylation of IRF3

doi: 10.1084/jem.20170856

Figure Lengend Snippet: NSD3 directly binds to IRF3 and methylates K366 of IRF3. (A) HEK293T cells were cotransfected with Myc-NSD3– and FLAG-IRF3–expressing plasmids and were infected 24 h later with VSV (1 MOI) for 8 h. Cell lysates were subjected to IP with anti-Myc or anti-FLAG and then immunoblotted with the indicated antibodies. (B) Mouse PMs were infected with VSV (1 MOI) for the indicated times. Cell lysates were subjected to IP with anti-IRF3 antibody and then immunoblotted with the indicated antibodies. (C) HEK293T cells were cotransfected with FLAG-IRF3 and/or Myc-NSD3 expression vectors and infected 24 h later with VSV (1 MOI) for 8 h. K366 methylation was immunoprecipitated using anti-FLAG antibody and then detected by immunoblot analysis. (D) PMs derived from NSD3 fl/fl lyz2-cre + or NSD3 fl/fl lyz2-cre − mice were infected by VSV (1 MOI), and K366 methylation was immunoprecipitated using anti-IRF3 antibody and then detected by immunoblot analysis. (E) MEF cells from NSD3 fl/fl lyz2-cre + or NSD3 fl/fl lyz2-cre − mice were transfected with WT NSD3 or NSD3 mutants by FuGENE HD transfection reagent for 24 h and then infected with VSV (1 MOI) for 8 h and subjected to IP and immunoblot analysis for K366 methylation. (F) Immunoprecipitated IRF3 from HEK293T cells overexpressing FLAG-IRF3 was used as substrate and subjected to in vivo methylation kinase assay using purified NSD3(SET) protein. Immunoblot assay was performed with the indicated antibodies. (G) Purified His-tagged IRF3 protein was used as substrate in the in vitro kinase assay as in F. Immunoblots are representative of three independent experiments. WCL, whole cell lysate.

Article Snippet: The polyclonal antibody against IRF3 K366 monomethylation (IRF3 K366me1) was custom produced by Abmart.

Techniques: Expressing, Infection, Methylation, Immunoprecipitation, Western Blot, Derivative Assay, Transfection, In Vivo, Kinase Assay, Purification, In Vitro

Journal: The Journal of Experimental Medicine

Article Title: The methyltransferase NSD3 promotes antiviral innate immunity via direct lysine methylation of IRF3

doi: 10.1084/jem.20170856

Figure Lengend Snippet: NSD3 enhances IRF3 transcriptional activity. (A and B) Luciferase activity assay in lysates of VSV-infected HEK293T cells cotransfected with different doses of NSD3-expressing plasmids with or without IRF3 plasmid (A) and transfected with NSD3 or its transcant mutants together with IRF3 (B) with Ifnb Luc reporter plasmid, and pTK-Renilla-luciferase. (C) ChIP analysis of H3K36 methylation (including H3K36me1, H3K36me2, and H3K36me3) to the Ifnb promoter in VSV-infected PMs derived from NSD3 fl/fl lyz2-cre + or NSD3 fl/fl lyz2-cre − mice. Med, control medium. (D) HEK293T cells transfected with NSD3 or its transcant mutants together with IRF3 or IRF3 mutants, Ifnb Luc reporter plasmid, and pTK-Renilla-luciferase. 24 h later, the cells were infected with VSV (1 MOI) for 8 h, and Ifnb luciferase activity in the cell lysates was analyzed. (E) IFN-β production in MEF cells derived from IRF3-deficient mice transfected with NSD3 and IRF3 or IRF3(K366A) or IRF3(K366F) was detected by ELISA. Mock, NSDE empty control vector. Data are mean ± SEM and representative of three independent experiments (A and B, one-way ANOVA, n = 4–6 per group; C–E, unpaired, two-tailed Student’s t test). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: The polyclonal antibody against IRF3 K366 monomethylation (IRF3 K366me1) was custom produced by Abmart.

Techniques: Activity Assay, Luciferase, Infection, Expressing, Plasmid Preparation, Transfection, Methylation, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: The methyltransferase NSD3 promotes antiviral innate immunity via direct lysine methylation of IRF3

