Fig 2: 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.

Fig 3: 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-ß (3.28 to 3.75× 103 U/ml), or (C) IFN-ß (3.87 × 103 U/ml) and IFN-? (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; ß-actin served as an internal loading control. The following antibodies were used: anti-ATRX (39-f), anti-ß-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-ß (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 ?Cq-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 4: Suppressed innate immune response to HCMV infection in ATRX knockdown HFFs.(A) ATRX knockdown efficiency in siATRX cells was assessed by Western blotting; ß-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; ß-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 ?Cq-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-ß-actin (AC-15); (D) anti-phospho-IRF3 (Ser386) (EPR2346), anti-IRF3 (D6I4C); (B) anti-ATRX (D-5), anti-Daxx (M112).

Fig 5: ALT-FISH visualizes ALT activity changes induced by ASF1 or ATRX depletion. (A) Scheme of ALT induction by ASF1 knock-down in the ALT-prone cell line HeLa LT (long telomeres). (B) Western blot and corresponding quantification to verify ASF1A/B depletion upon siRNA-mediated knock-down (kd) in HeLa LT and the control cell line HeLa ST (short telomeres). ASF1 levels were normalized to tubulin and a matched sample transfected with a non-targeting siRNA control (ctrl). Bar plots represent the mean and SD of 5 independent knock-down experiments with P = 0.0001 (***) and P < 0.0001 (****) from a one-sample Student's t-test. (C) Quantification of CC level changes upon ASF1 kd with representative blots on top. Bar plot depicts the mean and SEM of five replicates. All values are expressed relative to an ALT+ U2OS reference sample (n.d., not detectable) with P > 0.05 (not significant, n.s.), P = 0.015 (*) and P = 0.0079 (**) from a Wilcoxon rank sum test. (D) Exemplary TelC ALT-FISH stained cells and quantification for the ASF1 knock-down. Data from untreated/wt cell lines are included for comparison and are the same as in Figure 3B. Count data were binned for better visualization and are depicted as percentage fraction of all analyzed cells for each condition. For TelG ALT-FISH data and un-binned spot count data see Supplementary Figure S4. Data were pooled from three independent knock-down experiments. (E) Schematic of ALT activity induction by ATRX knock-out in weakly ALT+ pedGBM NEM168 cells. (F) ATRX western blot confirming loss of ATRX in the two NEM168 CRISPR knock-out clones B5 and F2 versus the parental cell line (wt). HeLa (ATRX wt) and U2OS (ATRX-deficient) are included as references. (G) CC level changes upon ATRX knock-out with representative blots shown on top. Bar plots depict the mean and SEM of three independent replicates. All values are expressed relative to an ALT+ U2OS reference sample. The P-values for the indicated comparisons were calculated using Wilcoxon rank sum test. (H) TelC and TelG ALT-FISH staining and nuclear spot number quantification for NEM168 wt and the two ATRX knock-out clones (B5, F2). Data were pooled from 2–4 independent replicates (cell numbers given below). Adjusted P-values from a pair-wise Wilcoxon rank sum test with BH correction were < 2e–16 (****). All boxplots depict the median with first and third quartile of the distribution. All scale bars are 5 µm.