Fig 1.
Identification of SENP7 as a new regulator of cGAS-STING signaling.
(A) IFN-βluciferase and pTK-Renilla reporters were transfected into THP1 cells together with the indicated siRNA. Forty-eight hours after transfection, cells were stimulated with HSV-1 60mer for eight hours before luciferase reporter assays were performed. (B-E) MEFs transfected with the indicated siRNAs were stimulated with poly(dA:dT) (B, C) or ISD (D, E). Induction of Ifnb, Ifna4 and Cxcl10 mRNAs was measured by quantitative PCR (B, D). IFN-β and IFN-α4 production was assayed by ELISA (C, E). (F, G) Senp7-/- MEFs were generated by CRISPR-Cas9-mediated targeting. Induction of Ifnb and Ifna4 mRNAs was measured by quantitative PCR after ISD stimulation (F, upper panel). The immunoblots in (F, lower panel) show the ablation of SENP7 in Senp7-/- MEFs. And the supernatants were collected and assayed for IFN-β and IFN-α4 production by ELISA (G). (H, I) HEK293 cells were transfected with the nonspecific control (N.C.) or SENP7 siRNAs together with the indicated reporter plasmids. Forty-eight hours after transfection, the cells were infected with SeV (H) or treated with TNF- α (I) before luciferase assays were performed. Graphs show the mean ± s.d. and data (B-I) shown are representative of three independent experiments. n.s., not significant; *P < 0.05; **P < 0.01 (two-tailed t-test).
Fig 2.
De-SUMOylation activity of SENP7 is essential in potentiating STING signaling.
(A) MEFs transfect with empty vector (EV) or vector for the expression of Flag-tagged wild-type SENP7 or SENP7 C992S. Induction of Ifnb and Ifna4 mRNAs was measured by quantitative PCR after ISD stimulation. (B) MEFs were treated as in (A), and the supernatants were collected and assayed for IFN-β and IFN-α4 production by ELISA. (C) MEFs were transfected with the nonspecific control (N.C.) or Senp7 siRNA and then rescued with the indicated siRNA-resistant SENP7 constructs. After ISD stimulation, induction of Ifnb and Ifna4 mRNA was measured by quantitative PCR. (D) MEFs were treated as in (C), and the supernatants were collected and assayed for IFN-β and IFN-α4 production by ELISA. Graphs show the mean ± s.d. and data shown are representative of three independent experiments. n.s., not significant; **P < 0.01 (two-tailed t-test).
Fig 3.
SENP7 regulates STING signaling through direct interaction with cGAS.
(A) The indicated siRNAs were transfected into HEK293T cells together with IFN-β-luciferase and pTK-Renilla reporter plasmids. 48 hr after transfection, cells were transfected again with cGAS, STING, RIG-I CARD, MAVS, TBK1 or IRF3-5D for sixteen hours before luciferase assays were performed. (B) MEFs transfected with the indicated siRNAs were treated or not with ISD for various time periods, and cell extracts were analyzed for TBK1 phosphorylation. (C) MEFs transfected with the indicated siRNAs were treated or not with ISD for various time periods, and cell extracts were analyzed for IRF3 phosphorylation and IRF3 dimerization by SDS-PAGE and native PAGE, respectively. (D, E) MEFs transfected with the indicated siRNAs were treated with ISD for 4 hours, stained with the antibody against IRF3, and imaged by confocal microscopy (D). Cells with nuclear IRF3 staining are counted as a percentage of total cells (n = 100 cells per sample) (E). Scale bars represent 50 μm. (F, G) HEK293T cells were transfected with the indicated plasmids. 24 hr after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody and then immunoblotted with the indicated antibodies. (H) MEFs were infected or not with HSV-1 for the indicated time periods, and the cell lysates were immunoprecipitated with an anti-SENP7 antibody or normal IgG, and then immunoblotted with the indicated antibodies. (I) HeLa cells were infected or not with HSV-1 for the indicated time periods, stained with indicated antibodies and imaged by confocal microscopy. Graphs show the mean ± s.d. and data shown are representative of three independent experiments. n.s., not significant; **P < 0.01 (two-tailed t-test).
Fig 4.
SENP7 modulates the dynamic equilibrium of cGAS SUMOylation.
(A) Flag-tagged cGAS were transfected into HEK293T cells along with HA-tagged SUMO-1 or HA-tagged SUMO-2/3. 36 hr after transfection, cell lysates were immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were denatured and reimmunoprecipitated with an anti-Flag antibody, and then analyzed by immunoblotting with the indicated antibodies. (B) HEK293T cells were transfected with the indicated plasmids. 36 hr after transfection, cell lysates were subjected to a two-step immunoprecipitation and then immunoblotted with the indicated antibodies. (C) The SUMOylation reaction mixture contains E1, E2, SUMO and cGAS as indicated. After incubation for 120 min, the mixture was detected by immunoblotting with Flag antibody. (D) BMDMs were infected or not with HSV-1 for the indicated time periods, and the lysates were subjected to denaturing immunoprecipitation with an anti-cGAS antibody or normal IgG, and then analyzed by immunoblotting with the indicated antibodies. (E, F) Flag-tagged mouse cGAS or its mutants were individually transfected into HEK293T cells along with HA-tagged SUMO-2/3. Cell lysates were subjected to a two-step immunoprecipitation, and then immunoblotted with the indicated antibodies. K3A denotes cGAS with lysine residues 335/ 372/ 382 mutated to alanine. (G, H) HEK293T cells were transfected with the indicated plasmids. 36 hr post-transfection, cell lysates were subjected to a two-step immunoprecipitation and then immunoblotted with the indicated antibodies. (I) HEK293T cells were transfected with the indicated siRNAs. 36 hr later, Flag-cGAS and HA-SUMO-3 were transfected into the knockdown cells. Cell lysates were subjected to a two-step immunoprecipitation with Flag antibody and immunoblotted with the indicated antibodies. (J) MEFs were transfected with the indicated siRNAs and the cell lysates were subjected to denaturing immunoprecipitation with an anti-cGAS antibody or normal IgG, and then analyzed by immunoblotting with the indicated antibodies. (K) The deSUMOylation reaction mixture contains SUMOylated cGAS and recombinant WT or catalytically-dead SENP7 as indicated. After incubation for 30 min, the mixture was detected by immunoblotting with the indicated antibodies. All data shown are representative of three independent experiments.