doi: 10.1084/jem.20170856

Figure Lengend Snippet: NSD3 increases viral infection–triggered IRF3 phosphorylation in nucleus dependent on K366 methylation. (A–D) PMs –from NSD3 fl/fl lyz2-cre + or NSD3 fl/fl lyz2-cre − mice were infected with VSV (1 MOI) for the indicated times. IRF3 phosphorylation in total cell lysates (A), IRF3 dimerization (B), nuclear translocation (C), and IRF3 phosphorylation (D) in nucleus or cytoplasm fractions were detected by immunoblot analysis. (E) HEK293T cells were cotransfected with NSD3 together with WT IRF3. 24 h later, cells were infected with VSV (1 MOI) for 8 h. The cellular extracts were divided into nuclear and cytosolic fractions and subjected to IP and immunoblot analysis for K366 methylation. (F and G) HEK293T cells (F) and MEF cells from IRF3-deficient mice (G) were cotransfected with NSD3 together with WT IRF3 or mutants. 24 h later, the cells were infected with VSV (1 MOI) for 8 h and subjected to IP and immunoblot analysis for K366 methylation and IRF3 phosphorylation. (H) Immunoprecipitated IRF3 from HEK293T cells overexpressing IRF3 or IRF3 mutants (IRF3 K388A) was used as substrate and subjected to in vitro methylation kinase assay using purified NSD3(SET) protein. Immunoblot analysis was performed for K366 methylation. Immunoblots are representative of three independent experiments. WCL, whole cell lysate.

Article Snippet: The polyclonal antibody against IRF3 K366 monomethylation (IRF3 K366me1) was custom produced by Abmart.

Techniques: Infection, Phospho-proteomics, Methylation, Translocation Assay, Western Blot, Immunoprecipitation, In Vitro, Kinase Assay, Purification

Journal: The Journal of Experimental Medicine

Article Title: The methyltransferase NSD3 promotes antiviral innate immunity via direct lysine methylation of IRF3

doi: 10.1084/jem.20170856

Figure Lengend Snippet: NSD3-mediated IRF3 K366 methylation disrupts the association between IRF3 and PP1cc. (A) PMs were infected with VSV (1 MOI) for the indicated times and subjected to immunoblot analysis by the indicated antibodies. (B and C) HEK293T cells were transiently transfected with FLAG-IRF3 together with Myc-NSD3 or HA-PP1cc. 24 h later, the cells were infected with VSV (1 MOI) for the indicated times and subjected to IP and immunoblot analysis (B) and confocal microscopy (C) for the interaction of IRF3 and PP1cc and IRF3 phosphorylation in nucleus; numbers below lanes indicate densitometry of interacted PP1cc relative to that of total HA-PP1cc. Bars, 15 µm. (D) PMs from NSD3 fl/fl lyz2-cre + or NSD3 fl/fl lyz2-cre − mice were infected with VSV (1 MOI) for 8 h and then subjected to IP and immunoblot analysis for the interaction of IRF3 and PP1cc and IRF3 phosphorylation in nucleus; numbers below lanes indicate densitometry of interacted PP1cc and total NSD3 relative to that of LaminA/C. (E and F) PMs from NSD3 fl/fl lyz2-cre + or NSD3 fl/fl lyz2-cre − mice were transfected with PP1cc siRNA for 48 h and infected with VSV (1 MOI) for 8 h and then subjected to immunoblot analysis (E) and ELISA analysis (F). si-Non represents nonsense sequence as control siRNA. (G) HEK293T cells were transiently transfected with FLAG-IRF3 or mutants together with Myc-NSD3 or HA-PP1cc. 24 h later, the cells were infected with VSV (1 MOI) for the indicated times and subjected to IP and immunoblot analysis for the interaction of IRF3 and PP1cc and IRF3 phosphorylation in nucleus. (H) Purified His-tagged IRF3 or mutants expressing protein were used as substrate of purified NSD3(SET) protein in the vitro kinase assay and were incubated with PP1cc-GST protein for 6 h and immunoprecipitated with His antibody, and immunoblot assay was performed with the indicated antibodies. (I) HEK293T cells were transiently transfected with PP1cc together with IRF3 and mutant. 24 h later, the cells were infected with VSV (1 MOI) for 8 h and subjected to IP and immunoblot analysis for the interaction of IRF3 and PP1cc in nucleus. (J) PMs from WT mice were infected with VSV (1 MOI) for the indicated times and then subjected to IP and immunoblot analysis for the interaction of IRF3 and PP1cc and IRF3 phosphorylation and IRF3 methylation in nucleus. Immunoblots (A, B, D, E, and G–J) are representative of three independent experiments. Images are representative of two independent experiments (C). (F) Data are mean ± SEM and representative of three independent experiments. Unpaired, two-tailed Student’s t test. **, P < 0.01; ***, P < 0.001. WCL, whole cell lysate.

Article Snippet: The polyclonal antibody against IRF3 K366 monomethylation (IRF3 K366me1) was custom produced by Abmart.

Techniques: Methylation, Infection, Western Blot, Transfection, Confocal Microscopy, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Purification, Expressing, Kinase Assay, Incubation, Immunoprecipitation, Mutagenesis, Two Tailed Test