Topological stress triggers difficult-to-repair DNA lesions in ribosomal DNA with ensuing formation of PML-nucleolar compartment

  1. Laboratory of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague 142 20, Czech Republic
  2. Centre for Chromosome Biology, College of Science and Engineering, University of Galway, Galway, Ireland
  3. Genome Integrity Unit, Danish Cancer Society Research Center, Copenhagen, Denmark, Copenhagen DK-2100, Denmark
  4. Division of Genome Biology, Department of medical biochemistry and biophysics, Karolinska Institutet, Stockholm, Sweden

Editors

  • Reviewing Editor
    Akira Shinohara
    Osaka University, Suita/Osaka, Japan
  • Senior Editor
    Tony Yuen
    Icahn School of Medicine at Mount Sinai, New York, United States of America

Reviewer #1 (Public Review):

Summary:
This paper described the dynamics of the nuclear substructure called PML Nucleolar Association (PNA) in response to DNA damage on ribosomal DNA (rDNA) repeats. The authors showed that the PNA with rDNA repeats is induced by the inhibition of topoisomerases and RNA polymerase I and that the PNA formation is modulated by RAD51, thus homologous recombination. Artificially induced DNA double-strand breaks (DSBs) in rDNA repeats stimulate the formation of PNA with DSB markers. This DSB-triggered PNA formation is regulated by DSB repair pathways.

Strengths:
This paper illustrates a unique DNA damage-induced sub-nuclear structure containing the PML body, which is specifically associated with the nucleolus. Moreover, the dynamics of this PML Nucleolar Association (PNA) require topoisomerases and RNA polymerase I and are modulated by RAD51-mediated homologous recombination and non-homologous end-joining. This study provides a unique regulation of DSB repair at rDNA repeats associated with the unique-membrane-less subnuclear structure.

Weaknesses:
Although the PNA formation on rDNA repeat is nicely shown by cytological analysis, the biological significance of PNA in DSB repair is not fully addressed.

Reviewer #2 (Public Review):

In this manuscript, the authors aim to study the PML-nucleoli association (PNAs) by different genotoxic stress and to determine the underlying molecular mechanisms.

First, from a diverse set of genotoxic stress conditions (topoisomerases, RNA Pol I, rRNA processing, and DNA replication stress), the authors have found that the inhibition of topoisomerases and RNA Polymerase I has the highest PNA formation associated with p53 stabilization, gamma-H2AX, and PAF49 segregation. It was further demonstrated that Rad51-mediated HR pathway but not NHEJ pathway is associated with the PNA formation. Immuno-FISH assays show that doxorubicin induces DSBs (53BP1 foci) in rDNA and PNA interactions with rDNA/DJ regions. Furthermore, endonuclease I-Ppol induced DSB at a defined location in rDNA and led to PNAs.

Most claims by the authors are supported by the data provided. However, below weaknesses/concerns may need to be addressed to improve the quality of the study.

  1. Top2B toxin doxorubicin had the highest degree of elevating PNAs; however, Top2B-knockdown had almost no noticeable effects on PNAs. How to reconcile the different phenotypes targeting Top2B?

  2. To test the role of Rad51 and DNA-PKcs in the PNA formation, Rad51 inhibitor B02 and DNA-PKcs inhibitor NU-7441 were chosen to use in the study. To further exclude the possible off-target of B02 and NU-7441, siRNA-mediated knockdown of Rad51 and DNA-PKcs would be an appropriate complementary approach to the pharmaceutical inhibitor approach.

  3. Several previous studies have shown the activation of the nucleolar ATM-mediated DNA damage response pathway by I-Ppol-induced DSBs in rDNA. What is the role of nucleolar ATM in the regulation of PNAs?

Reviewer #3 (Public Review):

Summary:
Hornofova et al examined interactions between the nucleolus and promyelocytic leukemia nuclear bodies (PML-NBs) termed PML-nucleolar associations (PNAs). PNAs are found in a minor subset of cells, exist within distinct morphological subcategories, and are induced by cellular stressors including genotoxic damage. A systematic pharmacological investigation identified that compounds that inhibit RNA Polymerase 1 (RNAPI) and/or topoisomerase 1 or 2A caused the greatest proportion of cells with PNA. A specific RAD51 inhibitor (R02) impacted the number of cells exhibiting PNAs and PNA morphology. Genetic double-strand break (DSB) induction within the rDNA locus also induced PNA structures that were more prevalent when non-homologous end joining (NHEJ) was inhibited.