Fig 5.
The SUMOylation of cGAS impairs its activation.
(A) Modeled structure of the cGAS homo-oligomer; red, yellow and blue spheres indicate SUMOylation sites. (B) DNA-precipitation assay of cGAS alone or cGAS pre-modified with SUMO by incubating with SUMOylation reaction mixture containing E1, E2 and SUMO or de-SUMOylated cGAS by incubating with SENP7 and SENP7 C992S. (C, D) Confocal microscopy of HeLa cells transfected with cyanine dye-3 (Cy3) conjugated ISD (C), and co-localization of cGAS with the labeled ISD (C, D) (Pearson's correlation coefficient; D). Arrows indicate representative co-localization between ISD and cGAS. (E, F) N-terminal EGFP-tagged cGAS and C-terminal mcherry-tagged cGAS or cGAS K3A mutant plasmids were transfected into HeLa cells together with or without His-tagged SUMO. cGAS self-association was examined by FRET assay. Rectangle in red (bottom right image of each group) indicates photobleached area (E). (F), summary of results, presented relative to initial FRET. (G, H) The indicated siRNAs were transfected into HeLa cells. 48 hr later, cells were transfected again with N-terminal GFP-tagged cGAS and C-terminal mcherry-tagged cGAS plasmids together with or without His-tagged SUMO. cGAS self-association was examined by FRET assay. Rectangle in red (bottom right image of each group) indicates photobleached area (G). (H), summary of results, presented relative to initial FRET. (I) Cell lysates from HEK293T cells transfected with the indicated plasmids were immunoprecipitated with an anti-Flag antibody, and then immunoblotted with the indicated antibodies. (J, K) Reaction products from cGAS, SUMOylated cGAS and cGAS K3A (J) as well as WT or catalytically-dead SENP7-treated SUMOylated cGAS (K) were analyzed by ion exchange chromatography. 0.2 mg/mL Salmon sperm DNA was added to stimulate cGAS catalytic activity. Graphs show the mean ± s. d. and data shown are representative of three independent experiments. **P < 0.01 (two-tailed t-test). Scale bars, 25 μm.
Fig 6.
SENP7 modulates innate immune defense against HSV-1 and Listeria monocytogenes invasion.
(A-D) MEFs transfected with the indicated siRNAs were infected with HSV-1 (A, B) or Listeria monocytogenes (C, D). Induction of Ifnb, Ifna4 and Cxcl10 mRNAs was measured by quantitative PCR (A, C). IFN-β and IFN- α 4 production was assayed by ELISA (B, D). (E) MEFs transfected with the nonspecific control (N.C.) or SENP7 siRNAs were treated or not with ISD. Equal volumes of culture supernatants from these treatments were applied to fresh MEF cells, followed by HSV-1 infection. The proliferation of cells was examined by crystal violet staining. Scale bars represent 200 μm. (F) MEFs transfected with the nonspecific control (N.C.) or SENP7 siRNAs were infected with HSV-1. The titers of HSV-1 were determined by standard plaque assay. (G) HSV-1-GFP replication in MEFs transfected with the indicated siRNAs was visualized by fluorescence microscopy. (H) MEFs transfected with the nonspecific control (N.C.) or SENP7 siRNAs were infected with Listeria monocytogenes. Counts of intracellular bacteria were determined by CFU assay. Graphs show the mean ± s.d. and data shown are representative of three independent experiments. **P < 0.01 (two-tailed t-test).
Fig 7.
SENP7 is indispensable for the innate defense against HSV-1 infection.
(A) Immunoblot analysis of SENP7 in lysates of liver, kidney and spleen from mice at 48 hr after transfection with Senp7 or nonspecific (N.C.) shRNA. (B) Survival of mice (n = 9 per group) transfected with Senp7 or N.C. shRNA and 48 hr later injected intravenously with HSV-1. P = 0.0014 (Mantel-Cox test). (C) ELISA of IFN-β and IFN-α4 in serum from mice (n = 5 per group) transfected with Senp7 or N.C. shRNA and 48 hr later injected intravenously with HSV-1, assessed 6 hr after HSV-1 infection. (D) Quantitative PCR of relative Ifn-b, Ifn-a4 and Cxcl10 mRNA in livers from mice (n = 5 per group) transfected with Senp7 or N.C. shRNA and 48 hr later injected intravenously with HSV-1, assessed 6 hr after HSV-1 injection. (E, F) Brains of the HSV-1-infected mice (n = 6 each) were harvested for immunohistochemistry (IHC) analysis by an anti-HSV-1 antibody. Tissue sections were visualized by microscopy (E). Percentages of cells infected by HSV-1 were quantified (F). (G, H) Quantitative PCR of relative Ifn-b mRNA in spleen (G) or kindey (H) from mice (n = 5 per group) transfected with Senp7 or N.C. shRNA and 48 hr later injected intravenously with HSV-1, assessed 6 hr after HSV-1 injection. Graphs show the mean ± s.d. and data shown are representative of at least two experiments. *P < 0.05, **P < 0.01 (two-tailed t-test). Scale bars, 50 μm.