PNAs formation after diverse stress-inducing stimuli and topoisomerase downregulation.

(A) Structural types of PML-nucleolar associations (PNAs) – ‘bowls’, ‘funnels’, ‘balloons’, and PML-nucleolus-derived structures (PML-NDS), occurring in RPE-1hTERTcells after treatment with 0.75 μM doxorubicin detected by indirect IF with anti-PML antibody (green) and anti-B23 (red, for nucleoli visualization) and DAPI (blue). (B) Quantification of the percentage of RPE-1hTERT cells containing PNAs, 48 hours after treatment with various stress-inducing stimuli. The stress stimuli were divided into five categories according to their mechanism of action (see Supplementary Table 1): 1) poisons/inhibitors of topoisomerases, 2) treatments inducing the inhibition of RNAPI, 3) inhibitors of pre-RNA processing, 4) inductors of replication stress, and 5) other stressors. p53 stabilization, γH2AX foci formation, PAF49 segregation, and TOP1 or TOP2A decline have been assessed for each treatment. The concentration and abbreviation used: DOXO, doxorubicin (0.75 µM); BMH-21 (0.5 µM); CPT, camptothecin (50 µM); TPT, topotecan (50 µM); ACLA, aclarubicin (0.05 µM); OXLP, oxaliplatin (10 µM); AMD, actinomycin D (10 nM); ETP, etoposide (50 µM); CX-5461 (5 µM); IR, ionizing radiation (10 Gy); APH, aphidicolin (0.4 µM); 5-FU (200 µM); HU, hydroxyurea (100 µM), IFNγ (5 ng/mL); MG-132 (10 µM); ROSC, roscovitine (20 µM); AA, acetic acid. (C) The pattern of PML (green) and B23 (red) in RPE-1hTERT cells visualized by indirect IF, 3 days post-transfection with siRNAs targeting TOP1 and TOP2A, or with non-targeting siRNA (siNT), respectively. (D) Quantification of the percentage of RPE-1hTERTcells containing PNAs 2, 3, and 6 days after transfection with esiTOP1 and esiTOP2A. (E) RPE-1hTERT cells were pre-treated with 10 nM AMD for 5 hours or with 5 μM CX-5461 for 2 hours to inhibit RNAPI. The cells were then treated with 0.375 μM or 0.75 μM doxorubicin or transfected with esiRNA targeting TOP1 for 48 hours. The bar graphs show the percentage of cells containing either PML-NDS or bowls/funnels/balloons for three independent experiments (graph 1), for a single experiment (graph 2), or for three biological replicates (graph 3). Results are presented as a mean ± s.d. Scale bar, 10 μm.

Inhibition of DNA repair augmented the PNAs formation.

RPE-1hTERT cells were treated with doxorubicin and three concentrations of B02 or with etoposide after downregulation of TDP2 by RNA interference. After the treatment, the PML (green) and nucleolar marker B23 (red) were visualized by indirect IF and wide-field fluorescent microscopy, and a number of nuclei with PNAs were analyzed. (A) The bar graph represents the percentage of nuclei with PNAs after 2-day-long treatment with doxorubicin (0.375 µM or 0.56 µM), three concentrations of B02 (5, 10, and 20 µM), and corresponding concentrations of DMSO as a mock. (B) The bar graph represents the distribution of individual types of PNAs after the same treatments as shown in (A). (C) Representative cells after 2-day-long treatment with 0.375 µM doxorubicin combined with 20 µM B02 or 0.1% DMSO (mock). (D) Representative cells after 2-day-long treatment with 0.56 µM doxorubicin combined with 20 µM B02 or 0.1% DMSO (mock). (E) The bar graph represents the percentage of nuclei with PNAs after 4 days of recovery from 2-day-long treatments with doxorubicin (0.375 µM or 0.56 µM) together with three concentrations of B02 (5, 10, and 20 µM), and corresponding concentrations of DMSO. (F) Representative cells after 4 days of recovery from 2-day-long treatment, with 0.375 µM doxorubicin combined with 20 µM B02 or 0.1% DMSO (mock). (G) Representative cells after 4 days of recovery from 2-day-long treatment with 0.56 µM doxorubicin combined with 20 µM B02 or 0.1% DMSO (mock). (H) The bar graph represents the percentage of nuclei with PNAs after 2-day-long treatment with 5 µM etoposide in cells where TDP2 was downregulated by RNA interference. In all experiments, at least three biological replicates were evaluated. Results are presented as a mean ± s.d. Student’s t-test was used for statistical evaluation. Asterisks indicate the following: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, 20 μm.

PNAs encircle rDNA and DJ loci containing DNA DSB after doxorubicin treatment.

