The SP140-RESIST pathway regulates interferon mRNA stability and antiviral immunity

Abstract

Type I interferons (IFN-Is) are essential for antiviral immunity but must be tightly regulated^1-3. The conserved transcriptional repressor SP140 inhibits interferon beta (Ifnb1) expression via an unknown mechanism^4,5. Here we report that SP140 does not directly repress Ifnb1 transcription. Instead, SP140 negatively regulates Ifnb1 mRNA stability by directly repressing the expression of a previously uncharacterized regulator we call RESIST (REgulated Stimulator of Interferon via Stabilization of Transcript, previously annotated as Annexin-2 Receptor). RESIST promotes Ifnb1 mRNA stability by counteracting Ifnb1 mRNA destabilization mediated by the Tristetraprolin (TTP) family of RNA-binding proteins and the CCR4-NOT deadenylase complex. SP140 localizes within nuclear bodies, punctate structures that play important roles in silencing DNA virus gene expression in the nucleus⁴. Consistent with this observation, we found that SP140 inhibits replication of the gammaherpesvirus MHV68. The antiviral activity of SP140 was independent of its ability to regulate Ifnb1. Our results establish dual antiviral and interferon regulatory functions for SP140. We propose that SP140 and RESIST participate in antiviral effector-triggered immunity^6,7.

Extended Data Figure 1.. SP140 predominantly represses chromatin accessibility and binds genes involved in development, although these genes are not differentially expressed in the absence of SP140.

a. RT-qPCR for Sp140^−/− BMMs transduced with indicated retroviral constructs, treated with 100 μg/mL DMXAA for 8 hours. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant, one-way ANOVA with Dunnett’s T3 post-hoc correction. b. Top 10 GO terms for genes bound by HA-SP140 in anti-HA CUT&RUN. c. Volcano plot of differentially accessible ATAC-seq peaks in DMXAA-treated Sp140^−/− vs. B6 BMMs, filtered by genes that are also bound by HA-SP140 in anti-HA CUT&RUN. Blue dots indicate genes with log₂ fold change < −1 and adjusted p (padj) < 0.05, and red dots indicate genes with log₂ fold change > 1 and padj < 0.05. for differential ATAC-seq peak accessibility in Sp140^−/− BMMs treated with DMXAA. d. Alignment of reads from HA-SP140 or untagged SP140 anti-HA CUT&RUN, and RNA-seq/ATAC-seq of Sp140^−/− and B6 BMMs treated with DMXAA at Hoxa9. e. GIGGLE similarity score for overlap of HA-SP140 CUT&RUN peaks with publicly available ChIP-seq datasets for indicated histone marks. f. Table of adjusted p (padj) and log2 fold change for “lineage-inappropriate SP140-regulated” genes from RNA-seq of DMXAA-treated Sp140^−/− and B6 BMMs.

Extended Data Figure 2.. SP140 does not bind the Ifnb1 locus or known regulatory elements.

a. Alignment of reads at Ifnb1 from HA-SP140 or untagged SP140 anti-HA CUT&RUN, ATAC-seq of DMXAA-treated Sp140^−/− and B6 BMMs, and RNA-seq of DMXAA-treated Sp140^−/− and B6 BMMs. b. Alignment of reads from HA-SP140 or untagged SP140 anti-HA CUT&RUN, and ATAC-seq/RNA-seq of Sp140^−/− and B6 BMMs treated with DMXAA, at the Ifnb1 regulatory elements ICE^,, FIRE, and the MRE.

Extended Data Figure 3.. ANXA2R/RESIST expression in humans and mice, assessment of binding to Annexin 2, and generation of Gm21188/Resist1^−/− Gm36079/Resist2^−/− mice.

a. Expression values (DESeq2 normalized values) for Gm21188 (Resist1) for all cell types with high expression (>80 for expression value). Data from immgen.org. b. Expression of ANXA2R in human PBMC single-cell RNAseq data from Immune Cell Atlas (data from https://singlecell.broadinstitute.org/single_cell/study/SCP345/ica-blood-mononuclear-cells-2-donors-2-sites?scpbr=immune-cell-atlas#study-summary) c. Pull-down assay of recombinant STREP-ANXA2R and STREP-SMARCA3 (residues 26–39) upon incubation with ANXA2-S100A. For gel source data, please see Supplementary Figure 1. d. Schematic of Resist1/2 (Gm21188/Gm36079) locus with protein coding sequences indicated with purple arrows and gRNA targeted sequence indicated in blue. Resist2 contains a SNP within the gRNA targeting sequence (in bold). WT traces are indicated at guide-targeted regions for both Resist1 and Resist2. Sequence traces from Sp140^−/−Resist1^−/−Resist2^−/− mice (KO) are below with indicated mutations in Resist1/2.

