Single-nucleotide methylation specifically represses type I interferon in antiviral innate immunity

Abstract

Frequent outbreaks of viruses have caused a serious threat to public health. Previous evidence has revealed that DNA methylation is correlated with viral infections, but its role in innate immunity remains poorly investigated. Additionally, DNA methylation inhibitors promote IFN-I by upregulating endogenous retrovirus; however, studies of intrinsically demethylated tumors do not support this conclusion. This study found that Uhrf1 deficiency in myeloid cells significantly upregulated Ifnb expression, increasing resistance to viral infection. We performed whole-genome bisulfite sequencing and found that a single-nucleotide methylation site in the Ifnb promoter region disrupted IRF3 recruitment. We used site-specific mutant knock-in mice and a region-specific demethylation tool to confirm that this methylated site plays a critical role in regulating Ifnb expression and antiviral responses. These findings provide essential insight into DNA methylation in the regulation of the innate antiviral immune response.

Graphical abstract

Figure 1.

Distinct strains of influenza virus lead to different levels of DNA methylation and IFN-I induction. (A and B) Viral loads (A) and mRNA levels of Ifnb (B) induced by different influenza viruses (1 MOI for 6 h) in PBMCs from healthy donors (n = 32), as measured by qPCR. (C) Pyrosequencing results of the methylation levels in the whole genome of PBMCs from healthy donors infected with different influenza virus strains. (D) The expression of the indicated genes in PBMCs from healthy donors (n = 32) infected with different influenza viruses (1 MOI for 6 h) in vitro was measured by qPCR. The data from the qPCR assay are presented as fold changes relative to the actin mRNA levels. All data are representative of at least three independent experiments. The data are presented as means ± SEMs. The significance of differences was determined using a t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Figure S1.

Uhrf1 did not affect the development and homeostasis of the immune system. (A) Genotyping PCR of myeloid cell-conditional Uhrf1 WT (Uhrf1^+/+lyz2^Cre/+), Uhrf1^MKO (Uhrf1^fl/fllyz2^Cre/+), and heterozygous (Uhrf1^fl/+lyz2^Cre/+) mice to amplify WT and floxed alleles and Cre-specific primers for the Lyz2^Cre DNA. (B and C) Flow cytometry analysis of macrophages (CD11b⁺F4/80⁺) and neutrophils (CD11b⁺GR-1⁺) in BM and spleen (Spl) from 6–8-wk-old WT and Uhrf1^MKO mice (n = 3). (D–G) Flow cytometry analysis of the absolute numbers of different immune cells in the spleen (D and E) and naive and memory T cells (F and G) from WT or USP16^MKO mice (n = 3). All FACS data are presented in a representative plot and summary graph of the subpopulation percentages. All data are representative of three independent experiments. The bars and error bars show the means ± SEMs. Significance was determined by a two-tailed Student’s t test. BMC, BM cells; CM, central memory; EM, effector memory; iLN, inguinal LN; M, total memory; N, naïve; SP, splenocytes.

Figure 2.

Uhrf1 deficiency potentiates the antiviral immune response. WT and Uhrf1^MKO mice (6–8 wk) were i.n. infected with a sublethal dose (0.1 HA) of H5N1 influenza virus. (A and B) The body weight loss (A) and survival rate (B) were measured for 14 d (n = 20). (C) H&E staining of lung tissue sections on days 2 and 5 after infection. Inflammation scores are presented as a bar graph (n = 5). Scale bar, 200 µm. (D) ELISA for IFN-β in the sera of WT and Uhrf1^MKO mice infected with H5N1 influenza virus on days 2 and 5 (n = 3). (E) The viral titers in the lung were quantified on day 2 using the TCID₅₀ assay (n = 10). (F–J) WT and Uhrf1^MKO mice (6–8 wk) were i.n. infected with a sublethal dose (0.1 HA) of PR8. The body weight loss (F) and survival rate (G) were measured for 14 d (n = 12). H&E staining of lung tissue sections (H; n = 5) and IFN-β in serum (I; n = 4) was measured on days 2 and 5 after infection. Scale bars, 200 µm. (J) The viral titers in the lung were quantified on day 2 using the TCID₅₀ assay (n = 10). (K–M) Survival rate (K; n = 12), viral titer (L; n = 8), and IFN-β in serum (M; n = 4) of WT and Uhrf1^MKO mice intravenously injected with HSV-1 (3 × 10⁶ PFU per mouse). (N–P) WT and Uhrf1^MKO mice bred to the Ifnar1^−/− background were i.n. infected with a sublethal dose (0.1 HA) of PR8. The survival rate (N; n = 16), viral titer (O; n = 5), and IFN-β in serum (P; n = 4) were monitored for 14 d. WT or Uhrf1-deficient BMDMs were infected with GFP-expressing VSV (VSV-GFP) at an MOI of 0.1 for 24 h. (Q) The data are presented as a representative picture, showing the infected (GFP⁺) and total (bright-field) cells. Scale bar, 1,000 µm. (R) Summary graph of flow cytometric quantification of the infected cells. All the data are representative of at least three independent experiments. The data are presented as means ± SEMs. The significance of differences was determined by a t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Figure S2.