RPE-1hTERT cells were treated with 0.75 µM doxorubicin for 2 days and recovered from the treatment for one and four days. The proliferating cells were used as a control. The localization of rDNA, DJ, PML, and 53BP1 was analyzed using immuno-FISH staining and confocal microscopy. (A) The scheme of a human acrocentric chromosome. The position of probes used for the detection of the rDNA locus (blue) and DJ locus (grey) is shown. (B) The representative nuclei and the nucleoli with and without PNAs in control and treated cells are shown. rDNA (blue), DJ (white), PML (green), and 53BP1 (red). (C) The extent of PML-rDNA and PML-DJ size-based colocalization calculated for individual nucleoli of treated and untreated cells with respect to the presence of PNAs is shown as a scatter plot. The median with an interquartile range is shown. The colocalization was calculated using Fiji(ImageJ)/ Mosaic/Segmentation/Squassh plugin. The number of analyzed nucleoli in each group was: ctrl (n=26); 2 days + PNAs (n=28); 2 days without PNAs (n=18); 1-day-long recovery + PNAs (n=38); 1-day-long recovery without PNAs (n=20); 4-day-long recovery + PNAs (n=23); 4-day-long recovery without PNAs (n=24). (D) The extent of 53BP1-rDNA and 53BP1-DJ size-based colocalization calculated for individual nucleoli of treated and untreated cells with respect to the presence of PNAs is shown as a scatter plot. The median with interquartile range is shown. The same collection of nucleoli was used as presented in (C). (E) The example of analysis that was used to identify whether rDNA/DJ with DNA DSB colocalized with PNAs. The images of the representative nucleoli (2 days doxorubicin, 1- and 4-days recovery) after deconvolution, segmentation, and its 3D model are shown. Note, that the signal of all observed markers was identified as a unique 3D object. rDNA (blue), DJ (white), PML (green), and 53BP1 (red). (F) The combined bar graph shows the percentage of PNAs containing rDNA/DJ with DNA DSB, rDNA/DJ without DNA DSB, and PNAs in which the rDNA/DJ signal was not detected. The number of analyzed nucleoli: 2 days doxorubicin (n=28), 1-day-long recovery (n=39), 4-day-long recovery (n=21). Student’s t-test was used for statistical evaluation. Asterisks indicate the following: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Scale bars, 10 μm (nuclei in B and E) and 5 μm (nucleoli in B).

The DNA damage introduced into the rDNA locus by endonuclease I-PpoI induces PML-NDS.

(A) Scheme of inducible expression of endonuclease I-PpoI in RPE-1hTERT cells and the position of I-PpoI cleavage site in rDNA locus. Tetracycline-inducible promoter (P-TRE3GS), destabilization domain (FKBP), intergenic spacer (IGS), external transcribed spacer (ETS), internal transcribed spacer (ITS). (B) Scheme of the experimental setup used in all experiments presented. Briefly, I-PpoI was activated by doxycycline and Shield for 24 hours, then the medium was exchanged, and the cells were analyzed upon the recovery phase (0, 1, 2, and 5 days). (C) The representative images obtained by indirect IF and confocal microscopy (Stellaris) show the localization of 53BP1 (a marker of DNA DSB, red), and PML (green) upon 1-day-long activation of I-PpoI and during recovery from I-PpoI insult. DAPI (blue) and TOTO-3 (white) marked the nucleus and nucleolus, respectively. Only one layer from the several sections is presented. (D) The level of DNA DSB upon the recovery from I-PpoI insult was obtained by detecting the 53BP1 (a marker of DNA DSB) by indirect IF and high-content microscopy unit (ScanR). The histogram represents the frequency of nuclei (%) with the same number of 53BP1 foci. The bin center used for analysis was 2. (D) The number of nuclei with PNAs upon the recovery from I-PpoI insult was obtained by detection of PML using indirect IF and a high-content microscopy unit (ScanR). The quantification was done manually by the evaluation of PML localization in more than 200 nuclei. The bar graph represents the percentage of nuclei with PNAs. Results are presented as a mean ± s.d. obtained from three biological replicates. (F) The representative images obtained by indirect IF and confocal microscopy (Stellaris) show the correlation between the localization of B23 (red) in PML-NDS (PML, green) upon the recovery from I-PpoI insult. The nucleus and nucleolus were marked by DAPI (blue), and TOTO-3 (white), respectively. Only one layer from the several sections is presented. (G) The representative images obtained by indirect IF and wide-field microscopy show the correlation between the localization of DHX9 (red) and PML-NDS (PML, green) upon the recovery from I-PpoI insult. The nucleus and nucleolus were marked by DAPI (blue), and TOTO-3 (white), respectively. (H-I) The representative images obtained by indirect IF and confocal microscopy (Stellaris) show the localization of UBF (H), a marker of rDNA; red) or PAF49 ((I), a subunit of RNAPI; red) in PML- NDS (PML, green) upon the recovery from I-PpoI insult. One layer of the nucleus and three sequential layers of nucleolus with PML-NDS are presented. The nucleus and nucleolus were marked by DAPI (blue) and TOTO-3 (white), respectively. Scale bars, 10 μm (nuclei C, F, G, H, and I) and 5 μm (nucleoli H and I).