Strengths:
PNA are morphologically distinct and readily visualized. The imaging data are high quality, and rDNA is amenable to studying nuclear dynamics. Specific induction of rDNA damage is a strong addition to the non-specific pharmacological damage characterized early in the manuscript. These data nicely demonstrate that rDNA double-strand breaks undermine PNA formation. Figure 1 is a comprehensive examination and presents a compelling argument that RNAPI and/or TOP1, TOP2A inhibition promote PNA structures.

Weaknesses:
The data are limited to fixed fluorescent microscopy of structures present in a minority of cells. Data are occasionally qualitative and/or based upon interpretation of dynamic events extrapolated from fixed imaging. This study would benefit from live imaging that captures PNA dynamics.

Cell cycle and cell division are not considered. Double-strand break repair is cell cycle dependent, and most experiments occur over days of treatment and recovery. It is unclear if the cultures are proliferating, or which cell cycle phase the cells are in at the time of analysis. It is also unclear if PNAs are repeatedly dissociating and reforming each cell division.

The relationship of PNA morphologies (bowl, funnel, balloon, and PML-NDS) also remains unclear. It is possible that PNAs mature/progress through the distinct morphologies, and that morphological presentation is a readout of repair or damage in the rDNA locus. However, this is not formally addressed.

An I-Ppol targeted sequence within the rDNA locus suggests 3D structural rearrangement following damage. An orthogonal approach measuring rDNA 3D architecture would benefit comprehension. Following I-Ppol induction, it is possible that cells arrest in a G1 state. This may explain why targeting NHEJ has a greater impact on the number of 53BP1 foci and should be investigated.

Conclusions: PNAs are a phenomenon of biological significance and understanding that significance is of value. More work is required to advance knowledge in this area. The authors may wish to examine the literature on APBs (Alt-associated PML-NBs), which are similar structures where telomeres associate with PML-NBs in a specific subset of cancers. It is possible that APBs and PNAs share similar biology, and prior efforts on APBs may help guide future PNA studies.

Author Response

First of all, we would like to thank you for the opportunity to get the three valuable sets of comments on our work from the reviewers and the important summary from the Chief Editor. If we understand correctly, at this moment, we are expected to check for any factual errors, and our response at this stage will affect the choice of which reviewer’s comment will be published as a part of the reviewed Preprint. If so, we want to comment on some of the reviewer's points (Part A). These are not factual errors but more misunderstandings that need to be corrected. Furthermore, it depends on your decision whether it will be a part of the response or not. In Part B, we will address the reviewer's comments.

Part A:

  1. Reviewers #1 and #3 missed our originally already reported PNAs dynamics based on live-cell imaging (mainly Reviewer #3 stressed that the dynamic we present is extrapolated from fixed imaging). We previously published the detailed dynamics of PNAs as detected by live-cell imaging (Imrichova, Aging 2019, doi: 10.18632/aging.102248. Epub 2019 Sep 7). It seems that we have not sufficiently highlighted this important aspect in the present eLife manuscript, despite in the Introduction part, we have described the dynamic transitions between the individual PNAs types/stages, yet without explicitly emphasizing that such dynamic insights were deduced from our live-cell imaging experiments.

  2. Reviewer#2 asked us to reconcile the different phenotypes after RNAi of TOP2A (KD induces PNAs) and TOP2B (KD does not induce PNAs), vis a vis the fact that the TOP2B-targeting drug -doxorubicin is a strong inducer of PNAs formation. We would like to stress that doxorubicin is not a specific poison of TOP2B (e.g., Atwal 2019; DOI: 10.1124/mol.119.117259). It can poison (at low concentration) or inhibit (at high concentration) all subtypes of topoisomerase 2. In other words, doxorubicin targets a wider spectrum of type 2 topoisomerases and hence can limit any potential redundant roles of the individual subtypes, which, on the other hand, can manifest under conditions when only a specific one member is depleted genetically. We have further discussed this interesting issue in the discussion presented in our manuscript, and we believe there is no discrepancy, due to the wider impact of doxorubicin and an apparently more dominant role of TOP2A than TOP2B in preventing PNAs.

