Fig 1.
Suppressed innate immune response to HCMV infection in ATRX knockdown HFFs.
(A) ATRX knockdown efficiency in siATRX cells was assessed by Western blotting; β-actin served as an internal loading control. (B) For further characterization of the siATRX cells, ATRX and its interaction partner Daxx were immunostained. 4’,6-diamidino-2-phenylindole (DAPI) was counterstained to visualize cellular nuclei. (C) To determine ATRX mRNA levels, total RNAs were isolated from ATRX knockdown HFFs (siATRX) and respective control HFFs (siC) and RT-qPCR was performed. (D+E) ATRX knockdown HFFs and respective control HFFs were either mock-infected or infected with the laboratory HCMV strain AD169 (MOI of 1). (D) At 6 and 8 hpi, cells were harvested for Western blot analyses to determine IRF3 phosphorylation; β-actin served as an internal loading control. Signal intensities were quantified relative to infected siC cells at 6 hpi (lane 2). (E) Additionally, total RNAs were isolated at 8 hpi and RT-qPCR was performed to determine IFNB1 induction. (C+E) Depicted values were calculated from triplicates relative to (C) untreated or (E) mock-infected siC cells using GAPDH as a housekeeping gene and are shown as mean ± SD. One out of (C) four or (E) two independent experiments is shown. Statistical analysis was performed with respective ΔCq-values using a student’s t-test (unpaired, two-tailed); *p<0.05, **p<0.01. (A+B+D) The following antibodies were used: (A+D) anti-ATRX (39-f), anti-β-actin (AC-15); (D) anti-phospho-IRF3 (Ser386) (EPR2346), anti-IRF3 (D6I4C); (B) anti-ATRX (D-5), anti-Daxx (M112).
Fig 2.
Repression of the cGAS-STING DNA sensing pathway in ATRX knockdown HFFs.
ATRX knockdown HFFs (siATRX) and respective control cells (siC) were treated with (A-C) 0.1 μg/ml poly(dA:dT) and (B+C) DMSO or (D-F) 50 μg/ml 2’3’-cGAMP for 24 h. (A+D) Cells were harvested for Western blot analyses to determine the phosphorylation of IRF3 and TBK1; β-actin served as an internal loading control. Signal intensities were quantified relative to treated siC cells (lane 2). The following antibodies were used: anti-phospho-IRF3 (Ser386) (EPR2346), anti-IRF3 (D6I4C), anti-phospho-TBK1 (Ser172) (D52C2), anti-TBK1 (D1B4), anti-β-actin (AC-15); (A) anti-ATRX (39-f); (D) anti-ATRX (HPA001906). (B+E) Total RNAs were isolated and RT-qPCR was performed to determine IFNB1 induction. Depicted values were calculated from triplicates relative to untreated siC cells using GAPDH as a housekeeping gene and are shown as mean ± SD. One out of (B) three or (E) two independent experiments is shown. Statistical analysis was performed with respective ΔCq-values using a student’s t-test (unpaired, two-tailed); **p<0.01, ***p<0.001. (C+F) Supernatants of stimulated cells were harvested and mixed with HEK-Blue IFN-α/β cells. Type I IFNs in the supernatants were quantitated by measuring SEAP activity produced by HEK-Blue IFN-α/β cells using QUANTI-Blue. Depicted values are derived from triplicates and are shown as mean ± SD. Statistical analysis was performed using a student’s t-test (unpaired, two-tailed); *p<0.05, **p<0.01.
Fig 3.
ATRX interacts with IRF3 and modulates IRF3 activity.