Extended Data Figure 4.. AlphaFold predictions suggest RESIST likely binds the CNOT1 M-HEAT domain.

a. Schematic of CCR4-NOT subunit and RESIST complexes produced by Alphafold Multimer. b. Aligned AlphaFold models of RESIST with the CNOT1 M-HEAT domain. c. The top-scoring structural prediction of the RESIST and CNOT1 M-HEAT complex, colored by pLDDT. d. AlphaFold PAE plot of predicted RESIST and CNOT1 M-HEAT complex. Plot generated with PAEViewer. e. Depiction of RESIST binding to a hydrophobic patch on the CNOT-1 M-HEAT domain (CNOT1 colored by hydrophobicity).

Extended Data Figure 5.. AlphaFold predictions suggest RESIST likely binds the CNOT9 subunit.

a. Aligned structural predictions of RESIST interactions with CNOT9. b. Second-ranked Alphafold structural prediction of RESIST and CNOT9 complex colored by pLDDT. c. AlphaFold PAE plot of predicted RESIST and CNOT9 complex. Plot generated with PAEViewer. d. Depiction of RESIST binding to multiple hydrophobic patches on CNOT9 (CNOT9 colored by hydrophobicity).

Extended Data Figure 6.. AlphaFold predictions suggest RESIST likely does not bind CNOT11.

a. Aligned AlphaFold structure predictions of RESIST with the CNOT1 NMIF4G-NHEAT domains, CNOT10, and CNOT11. b. Highest-scoring AlphaFold structure prediction of RESIST and CNOT1/CNOT10/CNOT11 complex colored by pLDDT. c. AlphaFold PAE plot of predicted RESIST and CNOT1/CNOT10/CNOT11 complex. Plot generated with PAEViewer.

Extended Data Figure 7.. Assessment of CCR4-NOT subunit, TTP family, and ROQUIN1/2 knockout efficiency and role of ROQUIN1/2 in Ifnb1 regulation in BMMs.

a. Immunoblot of B6 or Sp140^−/− BMMs electroporated with indicated gRNAs for indicated CCR4-NOT subunits for experiments shown in Fig. 4b. Actin blots represents loading controls. For gel source data, please see Supplementary Figure 1. b. Immunoblot of B6 or Sp140^−/− BMMs electroporated with indicated gRNAs for indicated TTP family members for experiments shown in Fig. 4f after 8 hours of 100 μg/mL DMXAA. Actin blots represents loading controls. For gel source data, please see Supplementary Figure 1. c. RT-qPCR for Ifnb1 in BMMs electroporated with NTC gRNAs or gRNAS targeting Rc3h1/Rc3h2 (genes encoding ROQUIN1/2) after 8 hours of 100 μg/mL DMXAA treatment. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant, one-way ANOVA with Dunnett’s T3 post-hoc correction. d. Knockout efficiency for Rc3h1/Rc3h2 in BMMs electroporated with gRNAs targeting Rc3h1 and Rc3h2 for results shown in c.

Extended Data Figure 8.. The TTP zinc finger domain (TZF) binds the Ifnb1 3’UTR in an ARE-dependent manner.

Top: binding curve of TZF to SYBR-Gold labelled Ifnb1 3’UTR RNA for either WT (orange) or ARE mutant (purple), quantified from bottom. Bottom: representative electrophoretic mobility shift assay (EMSA) of TZF to ARE-WT or mutant Ifnb1 3’UTR. For gel source data, please see Supplementary Figure 1.

Extended Data Figure 9.. Generation and validation of HA-Sp140 knock-in mice.

a. Schematic of gene-targeting strategy to generate HA-Sp140 knock-in mice and depiction of resulting HA-Sp140^+/+ founder line. b. Immunoblot of BMMs of indicated genotypes treated with 100 μg/mL DMXAA for 8 hours or 10 ng/mL IFNγ for 24 hours. Actin blot represents loading control for anti-SP140 blot, and sample processing control for anti-HA blot. For gel source data, please see Supplementary Figure 1. c. RT-qPCR for Ifnb1 from BMMs of indicated genotypes after 8 hours of 100 μg/mL DMXAA treatment. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant, one-way ANOVA with Dunnett’s T3 post-hoc correction. d. Immunofluorescence of B6 BMMs treated with 8 hours of 100 μg/mL DMXAA stained with anti-HA, anti-PML, and DAPI. Staining control for Fig. 5a. e. Immunofluorescence of B6 BMMs treated with 8 hours of 100 μg/mL DMXAA stained with anti-HA, anti-fibrillarin, and DAPI. Staining control for Fig. 5b.