Uhrf1 deficiency in myeloid cells caused an autoimmune feature. (A) Uhrf1^ER-Cre MEFs were incubated with DMSO or 4-OH for 72 h and then infected with VSV-GFP at a MOI of 0.1 for 24 h Data are presented as a representative picture, showing the infected (GFP⁺) and total (bright-field) cells. Scale bar, 200 µm (n = 3). (B) The summary graph of flow cytometric quantification of the infected cells (n = 3). (C) Immunofluorescence microscopy of the deposition of IgM and IgG in glomeruli (arrows) of kidney sections from 8-mo-old WT and Uhrf1^MKO mice. Original magnification, ×10. Scale bar, 1,000 µm. (D) ELISA of the autoantibodies antinuclear antigen (ANA) and antibody to double-stranded DNA (dsDNA) in the serum of these aging WT and Uhrf1^MKO mice (n = 4). (E) Flow cytometric analysis of the percentages of IFN-γ– and IL-17–producing CD4⁺ T cells in the spleens and inguinal LNs (iLNs) of WT and Uhrf1^MKO mice (n = 4). All FACS data are presented in a representative plot and summary graph of the subpopulation percentages. All data are representative of three independent experiments. The bars and error bars show the means ± SEMs. Significance was determined by two-tailed Student’s t test. *, P < 0.05, **, P < 0.01; ***, P < 0.005.

Figure 3.

Uhrf1 deficiency causes specific upregulation of IFN-I and ISGs. (A and B) Venn diagrams (A) and volcano graphs (B) illustrating the overlap of DEGs between WT and Uhrf1-deficient BMDMs stimulated with 20 µg/ml pI:C for 2 h. FC, fold change; NT, nontreatment. (C) Network visualization of gene ontology enrichment analysis of DEGs in Uhrf-deficient BMDMs in response to pI:C for 2 h. (D) KEGG analysis of these DEGs in Uhrf1-deficient BMDMs indicated pathways that differed significantly (in abundance). RLR, RIG-I–like receptor. (E and F) Heat map (E) and qPCR analysis (F) showing ISG expression in Uhrf1-deficient cells activated by pI:C for 2 h (n = 3). (G) qRT-PCR analysis of the indicated genes using WT or Uhrf1-deficient BMDMs stimulated with pI:C (n = 6). (H) The mRNA levels of Ifnb induced by different viruses in WT and Uhrf1-deficient BMDMs were measured by qPCR (n = 6). (I) ELISAs of IFN-β in the supernatants of WT or Uhrf1-deficient BMDMs stimulated with specific viruses (n = 3). All the data are representative of at least three independent experiments. The data from the qPCR assay are presented as fold changes relative to the actin mRNA levels. The data are presented as means ± SEMs. The significance of differences was determined by a t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Figure S3.

Uhrf1 is not required for the proinflammatory cytokines production except Ifnb. (A) Heatmap showing the pI:C-stimulated DEGs in BMDMs from WT and Uhrf1^MKO mice. The DEGs were identified with a fold change of experimental sample to nontreated control (ES/NT) >2.0 or <0.5. (B) qRT-PCR analysis of the indicated genes using WT or Uhrf1-deficient BMDMs stimulated with 100 ng/ml LPS (n = 6). (C) Ifnb mRNA in Uhrf1MKO BMDMs were measured by qPCR assay responding to 1 μg/ml R848 or 5 μM CpG (n = 4). (D) qRT-PCR analysis of the indicated genes using WT or Uhrf1-deficicent BMDMs stimulated with 10 ng/ml IL-4 (n = 3). (E) Uhrf1^ER-Cre MEFs were incubated with DMSO or 4-OH for 72 h, and we examined the genotyping by amplifying WT and KO alleles. qRT-PCR analysis of the Uhrf1 mRNA level. (F) qRT-PCR analysis of the indicated genes using WT or Uhrf1-deficient MEFs generated as above stimulated with pI:C plus 4-OH (n = 4). (G–J) WT and DC-conditional Uhrf1 KO (Uhrf1^DKO) mice (6–8 wk) were i.n. infected with a sublethal dose (0.1 HA) of H5N1 influenza virus. Body weight loss (G) and survival rate (H) were measured for 14 d (n = 17). (I) Viral titers in the lung were quantified on day 2 by a TCID₅₀ assay (n = 10). (J) ELISA for IFN-β in the sera of WT and Uhrf1^MKO mice infected with H5N1 influenza virus on days 2 and 5 (n = 4). (K) qRT-PCR analysis of the indicated genes using WT or Uhrf1-deficient BM-derived DCs stimulated with 100 ng/ml LPS or 1 MOI VSV. Data in the qPCR assay are presented as fold relative to the actin mRNA level. All data are representative of at least three independent experiments. Data are presented as means ± SEMs. The significances of differences were determined by a t test. *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Figure 4.