Stable ATRX knockdown HFFs (siATRX) and respective control HFFs (siC) were (A) treated with DMSO and 0.1 μg/ml poly(dA:dT) for 24 h, (B) treated with 50 μg/ml 2’3’-cGAMP for 24 h, (C) infected with the laboratory HCMV strain AD169 (MOI of 1) for 8 h or (D) treated with 3.28 × 103 U/ml IFN-β for 24 h. (E+F) Alternatively, cells were treated with 0.1 μg/ml poly(dA:dT) and 3 μM or 10 μM Ruxolitinib for 24 h. (A-E) Total RNAs were prepared and RT-qPCR was performed to determine transcription of the IRF3-responsive gene ISG54. Depicted values were calculated from triplicates relative to untreated siC cells using ACTB or GAPDH as housekeeping genes and are shown as mean ± SD. One out of at least two independent experiments is shown. Statistical analysis was performed with respective ΔCq-values using a student’s t-test (unpaired, two-tailed); n.s.: not significant, **p<0.01, ***p<0.001, ****p<0.0001. (F) Cells were harvested for Western Blot analyses to determine STAT2 expression; β-actin served as internal loading control. The following antibodies were used: anti-ATRX (HPA001906), anti-STAT2 (A7), anti-β-actin (AC-15). (G-I) HEK293T cells were (G) stimulated with 0.5 μg/ml poly(I:C) or (I) transfected with a plasmid coding for GFP-V5-IRF3. After 24 h, cells were immunoprecipitated with an ATRX antibody (D1N2E) or an IgG isotype control antibody. The following antibodies were used for detection of proteins in the immunoprecipitate (IP) and in the whole cell lysate (lysate): ATRX (HPA001906), Daxx (E94) and IRF3 (D6I4C). (H) Quantification of co-immunoprecipitated IRF3 relative to total IRF3 levels in the lysate control. Signal intensities were calculated relative to the IgG isotype control. Data were obtained from four independent experiments and are shown as mean ± SD. Statistical analysis was performed using a student’s t-test (one sample, two-tailed); **p<0.01.
Fig 4.
Repression of the RIG-I/MDA-5 signaling pathway in ATRX knockdown HFFs.
Stable ATRX knockdown HFFs (siATRX) and respective control cells (siC) were treated with (A) 0.5 μg/ml poly(I:C) HMW and poly(I:C) LMW, (B) 0.5 μg/ml poly(I:C) HMW or (C) 0.1 μg/ml poly(I:C) HMW for 24 h. (A) Cells were harvested for Western blotting to determine the phosphorylation of IRF3 and TBK1; β-actin served as internal loading control. Signal intensities were quantified relative to poly(I:C) HMW-treated siC cells (lane 2). The following antibodies were used: anti-ATRX (39-f), anti-phospho-IRF3 (Ser386) (EPR2346), anti-IRF3 (D6I4C), anti-phospho-TBK1 (Ser172) (D52C2), anti-TBK1 (D1B4), anti-β-actin (AC-15). (B) Total RNAs were prepared and RT-qPCR was performed to determine IFNB1 induction. Depicted values were calculated from triplicates relative to untreated siC cells using GAPDH as a housekeeping gene and are shown as mean ± SD. One out of two independent experiments is shown. Statistical analysis was performed with respective ΔCq-values using a student’s t-test (unpaired, two-tailed); *p<0.05. (C) Supernatants were harvested and mixed with HEK-Blue IFN-α/β cells. Type I IFNs in the supernatants were quantitated by measuring SEAP activity produced by HEK-Blue IFN-α/β cells using QUANTI-Blue. Depicted values were derived from triplicates and are shown as mean ± SD. Statistical analysis was performed using a student’s t-test (unpaired, two-tailed); ***p<0.001.
Fig 5.
Impaired ISG expression in ATRX knockdown HFFs.
(A-C) ATRX knockdown HFFs (siATRX) and respective control cells (siC) were treated with (A) 0.1 μg/ml poly(dA:dT), (B) IFN-β (3.28 to 3.75× 103 U/ml), or (C) IFN-β (3.87 × 103 U/ml) and IFN-γ (0.2 μg/ml) for 24 h. Subsequently, total RNAs were prepared and RT-qPCR was performed to determine transcription of the ISGs (A) CCL8, OASL and MX1, (B) CCL8, TNFSF10 and MX1 or (C) CCL8, CCL7, CD38 and CIITA. (D) HFFs with a doxycycline-inducible expression of an shRNA targeting ATRX were either left untreated (−) or treated with 500 ng/ml doxycycline (dox) for 14 days (d). Subsequently, cells were harvested for Western blot analyses to determine the knockdown efficiency; β-actin served as an internal loading control. The following antibodies were used: anti-ATRX (39-f), anti-β-actin (AC-15). kd: knockdown (E) Doxycycline-inducible ATRX knockdown HFFs were either left untreated (−dox) or treated with 500 ng/ml dox for 14 d to induce an ATRX knockdown, followed by stimulation with IFN-β (3.75 × 103 U/ml) for 24 h. Subsequently, total RNAs were prepared and RT-qPCR was performed to determine transcription of the ISGs CCL8, TNFSF10 and MX1. (A+B+C+E) Depicted values were calculated from triplicates relative to untreated control cells using GAPDH as a housekeeping gene and are shown as mean ± SD. One out of at least two independent experiments is shown. Statistical analysis was performed with respective ΔCq-values using a student’s t-test (unpaired, two-tailed); n.s.: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Fig 6.