  3. We are aware that the biological significance of the interaction of PML with nucleolus has not been fully solved yet. At this moment, we can conclude that PNAs recognize and sequester the damaged/aberrant rDNA from active nucleolus. This novel sorting mechanism might be necessary for maintaining the integrity of the repetitive rDNA loci that might otherwise be altered or lost during complex recombinational rDNA repair. Importantly, we also identified substances (mostly chemotherapeutics) that cause rDNA damage. Given that PML is a multifaceted protein involved in diverse processes; PML depletion might affect several stress-related processes. The rDNA quality/quantity analysis is also highly challenging because of the high number of rDNA copies (200-400). As preparing such an experimental model/s is difficult and time-consuming, addressing this issue in more detail will be a part of our follow-up work. Nevertheless, we will perform the bulk of the experiments recommended by the reviewers, to strengthen the conclusions of this manuscript, as follows: A) We will explore whether the PNAs formation is linked to some specific cell cycle phase; B) To strengthen the experiments with inhibition of NHEJ (DNA PKi) and HR (B02i), we will perform the RNA interference or use some other inhibitor/s operating through a distinct mechanism yet targeting the same repair process; C) We will analyze the recovery from I-PpoI treatment and assess cell proliferation, ability to form colonies, and the presence of senescent cells.

Part 2

Reviewer #1 (Public Review):

Summary:

This paper described the dynamics of the nuclear substructure called PML Nucleolar Association (PNA) in response to DNA damage on ribosomal DNA (rDNA) repeats. The authors showed that the PNA with rDNA repeats is induced by the inhibition of topoisomerases and RNA polymerase I and that the PNA formation is modulated by RAD51, thus homologous recombination. Artificially induced DNA double-strand breaks (DSBs) in rDNA repeats stimulate the formation of PNA with DSB markers. This DSB-triggered PNA formation is regulated by DSB repair pathways.

Strengths:

This paper illustrates a unique DNA damage-induced sub-nuclear structure containing the PML body, which is specifically associated with the nucleolus. Moreover, the dynamics of this PML Nucleolar Association (PNA) require topoisomerases and RNA polymerase I and are modulated by RAD51-mediated homologous recombination and non-homologous end-joining. This study provides a unique regulation of DSB repair at rDNA repeats associated with the unique-membrane-less subnuclear structure.

Weaknesses:

Although the PNA formation on rDNA repeat is nicely shown by cytological analysis, the biological significance of PNA in DSB repair is not fully addressed.

At this moment, we cannot mechanistically fully elucidate the biological significance of this peculiar process. However, our data shows that the dynamic interaction of PML with nucleolus can sequester damaged rDNA from reactivating nucleolus. We propose that in this way, the actively transcribed intact rDNA is protected from possible detrimental interaction with the defective, PNAs-sequestered rDNA, most likely to avoid the harmful intra- and inter-chromosomal recombination events that would otherwise likely occur during recombinational repair of the damaged rDNA, as the rDNA repeats present on 5 chromosomes are repetitive. Thus, this novel sorting mechanism might help sustain repetitive rDNA loci integrity.

Reviewer #2 (Public Review):

In this manuscript, the authors aim to study the PML-nucleoli association (PNAs) by different genotoxic stress and to determine the underlying molecular mechanisms.

First, from a diverse set of genotoxic stress conditions (topoisomerases, RNA Pol I, rRNA processing, and DNA replication stress), the authors have found that the inhibition of topoisomerases and RNA Polymerase I has the highest PNA formation associated with p53 stabilization, gamma-H2AX, and PAF49 segregation. It was further demonstrated that Rad51-mediated HR pathway but not NHEJ pathway is associated with the PNA formation. Immuno-FISH assays show that doxorubicin induces DSBs (53BP1 foci) in rDNA and PNA interactions with rDNA/DJ regions. Furthermore, endonuclease I-Ppol induced DSB at a defined location in rDNA and led to PNAs.

Most claims by the authors are supported by the data provided. However, below weaknesses/concerns may need to be addressed to improve the quality of the study.

  1. Top2B toxin doxorubicin had the highest degree of elevating PNAs; however, Top2B-knockdown had almost no noticeable effects on PNAs. How to reconcile the different phenotypes targeting Top2B?
  1. We thank the reviewer for this comment and below explain why there is no discrepancy in the observed phenotypes. Doxorubicin is not a specific poison of TOP2B (e.g., Atwal 2019; DOI: 10.1124/mol.119.117259). It can poison (stabilize ternary complex at low concentration) or inhibit (e.g., defects in decatenation at high concentration) all subtypes of topoisomerase 2. It intercalates DNA (alteration of DNA torsion; histone eviction) and elevates oxidative stress. Therefore, the observed effect of doxorubicin reflects its broader impact, also beyond inhibition of Top2B: as doxorubicin targets a wider spectrum of type 2 topoisomerases and hence can limit any potential redundant roles of the individual subtypes (which on the other hand can manifest under conditions when only one specific member is depleted genetically), thereby causing a robust induction of PNAs. We have further discussed this issue in the Discussion section of our manuscript, and we believe there is no discrepancy, in the observed phenotypes due to the wider impact of doxorubicin and an apparently more dominant role of TOP2A than TOP2B (both of which are impacted to some extent by doxorubicin) in preventing PNAs.
  1. To test the role of Rad51 and DNA-PKcs in the PNA formation, Rad51 inhibitor B02 and DNA-PKcs inhibitor NU-7441 were chosen to use in the study. To further exclude the possible off-target of B02 and NU-7441, siRNA-mediated knockdown of Rad51 and DNA-PKcs would be an appropriate complementary approach to the pharmaceutical inhibitor approach.