Inhibition of ATM, ATR, and RAD51 suppressed the formation of I-PpoI-induced PML-NDS.

(A) The scheme of the experimental setup used in the experiment shown in (B) is presented. Briefly, 24 h after seeding of RPE-1hTERT-I-PpoI cell (clone 1H4), the ATM inhibitors (1 µM KU-60019 or 6 µM KU-55933) or ATR inhibitor (0.2 µM VE-822) were added 1 hour before the activation of I-PpoI expression. (B) The number of nuclei with PNAs upon the I-PpoI insult and simultaneous inhibition of ATM or ATR was obtained by detection of PML using indirect IF and a high-content microscopy unit (ScanR). The quantification was done manually evaluating PML localization in more than 200 nuclei. The bar plot represents the percentage of nuclei with PNAs. (C) The scheme of the experimental setup used in experiments shown in (D and E and Supplementary Figures 5B and C) is presented. Briefly, 24 h after seeding I-PpoI was activated in RPE-1hTERT-I-PpoI cell clones 1A11 and 1H4 for 24 hours. The inhibitors of DNA PK (1 µM NU-7441) or RAD51 (10 µM B02) were applied individually or both together at the time of I-PpoI activation. After 24 hours, the medium was exchanged, and the cells were analyzed during the recovery phase (0, 1, 2, and 5 days). (D) During the recovery phase, the level of DNA DSB was quantified by the detection of 53BP1 (a marker DNA DSB) using indirect IF and a high-content microscopy unit (ScanR). The data from three independent biological replicates (values for 200 nuclei) were pooled together and represented as histograms showing the frequency of nuclei (%) with the same number of 53BP1 foci. The bin center used for analysis was 2. (E) The number of nuclei with PNAs upon the recovery from I-PpoI insult and simultaneous inhibition of particular DNA damage repair pathway was obtained by detection of PML using indirect IF and ScanR. The quantification was done manually by the evaluation of PML localization in more than 200 nuclei. (F) The scheme of the experimental setup used in experiments shown in (G, H, and I) is presented. Briefly, the RPE-1hTERT-I-PpoI (1H4) were transfected by interfering RNA upon seeding. After 24 h, the I-PpoI was activated for 24 h. Then, the medium was changed, and cells recovered from rDNA damage for 0 and 1 day. The control cells were treated with DMSO as a mock simultaneously. (G). The level of DNA DSB was quantified by the detection of γH2AX (a marker DNA DSB) using indirect IF and ScanR. The data from three independent biological replicates (values for 200 nuclei) were pooled together and represented as histograms showing the frequency of nuclei (%) with the same number of γH2AX foci. The number of nuclei with PNAs upon the recovery from (H) inhibition of a particular DNA damage repair pathway by RNA interference and without the activation of I-PpoI or (I) when I-PpoI was activated together with the inhibition of a particular DNA damage repair pathway was obtained by detection of PML using indirect IF and ScanR. The quantification was done manually by the evaluation of PML localization in more than 200 nuclei. The bar plot represents the percentage of nuclei with PNAs. Results are presented as a mean ± s.d. obtained from three biological replicates. Asterisks indicate the following: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

The resected DNA is present in both G1 and S/G2 cells and colocalizes with I-PpoI- induced PNAs.