Uhrf1 does not affect canonical antiviral signal transduction. (A–E) The indicated proteins in cytoplasmic (CE) and nuclear (NE) extracts (A–C) or whole-cell lysates (D and E) of WT and Uhrf1-deficient BMDMs were measured by IB analysis. (F and G) Specific virus-induced phosphorylation of TBK1 and IRF3 in whole-cell lysates was measured by IB analysis. (H) IB analysis of monomeric and dimeric IRF3 (top blot), total IRF3, Uhrf1, and actin (bottom) in HEK293T cells transfected with empty vector or an expression plasmid for Uhrf1 and then infected with PR8 for various times. IB analysis of monomeric and dimeric IRF3 (top blot), total IRF3, Uhrf1, and actin (bottom) in HEK293T cells transfected with empty vector or an expression plasmid for Uhrf1 and then infected with PR8 for various times. All the data are representative of at least three independent experiments.

Figure 5.

Uhrf1 deficiency removes the methylation on the Ifnb promoter. (A) HEK293T cells were transfected with an Ifnb-luciferase reporter plasmid in the presence or absence of the indicated Uhrf1 expression plasmids (n = 4). Luciferase assays were performed, and the data are expressed as fold changes based on the empty vector group 36 h after transfection. (B) BM cells were incubated with 1 µM 5-AZA for 5 d (long). Differentiated macrophages were incubated with 1 µM 5-AZA for 24 h (short). The mRNA levels of Ifnb induced by pI:C in 5-AZA–pretreated WT and Uhrf1-deficient BMDMs were measured by qPCR (n = 5). (C) Methylation level (percentage) in the genomes of WT and Uhrf1-deficient BMDMs with the gene features of interest, including the gene body (all exons and introns), promoter, first exon, first intron, the rest of the exons, and the rest of the introns. (D) Epigenome density plot for CG, CHG, and CHH methylation contexts in chromosome 4 from WT and Uhrf1-deficient BMDMs. (E and F) Venn diagrams illustrating the overlap of DEGs whose promoters exhibited significant demethylation in pI:C-stimulated Uhrf1-deficient BMDMs (E), and correlation between the expression of these DEGs and methylation level on their promoter (F). (G and H) Heat map and KEGG analysis of these overlapping DEGs selected as above. (I) Genome Browser snapshots of the DNA methylation levels near Ifnb and other proinflammatory cytokine promoters in mice. (J) Sequence alignment of single-nucleotide methylated sites on Ifnb promoters of different species. The red box indicates the methylated site. (K) Pyrosequencing graphic results of the methylation levels of the Ifnb promoter region. The ratio of thymine (T) to cytosine (C) at each CpG position (gray) to determine the percentage of DNA methylation (n = 17). (L) Correlation between Ifnb and the methylation level on its promoter in PBMCs from healthy donors infected with different strains of the influenza virus. The data from the qPCR assay are presented as fold changes relative to the actin mRNA levels. All the data are representative of at least three independent experiments. The data are presented as means ± SEMs. The significance of differences was determined by a t test. *, P < 0.05.

Figure S4.

The transcriptome caused by Uhrf1 deficiency under nontreated conditions. (A) BMDMs were incubated with 10 μM TSA for 24 h. mRNA levels of Ifnb induced by pI:C WT and Uhrf1-deficient BMDMs were mentored by qPCR (n = 5). (B) BM cells were incubated with 1 μM 5-AZA for 5 d (long). Differentiated macrophages were incubated with 1 μM 5-AZA for 24 h (short). mRNA levels of Il6 and Il12b induced by pI:C in 5-AZA–pretreated WT and Uhrf1-deficient BMDMs were mentored by qPCR (n = 5). (C and D) Epigenome density plot for CG, CHG, and CHH methylation contexts in global genomes the genes feature of interest in WT and Uhrf1-deficient BMDMs, including the gene body (all exons and introns), promoter, first exon, first intron, the rest of the exons, and the rest of the introns. (E and F) Venn diagrams illustrating the overlap of DEGs whose promoter performed a significant demethylation in nontreated Uhrf1-deficient BMDMs (E). Correlation between these DEGs expression and methylation level on their promoter (F). (G and H) Heat map and KEGG analysis of these overlapped DEGs selected as above. Data in the qPCR assay are presented as fold relative to the actin mRNA level. All data are representative of at least three independent experiments. Data are presented as means ± SEMs. The significances of differences were determined by a t test. *, P < 0.05.