Extended Data Figure 10.. Gating strategy for BMMs infected with GFP-encoding viruses, and measurement of Ifnb1 transcripts in B6 and Sp140^−/− BMMs upon infection with MHV68-GFP.

a. Representative flow plots and gating strategy for BMMs infected with viruses encoding GFP. Experiment shown is for B6 BMMs infected with MHV68-GFP, MOI 3, for 24 hours. b. RT-qPCR of BMMs 8 hours after infection with MHV68-GFP, MOI of 1. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant, two-tailed t-test with Welch’s correction.

Figure 1.. Ifnb1 mRNA is stabilized in the absence of SP140.

a. Ifnb1 RT-qPCR in BMMs treated for 4 or 8 hours with 10 ng/mL LPS, or 100 μg/mL poly(I:C) or DMXAA. p values (* = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant), calculated with two-tailed t-tests using Welch’s correction and FDR correction. b. Ifnb1 RT-qPCR from BMMs treated with 100 μg/mL DMXAA at indicated timepoints. p values (* = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant) calculated with a two-way ANOVA and Šidák’s multiple comparison correction. c. Roadblock RT-qPCR of BMMs treated with 4SU 2 hours after treatment with 100 μg/mL DMXAA. p values (* = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant) calculated with a two-way ANOVA and Šidák’s multiple comparison correction. d. ELISA for IFNβ protein levels in supernatants of BMMs treated for 24 hours with 100 μg/mL poly(I:C) or DMXAA. p values (* = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant), calculated with two-tailed t-tests using Welch’s correction.

Figure 2.. Resist1 (Gm21188) and Resist2 (Gm36079) are repressed by SP140 and correlate with increased Ifnb1 transcript in Sp140^−/− cells.

a. Volcano plot of differentially expressed genes (DEGs) from RNA-seq of DMXAA-treated Sp140^−/−Ifnar^−/− vs. Ifnar^−/− BMMs. Red genes are upregulated in Sp140^−/− Ifnar^−/− BMMs with log2 fold change > 1 and adjusted p-value (padj) < 0.05. Blue genes are downregulated in Sp140^−/−Ifnar^−/− BMMs with log2 fold change > −1 and adjusted p-value < 0.05. The adjusted p-value for Sp140 is < 2.225074 e-308 and is graphed as −10*(adjusted p-value) of Mid1 for visualization. Resist2 (Gm36079) is not depicted on volcano plot as it is removed by the DeSeq2 independent filtering function for genes with low read counts. b. Table of maximum HA-SP140 CUT&RUN MACS2 signal value, maximum log2 fold change in chromatin accessibility from ATAC-seq of DMXAA-treated B6 and Sp140^−/− BMMs, and log2 fold change from RNA-seq of DMXAA-treated B6, Sp140^−/−, Ifnar^−/− and Sp140^−/−Ifnar^−/− BMMs, for significantly upregulated DEGs from a, as well as Sp140 and Resist2 (Gm36079) (ns = not significant). Cells are colored according to the column value. c. Alignment of reads at Resist1/2 locus from anti-HA CUT&RUN for DMXAA-treated BMMs transduced with HA-SP140 or SP140, and ATAC-seq/RNA-seq of DMXAA-treated B6 and Sp140^−/− BMMs. Alignments were visualized in the UCSC genome browser.

Figure 3.. RESIST binds the CCR4-NOT complex and drives Ifnb1 mRNA stabilization in Sp140^−/− macrophages.