(A) The I-PpoI expression was activated 24 hours after RPE-1hTERT-I-PpoI (1H4) seeding. The cells were harvested 6, 8, and 24 hours after activation of I-PpoI expression, and RAD51 and PML were detected by indirect IF. The number of RAD51 foci and total DAPI fluorescence in individual nuclei were evaluated using the ScanR software. The G1 and S/G2 cells were estimated according to the total DAPI fluorescence. The count of RAD51 per nucleus is shown for G1 and S/G2 cells using the Whiskers (box shows 10 – 90 percentiles; the black line is median). (B) The cells harvested after 24 hours of I-PpoI activation were stained for PML (green) and RAD51 (red). The characteristic nuclei with PNAs are shown. (C) The same experimental setup as described in (A) was used, but RPA2pS33 and PML were detected by indirect IF and ScanR. The number of RPA2pS33 foci and total DAPI fluorescence in individual nuclei were evaluated using the ScanR software. The G1 and S/G2 cells were estimated according to the total DAPI fluorescence. The count of RPA2pS33 per nucleus is shown for G1 and S/G2 cells using the Whiskers (box shows 10 – 90 percentiles; the black line is median). (D) PML and resected DNA (RPA2pS33) were detected after 24 hours of long activation of I-PpoI and after 1 day-long recovery using indirect IF and confocal microscopy (Stellaris). The whole nucleus and three separate layers of nucleolus are shown. Nuclei were stained by DAPI (blue) and nucleoli by TOTO3 (white). Scale bar, 10 µm (nucleus); 2 µm (nucleolus). (E) RPE-1hTERT-I-PpoI (1H4) were transfected by interfering RNA upon seeding. After 24 hours, the I-PpoI was activated by doxycycline and Shield for another 24 hours. Then, the medium was changed, and cells recovered for 1 day. The PML was detected by indirect IF and ScanR. ScanR analysis software divided the cells according to total DAPI fluorescence as a G1 and S/G2. Then, the gallery of nuclei was made for each group, and the number of nuclei with PNAs was manually calculated. The percentage of nuclei with PNAs presented in G1 and S/G2 is shown as a column graph. (F) The experimental setup and evaluation were like those in (E). Only the I-PpoI was not activated; instead, DMSO was added. (G) RPE-1hTERT cells were treated with 0.75 µM doxorubicin and EdU for 48 hours. Then, the PML was detected by indirect IF and EdU by ClickIt chemistry. ScanR analysis software divided the cells according to total DAPI fluorescence and EdU fluorescence into three groups: G1 (Edu-), S/G2 (Edu+), and G2 (EdU-). The scatter plot shows the total DAPI fluorescence and EdU mean fluorescence of individual nuclei and gates for the mentioned three groups. (H) Galleries of nuclei from gates shown in (G) were made, and the number of nuclei with PNAs was manually calculated. The percentage of nuclei with PNAs presented in G1(EdU-), S/G2(EdU+), and G2(EdU-) is shown as a column graph. (I) RPE-1hTERTwere transfected by interfering RNA targeting TOP2A and TOP1 upon seeding. After 2 days, PML was detected by indirect IF and ScanR. ScanR analysis software divided the cells according to total DAPI fluorescence as a G1 and S/G2. Then, the galleries of nuclei were made, and the number of nuclei with PNAs was manually calculated. The percentage of nuclei with PNAs presented in G1 and S/G2 is shown as a column graph. Scale bar, 10 µm (B, D – nucleus); 2 µm (D – nucleolus). Results are presented as a mean ± s.d. obtained from three biological replicates. Asterisks indicate the following: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

The schematic presentation of signals triggering the development of PNAs based on data from rDNA damage introduced by I-PpoI.

rDNA DSBs caused by the endonuclease I-PpoI are predominantly repaired by NHEJ in the nucleolar interior (28,57). Unrepaired rDNA lesions signal for ATM/ATR-dependent inactivation of RNA polymerase I and the translocation of damaged rDNA to the nucleolar periphery, forming a nucleolar cap (49,57). The damaged rDNA in the nucleolar cap is resected, suggesting repair by HDR (28,30). Our data indicate that I-PpoI-induced PNAs (PML-NDS) form as a late response to rDNA damage, occurring when RAD51 foci in the nucleolar periphery decline. Markers of DNA DSBs (γH2AX, 53BP1) and DNA resection (pRPA) still colocalize with PNAs, supporting the hypothesis that persistent rDNA DSBs incompatible with NHEJ or HDR signal for PNA development. The exact functions of PNAs are unknown, but we hypothesize they isolate damaged rDNA from active pre-rRNA transcription foci. We cannot exclude a link to alternative repair pathways for persistent rDNA damage. Notably, I-PpoI-induced PML-NDS are mainly present in the G1 cell cycle phase. Inhibiting ATM/ATR, which is essential for RNAPI inhibition, nucleolar cap establishment, and HDR, prevents their formation. RAD51 ablation negatively affects the occurrence of I-PpoI-induced PNAs. Importantly, the PNAs decline was present only in G1. The mechanisms by which RAD51 affects PNA establishment are unclear, but RAD51 promotes HR and inhibits alternative pathways such as SSA and A-EJ or regulates DNA end processing. Finally, this model describes the response to direct DNA damage, and we identified other treatments with equal or greater potential to induce PNAs. These treatments share common features, such as altering chromatin topology and inhibiting RNAPI. The scheme was created with BioRender.com.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.