Journal: Nature Communications

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

doi: 10.1038/s41467-018-05559-w

Figure Lengend Snippet: ZCCHC3 acts at the level of cGAS. a Effects of ZCCHC3-deficiency on cGAMP synthesis induced by transfected HSV120. Zcchc3 +/+ and Zcchc3 − / − MLFs were left untreated or treated with HSV120 (3 μg/ml) for 4 h, and then cell extracts containing cGAMP were separated by chromatography using a C18 column, the abundance of cGAMP was quantitated by mass spectrometry using SRM. The concentration of cGAMP (ng/ml) in Zcchc3 +/+ and Zcchc3 − / − MLFs was shown in the right histograph (mean ± SD, n = 3 technical repeats, **P < 0.01. b Effects of ZCCHC3-deficiency on transcription of downstream genes induced by cGAMP. Zcchc3 +/+ and Zcchc3 − / − MLFs were left un-treated or treated with 2′3′-cGAMP (0.2 mg/ml) for 4 h before qPCR analysis. c ZCCHC3 is associated with cGAS. HEK293 cells were transfected with the indicated plasmids before co-immunoprecipitation and immunoblotting analysis. d Colocalization of ZCCHC3 with cGAS. HeLa cells were transfected with GFP-ZCCHC3 and RFP-cGAS for 20 h before confocal microscopy. Scale bars, 10 μm. e PLA analysis of ZCCHC3 and cGAS interaction. Zcchc3 − / − MEFs reconstituted with an empty vector or Flag-ZCCHC3 were infected with HSV-1 or transfected with HSV120 (3 μg/ml) for 4 h before PLA analysis. Scale bars, 20 μm. f Endogenous ZCCHC3 is associated with cGAS in THP1 cells. THP1 cells were left un-infected or infected with HSV-1or transfected with HSV120 (3 μg/ml) for the indicated times before co-immunoprecipitation and immunoblotting analysis. Data are representative of at least two experiments with similar results

Article Snippet: Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Techniques: Transfection, Chromatography, Mass Spectrometry, Concentration Assay, Immunoprecipitation, Western Blot, Confocal Microscopy, Plasmid Preparation, Infection

Journal: Nature Communications

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

doi: 10.1038/s41467-018-05559-w

Figure Lengend Snippet: ZCCHC3 binds to dsDNA. a ZCCHC3 binds to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with the indicated biotinylated nucleic acids and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA. b ZCCHC3 binds to dsDNA through its C-terminal ZF domains. HEK293 cells were transfected with the indicated plasmids. Twenty hours after transfection, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads. The bound proteins were analyzed by immunoblots with anti-Flag. A schematic representation of ZCCHC3 and its truncation mutants was shown on the left. c ZCCHC3 and cGAS but not RIG-I bind to HSV-1 DNA of infected cells. HEK293 cells were transfected with HA-tagged ZCCHC3, cGAS, and RIG-I. Twenty hours after transfection, cells were infected with HSV-1for 3 h. Cell lysates were then immunoprecipitated with control IgG or anti-HA. The protein-bound DNAs were extracted and analyzed by qPCR analysis with primers corresponding to the indicated regions of HSV-1 genome. Positive ( + ) and negative (-) detections were shown at the top of the schematic presentation of the HSV-1 genome. A representative qPCR results were shown at the left. **P < 0.01 (unpaired t test). Data are representative of three experiments with similar results

Article Snippet: Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Techniques: Transfection, Incubation, In Vitro, Western Blot, Infection, Immunoprecipitation, Control

