Fig 2: UNC93B1V117L drives increased inflammation and TLR7 responses in mice.a, RT–qPCR analysis of Irf7 and Ifit1 mRNA in BMDMs from the indicated mice (n = 3). Mice were age matched to 8 months. Statistical analysis was done using unpaired Student’s t-tests. The exact P values are shown. b, Levels of phosphorylated NF-κB, JNK, P38, IRF5 and IRF7, as measured by immunoblotting, in lysates of BMDMs from the indicated mice (n = 3). Mice were age matched to 8 months. c, RNA-seq performed for UNC93B1V117L BMDMs compared with controls (UNC93B1WT) (n = 3 biological replicates). Mice were age matched to 8 months. Gene set enrichment analysis significantly associated with SLE. d, RT–qPCR analysis of Ifit1, Irf7, Isg-15 and Tnf mRNA expression in the BMDMs from the indicated mice (n = 3) after stimulation by 40 μg ml−1 of HMW poly(I:C), 2 μg ml−1 of R848 and 10 μg ml−1 of CPG-C for 24 h. Mice were age matched to 14 months. Statistical analysis was done using unpaired Student’s t-tests. The exact P values are shown. e, Production of CXCL1, CCL5 and IP-10 in the supernatant of mice BMDMs isolated from the indicated mice (n = 3), measured by CBA after stimulation by 40 μg ml−1 of HMW poly(I:C), 2 μg ml−1 of R848 and 10 μg ml−1 of CPG-C for 24 h. Mice were age matched to 14 months. Statistical analysis was done using unpaired, two-tailed Student’s t-tests and data are presented as the mean with s.d. The exact P values are shown.Source data

Fig 3: Endogenous IRF7 interacts with NF-κB-p65 and interferes with its transcriptional activation.A, IRF3 KO HT1080 cells expressing V5.IRF7 were infected with SeV for the indicated time, when co-immunoprecipitation analyses were performed, as indicated. B, IRF3 KO HT1080 cells expressing V5.IRF7 were treated with polyI:C (pIC) to activate TLR3 signaling for the indicated time, when co-immunoprecipitation analyses were performed, as indicated. C, IRF3 KO HT1080 cells expressing V5.IRF7 were infected with SeV for the indicated time, when co-immunoprecipitation analyses were performed from nuclear and cytosolic fractions, as indicated. D, Let1 cells were infected with SeV for the indicated time, when co-immunoprecipitation analyses were performed using endogenous IRF7 and NF-κB-p65 proteins. E, RAW264.7 cells, lentivirally expressing non-targeting (NT) or IRF7-specific shRNA, were infected with SeV, and endogenous IRF7 and p65 proteins were immunostained and analyzed by proximity ligation assay; scale bar represents 10 μm. F, RAW264.7 cells, lentivirally expressing NT or IRF7-specific shRNA, were infected with SeV for the indicated time and analyzed for p-p65, p65, p-IRF7 by immunoblot. Image J analyses of p-p65/p65 were performed and shown at the bottom of the panel. G, RAW264.7 cells, lentivirally expressing NT or IRF7-specific shRNA, were infected with SeV for the indicated time and the nuclear fractions were analyzed for p65 and IRF7 by immunoblot. IRF, interferon regulatory factor; SeV, Sendai virus.

Fig 4: SRSF7 is required for IRF7 expression(A) Secreted IFN-α/β in SCR and Srsf7 KD #2 RAW MΦs, expressed as relative light units generated by ISRE reporter cells over a time course of LPS treament.(B) RT-qPCR of Zbp1 in SCR and Srsf7 KD RAW MΦs 4 h post-IFN-β treatment (200 IU).(C) Immunoblot of ZBP1 and ACTIN over a time course of IFN-β treatment in SCR and Srsf7 KD #2 RAW MΦs. Quantification, right.(D) Immunoblot of pY701 STAT1, total STAT1, pS396 IRF3, total IRF3, and ACTIN in SCR and Srsf7 KD RAW MΦs over a time course of LPS treatment.(E) Differential expression of genes encoding innate transcription factor in untreated SCR vs. Srsf7 KD RAW MΦs.(F) As in (E) but for +Salmonella samples.(G) Integrated Genomics Viewer with Irf7 reads from SCR (black) and Srsf7 KD #2 (teal) RAW MΦs, untreated (top) and +Salmonella (bottom).(H) RT-qPCR of Irf7 over a time course of LPS stimulation in SCR and Srsf7 KD RAW MΦs.(I) Immunoblot of IRF7 and ACTIN protein levels over a time course of LPS stimulation in SCR and Srsf7 KD RAW MΦs. Quantification, right.(J) RT-qPCR of Irf7 over a time course of Mtb infection in SCR and Srsf7 KD #2 MΦs.(K) As in (J) but during VSV infection.(L) RT-qPCR of Irf7 in siNC or siSrsf7 MEFs 4 h post-LPS.(M) Immunoblot of IRF7 protein levels in GFP gRNA, Irf7 KO1, and Irf7 KO2 RAW MΦs at baseline. ACTIN loading control.(N) RT-qPCR of Zbp1 and Ifnb1 at baseline in Irf7 KO1 (blue) and GFP gRNA control (black) RAW MΦs.For all data, n = 3 ± SEM. Statistical significance was determined using two tailed unpaired Student’s t test.

Fig 5: SRSF7 interacts with the H4K20me1 histone methyltransferase KMT5a/SET8 to promote Irf7 transcription(A) Proteins with peptides that immunoprecipitated with 3×FLAG-SRSF7, but not 3×FLAG-GFP, -SRSF1, -SRSF3, -SRSF6, or -SRSF9, manually assigned to different functional groups.(B) IP of 3×FL-SRSF7 and 3×FL-GFP probed for endogenous KMT5a/SET8.(C) IP of 3×FL-SRSF7 and 3×FL-GFP probed for endogenous histone H4 or H4K20me1, ±RNase treatment of lysates prior to pull-down.(D) RT-qPCR and immunoblot of siNC- and siKmt5a-transfected RAW MΦs.(E) RT-qPCR of Irf7 and Ifit3b in siNC and siKmt5a RAW MΦs.(F) RT-qPCR of Kmt5a and Irf7 in siNC- and siKmt5a-transfected BMDMs.(G) As in (F) but at 4 h post-LPS (100 ng/mL).(H) pY701 STAT1 ChIP-qPCR at the Irf7 promoter in siNC and siKmt5a RAW MΦs.(I) H4K20me1 ChIP-qPCR in RAW MΦs, untreated and treated with 4 h LPS at Irf7. Data are represented as elution/input normalized to IgG and upstream primer set (−754).For all data, n = 3 ± SEM. Statistical significance was determined using two tailed unpaired Student’s t test.