Figure 6.

Targeting DNA methylation editing by dCas9-Tet1 activates IFN-I production. WT and Uhrf1-deficient BMDMs were stimulated for 9 h with specific virus or TLR agonists. (A and B) ChIP assays were performed and quantified by qPCR to detect the binding of IRF3 (A) or RNA polymerase (RNA pol; B) to the Ifnb promoter (n = 4). (C) EMSA of nuclear extracts of WT and Uhrf1-deficient BMDMs stimulated with Sendai Virus, as assessed using an unmethylated or methylated HRP-labeled Ifnb promoter probe. (D) Schematic graph of a catalytically inactive mutant Cas9 (dCas9) fused to Tet1 to remove DNA methylation modifications from the Ifnb promoter. (E) Bisulfite sequencing of WT MEFs transfected with dCas9-Tet1 (dC-T) plus a scrambled gRNA (sc gRNA) or four gRNAs targeting the Ifnb promoter region (target gRNA). (F) The mRNA levels of Ifnb induced by different viruses in MEFs described in E were measured by qPCR (n = 3). (G and H) MEFs described in B were infected with VSV-GFP at an MOI of 0.1 for 24 h. The data are presented as a representative picture, showing the infected (GFP⁺) and total (bright-field) cells (G; n = 3). Scale bar, 1,000 µm. Summary graph of flow cytometric quantification of the infected cells (H; n = 4). The data in the qPCR assay are presented as fold changes relative to the actin mRNA levels. All data are representative of at least three independent experiments. The data are presented as means ± SEMs. The significance of differences was determined by a t test. *, P < 0.05.

Figure S5.

A single-nucleotide methylation negative regulated Ifnb induction independent of chromatin assembly. (A) WT and Uhrf1-deficient BMDMs were stimulated for 9 h with VSV. ChIP assays were performed and quantified by qPCR to detect the enrichment of H4K16ac and H3K27ac on the different regions of the Ifnb promoter (n = 4). (B) Screenshot from a genome browser of peaks near Ifnb obtained from ATAC-seq. (C) The sequencing data of WT and Ifnb1^CpG(G-A) mice. (D) mRNA levels of indicated genes induced by 20 μg/ml pI:C in WT and Ifnb1^CpG(G-A) BMDMs were mentored by qPCR (n = 4). Data in the qPCR assay are presented as fold relative to the actin mRNA level. All data are representative of at least three independent experiments. Data are presented as means ± SEMs. The significances of differences were determined by a t test. *, P < 0.05.

Figure 7.

Ifnb production is inhibited by single-nucleotide methylation of its promoter. (A) Schematic picture showing the strategy for generating CpG mutant knock-in mice (Ifnb1^CpG(G-A)). (B) The mRNA levels of Ifnb induced by different viruses in WT and Ifnb1^CpG(G-A) BMDMs were measured by qPCR. ChIP assays were performed and quantified by qPCR to detect the binding of IRF3 to the WT or Ifnb1^CpG(G-A) promoters (n = 4). (C) WT and Ifnb1^CpG(G-A) mice (6–8 wk) were i.n. infected with a sublethal dose (0.1 HA) of H5N1 influenza virus (n = 4). (D and E) The body weight loss (D) and survival rate (E) were measured for 14 d (n = 18). (F) Viral titers in the lung were quantified on day 2 using the TCID₅₀ assay (n = 4). (G) ELISA for IFN-β in the sera of WT and Ifnb1^CpG(G-A) mice infected with H5N1 influenza virus on days 2 and 5 (n = 5). (H–K) WT and Uhrf1^MKO mice bred on the Ifnb1^CpG(G-A) background were i.n. infected with a sublethal dose (0.1 HA) of H5N1. The mRNA levels of Ifnb induced by different viruses in the indicated BMDMs were measured by qPCR (H; n = 3). The body weight loss (I; n = 8), survival rate (J; n = 8) for 14 d, and viral titer (K; n = 5) on day 2 are shown. The data in the qPCR assay are presented as fold changes relative to the actin mRNA levels. All data are representative of at least three independent experiments. The data are presented as means ± SEMs. The significance of differences was determined by a t test. *, P < 0.05.