Journal: Nature Communications

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

doi: 10.1038/s41467-018-05559-w

Figure Lengend Snippet: ZCCHC3 binds to dsDNA and facilitates the binding of cGAS to dsDNA. a ZCCHC3 enhances the binding of cGAS to dsDNA. HEK293 cells were transfected with the indicated plasmids. Twenty hours later, the cell lysates were incubated with biotinylated-HSV120 and streptavidin-Sepharose beads for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-HA and anti-ZCCHC3. b ZCCHC3-deficiency decreases the binding of cGAS to dsDNA in HEK293 cells. ZCCHC3-KO HEK293 clones were generated by the CRISPR-Cas9 method. ZCCHC3-KO and control HEK293 cells were transfected with HA-cGAS. Twenty hours after transfection, cells were collected for in vitro pull-down assays similarly as in a . c ZCCHC3-deficiency decreases the binding of endogenous cGAS to dsDNA in MLFs or MEFs. Zcchc3 +/+ and Zcchc3 − / − MLFs or MEFs were collected for in vitro pull-down assays. The bound proteins were then analyzed by immunoblots with anti-cGAS and anti-ZCCHC3. d MST measurement of binding affinities. Binding affinities between the indicated recombinant proteins as well as between the indicated recombinant proteins and synthetic dsDNA HSV60 were measured by MST. The purified recombinant proteins were stained with Coomassie blue (right gel). e Effects of reconstitution of ZCCHC3 or ZCCHC3 (1-340) on HSV-1-induced transcription of downstream genes in Zcchc3 − / − MLFs. Zcchc3 − / − MLFs were reconstituted with murine ZCCHC3 or ZCCHC3(1-340) by lentiviral-mediated gene transfer. The reconstituted MLFs were left un-infected or infected with HSV-1 for 6 h before qPCR analysis. f Effects of ZCCHC3 on transcription of downstream genes induced by HSV120 in cGas +/+ and cGas − / − L929 cells. cGas +/+ and cGas − / − L929 cells stably expressing ZCCHC3 were transfected with HSV120 (3 μg/ml) for 4 h before qPCR analysis. *P < 0.05, **P < 0.01 (unpaired t test). Data are representative of at least two experiments with similar results (mean ± SD, n = 3 independent samples in e , f )

Article Snippet: Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Techniques: Binding Assay, Transfection, Incubation, In Vitro, Western Blot, Clone Assay, Generated, CRISPR, Control, Recombinant, Purification, Staining, Infection, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: ZCCHC3 is a co-sensor of cGAS for dsDNA recognition in innate immune response

doi: 10.1038/s41467-018-05559-w

Figure Lengend Snippet: ZCCHC3 is important for cytosolic dsDNA-induced oligomerization of cGAS. a Effects of ZCCHC3 on self-association of cGAS. HEK293 cells were transfected with the indicated plasmids for 20 h. The cells were then left un-treated or treated with HSV120 for 4 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. b Effects of ZCCHC3-deficiency on self-association of cGAS. ZCCHC3-KO and control HEK293 cells were transfected with the indicated plasmids for 20 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c Effects of ZCCHC3-deficiency on oligomerization of cGAS induced by transfected VACV70. Zcchc3 +/+ and Zcchc3 − / − MLFs were left un-treated or treated with VACV70 for 4 h. Cell lysates were then fractionated by SDD-AGE and SDS-PAGE and analyzed by immunoblots with the indicated antibodies. Data are representative of three experiments with similar results

Article Snippet: Mouse monoclonal antibodies against HA (BioLegend, 901515; 1:2000), FLAG (Sigma, F3165; 1:2000) and β-actin (Sigma, A2228; 1:3000) and phosphor-IκBα (Cell Signaling Technology, 9246 S; 1:1000); rabbit monoclonal antibodies against cGAS (Cell Signaling Technology, 66546 S/31659 S; 1:1000), phosphor-Tyrosine701-STAT1 (Cell Signaling Technology, 9167 S; 1:1000) and phosphor-IRF3 (Cell Signaling Technology, 4947 S; 1:1000), phosphor-TBK1 (Abcam, ab109272; 1:1000) and TBK1(Abcam, ab40676; 1:2000), IRF3 (Santa Cruz Biotechnology, sc-33641; 1:1000) and STAT1 (Santa Cruz Biotechnology, sc-417; 1:1000) were purchased from the indicated manufacturers.

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, SDS Page