a. Immunoblot of input lysate or anti-HA immunoprecipitate for BMMs transduced with lentiviral RESIST constructs, stimulated with doxycycline and 100 μg/mL DMXAA for 5–7 hours. For gel source data, please see Supplementary Figure 1. b. Ifnb1 RT-qPCR from BMMs electroporated with non-targeting control (NTC) or Resist1/2 gRNA, stimulated for 8 hours with 100 μg/mL DMXAA or untreated (UT). Knockout efficiency in the experiment shown was 86–87% for Resist1 and 91% for Resist2. c. Ifnb1 RT-qPCR from BMMs of indicated genotypes, stimulated for 8 hours with 100 μg/mL DMXAA or untreated (UT). d. ELISA of culture supernatant from BMMs treated with 100 μg/mL DMXAA for 24 hours. e. Roadblock RT-qPCR of BMMs electroporated with indicated gRNAs and treated with 100 μg/mL DMXAA, followed by 4SU at 2hr. The estimated knockout efficiency in the experiment shown was 71% for Resist1 and 51–69% for Resist2. Asterisks indicate a significant difference in Ifnb1 transcript compared to B6 + NTC at each time point and are colored by condition compared to B6 + NTC. Black bars/asterisks indicate a comparison between Sp140^−/− + NTC and Sp140^−/− + Resist1/2 gRNA. f. Ifnb1 RT-qPCR from BMMs transduced with indicated lentiviral constructs, treated with doxycycline and 100 μg/mL DMXAA for 7 hours. g. Roadblock RT-qPCR for Ifnb1 from B6 BMMs transduced with indicated lentiviral constructs, stimulated with doxycycline and 100 μg/mL DMXAA at T0, followed by 4SU treatment at T2. Asterisks indicate a significant difference in Ifnb1 compared to mCherry at each time point. h. IFNB1 RT-qPCR from human BlaER1 monocytes overexpressing human RESIST constructs or mCherry, stimulated with ADU-S100 and doxycycline. Asterisks indicate a significant difference in IFNB1 transcript compared to mCherry-expressing cells. i. Colony forming units (CFU) from lungs of mice infected with Legionella pneumophila, 96 hours post-infection. Results represent 3 independent pooled experiments, including an infection with Sp140^−/−Resist1^+/+Resist2^+/+ and Sp140^–/–Resist1^−/−Resist2^−/− littermates. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant, calculated with one-way ANOVA tests with Dunnett’s T3 multiple comparison correction post-hoc (b, c, f) or FDR correction (d), or two-way ANOVA tests with Tukey’s post-hoc correction (e, g, h), or Mann-Whitney test (i).

Figure 4.. RESIST counteracts repression of IFN-I by TTP family proteins, a function which requires a RESIST C-terminal region and CNOT9

a. AlphaFold predictions of RESIST with CNOT1 M-HEAT and CNOT9. TTP peptide:CNOT1 M-HEAT is PDB code 4J8S. Roquin peptide:CNOT9 is PDB code 5LSW. N and C-terminal ends of RESIST are marked by “N” or “C” respectively. b. Ifnb1 RT-qPCR from BMMs electroporated with non-targeting control gRNA (NTC) or gRNAs targeting indicated CCR4-NOT subunits, treated for 8 hours with 100 μg/mL DMXAA. c. Strep pulldown of purified recombinant full-length human His-MBP-RESIST-Strep or His-MBP-Strep with human CNOT9 and CNOT1 (aa 1351–1588). First lane indicates purified CNOT9/CNOT1. d. Ifnb1 RT-qPCR for BMMs transduced with indicated lentiviral constructs, treated with doxycycline and DMXAA for 6 hours. Results include data also shown in Fig. 3f. e. Immunoblot for anti-HA IP of BMMs transduced with indicated constructs in d. RESIST construct is C-terminally tagged with HA. For gel source data, please see Supplementary Figure 1. f. Ifnb1 RT-qPCR from BMMs electroporated with indicated gRNAs, treated for 8 hours with 100 μg/mL DMXAA. g. Immunoblot for FLAG IP of FLAG-TTP (mouse) co-expressed with mouse HA-RESIST in HEK293Ts. For gel source data, please see Supplementary Figure 1. h. Schematic for how RESIST may interact with CCR4-NOT subunits CNOT1 and CNOT9 to mediate stabilization of Ifnb1 mRNAs. * = p < 0.05, ** = p < 0.005, ***= p < 0.0005, **** = p < 0.00005, ns = not significant, calculated with one-way ANOVA tests with Dunnett’s T3 multiple comparison correction post-hoc.

Figure 5.. SP140 is an antiviral NB protein that co-localizes with nucleoli

a. Left: Immunofluorescence of HA-Sp140^+/+ BMMs treated with DMXAA and stained with DAPI, anti-HA, and anti-PML. Right: quantification of overlap of HA-SP140 and PML NBs for 2 independent experiments. b. Immunofluorescence of HA-Sp140^+/+ BMMs treated with DMXAA and stained with DAPI, anti-HA, and anti-fibrillarin. Right: quantification of overlap of HA-SP140 and fibrillarin NBs for 2 independent experiments. c. Histograms of MHV68-GFP signal in MHV68-GFP infected BMMs, assessed by flow cytometry. Numbers represent MHV68-GFP⁺ as a percentage of live cells. d. Quantification of MHV68-GFP⁺ cells from c). e. Quantification of MHV68-GFP+ BMMs. f. Quantification of MCMV-GFP+ BMMS, assessed by flow cytometry. g. Quantification of Sendai-GFP+ BMMs, assessed by flow cytometry. h. Schematic for the proposed model of SP140 antiviral activity and RESIST-mediated stabilization of Ifnb1 transcript. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005, **** = p < 0.00005, ns = not significant, calculated with one-way ANOVA tests with FDR correction (d-g).