RNA-seq analysis of doxycycline-inducible ATRX knockdown HFFs stimulated with IFN-β.
(A) Schematic representation of the experiment. Doxycycline-inducible ATRX knockdown (ATRX kd) HFFs were either treated with 500 ng/ml doxycycline (dox) for 14 days (d) or left untreated as a control, followed by stimulation with IFN-β (3.28 × 103 U/ml) for 24 h. Subsequently, total RNAs were prepared and subjected to RNA-seq. Samples were divided in four groups of triplicates (untreated: wt; only IFN-β-treated: IFN; only dox-treated: dox; dox- and IFN-β-treated: doxIFN). (B) Venn-diagram of pairwise comparisons of the four groups. Differentially expressed genes were filtered to an adjusted p-value < 0.01. The following online tool was used: http://bioinformatics.psb.ugent.be/webtools/Venn/ (accessed 13 Sep 2021). (C) Homer de novo motif analysis of promoter regions of the differentially expressed genes from the comparison dox vs doxIFN. The top motif (p-value of 10−14) is shown (top) aligned with one of the matched known motifs (bottom). (D) Scatter plot of log2 fold changes from the comparison of dox vs doxIFN and wt vs IFN for every gene (n = 1893) with an adjusted p-value < 0.01. (E) Gene ontology (GO) terms of the category biological process for 85 corresponding proteins out of the top 100 hits with reduced log2 fold changes in dox vs doxIFN compared to wt vs IFN; FDR: false discovery rate. (F) Heat map of proteins belonging to the GO terms response to cytokine (GO:0034097) and cytokine-mediated signaling pathway (GO:0019221).
Fig 7.
Chromatin accessibility in stable ATRX knockdown HFFs stimulated with IFN-β determined by ATAC-seq.
(A) Schematic representation of the experiment. Stable ATRX knockdown HFFs (siATRX) and respective control HFFs (siC) were treated with IFN-β (2.22 × 103 U/ml) for 24 h or left untreated. Cryopreserved samples were subjected to ATAC-seq. Samples were divided in four groups of duplicates (untreated control HFFs: siC; IFN-β-treated control HFFs: siCIFN; ATRX knockdown HFFs: siATRX; IFN-β-treated ATRX knockdown HFFs: siATRXIFN). (B-E) Venn diagram of common and specific peaks of the comparison (B) siC vs siATRX, (C) siCIFN vs siATRXIFN, (D) siC vs siCIFN and (E) siATRX vs siATRXIFN. (F-I) Scatter plot of common peaks considering a FDR < 0.010 for the comparison (F) siC vs siATRX, (G) siCIFN vs siATRXIFN, (H) siC vs siCIFN and (I) siATRX vs siATRXIFN. Regions with a FC ≥ 1 or a FC ≤ −1 are highlighted in pink.
Fig 8.
Annotation of differentially open chromatin in stable ATRX knockdown HFFs stimulated with IFN-β.
(A) Gene annotations of regions with a FC ≥ 1 or a FC ≤ −1 in the comparison siC vs siATRX and siCIFN vs siATRXIFN. (B) GO-enrichment analysis of genes annotated to regions 1 to 5 kb prior to a TSS and promoter regions with a FC ≥ 1 in the comparison siCIFN vs siATRXIFN. Analysis was performed using the Gene ontology database. (C+D) Exemplary Integrative Genomics Viewer (IGV) tracks of the region annotated to (C) OAS1 showing less accessible chromatin in siATRX cells and (D) MX1 showing no change in chromatin accessibility. Both duplicates of the samples siCIFN and siATRXIFN are shown. (E) Homer de novo motif analysis of peaks annotated to promoter regions of the comparison siCIFN vs siATRXIFN. The top motif (p-value of 10−103) is shown (top) aligned with the matched known motifs (bottom).
Fig 9.
Enriched association of ATRX at target regions.
HFFs were stimulated with 0.1 μg/ml poly(dA:dT) for 16 h. Cells were fixed and subjected to ChIP with an anti-ATRX antibody (D1N2E) and IgG as control. Precipitation was analyzed by qPCR using primer pairs for rDNA (positive control), IFNB1 (promoter region), OAS1 (regulatory region), MX1 (regulatory region) and GAPDH (negative control). Results are shown as the percent of input immunoprecipitated by each antibody and normalized to the negative control (GAPDH), which was set to 1 (pink line). Depicted values were calculated from three independent experiments and are shown as mean ± SD.