We are grateful for this suggestion and will perform the recommended experiments the outcome of which will indeed help to exclude the possible off-target effects of B02 and NU-7441. We are now collecting/testing the necessary tools and will carry out these analyses proposed by the reviewer.

  1. Several previous studies have shown the activation of the nucleolar ATM-mediated DNA damage response pathway by I-Ppol-induced DSBs in rDNA. What is the role of nucleolar ATM in the regulation of PNAs?

We are aware of the relevant literature on ATM, and appreciate this question from the reviewer. During the revision of this manuscript, we will therefore address the role of ATM signaling in the phenomena that we report here. As ATM signaling is essential for the repression of pre-rRNA synthesis and the compaction of rDNA into the nucleolar caps in response to rDNA damage, we will complement this knowledge by testing to what extent might ATM inhibition affect the induction of PNAs/PML-NDS in our model and experimental settings.

Reviewer #3 (Public Review):

Summary:

Hornofova et al. examined interactions between the nucleolus and promyelocytic leukemia nuclear bodies (PML-NBs) termed PML-nucleolar associations (PNAs). PNAs are found in a minor subset of cells, exist within distinct morphological subcategories, and are induced by cellular stressors including genotoxic damage. A systematic pharmacological investigation identified that compounds that inhibit RNA Polymerase 1 (RNAPI) and/or topoisomerase 1 or 2A caused the greatest proportion of cells with PNA. A specific RAD51 inhibitor (R02) impacted the number of cells exhibiting PNAs and PNA morphology. Genetic double-strand break (DSB) induction within the rDNA locus also induced PNA structures that were more prevalent when non-homologous end joining (NHEJ) was inhibited.

Strengths:

PNA are morphologically distinct and readily visualized. The imaging data are high quality, and rDNA is amenable to studying nuclear dynamics. Specific induction of rDNA damage is a strong addition to the non-specific pharmacological damage characterized early in the manuscript. These data nicely demonstrate that rDNA double-strand breaks undermine PNA formation. Figure 1 is a comprehensive examination and presents a compelling argument that RNAPI and/or TOP1, TOP2A inhibition promote PNA structures.

Weaknesses:

The data are limited to fixed fluorescent microscopy of structures present in a minority of cells. Data are occasionally qualitative and/or based upon interpretation of dynamic events extrapolated from fixed imaging. This study would benefit from live imaging that captures PNA dynamics.

We believe this comment reflects a misunderstanding, for the following reason: We fully agree with the reviewer that live-cell imaging is critical to properly capture the dynamics of the PNAs formation and evolution, and apologize for not sufficiently highlighting that this was already presented in our previous study in which we described the existence and dynamics of PNAs over time, based on the live cell imaging that the reviewer correctly regards as important. In Imrichova et al. (doi: 10.18632/aging.102248. Epub 2019 Sep 7), we used live-cell imaging to describe the dynamics of forming PNAs and the transition between individual types, and we referred to this work in the Introduction section of our present manuscript. By those experiments, including the live-cell imaging, we showed that after the recovery of RNAPI transcription, which usually follows the washout (removal) of the DNA-damaging agents, the funnel-like PNAs are transformed into PML-NDS. These newly emerging PNAs (PML-NDS) are placed next to the reactivated nucleolus. To document this, we paste below the relevant part of the Introduction text that was included in our submitted manuscript (see below in italics). Nevertheless, we did not emphasize that the transition between individual types of PNAs was obtained using live-cell imaging of cells ectopically expressing PML-EGFP and B23-RFP. In the revised manuscript, we will include this critical information and will complement this by a scheme explaining the dynamics of PNAs transitions.

Copied text from our manuscript, relevant to this issue: Doxorubicin, a topoisomerase inhibitor and one of the PNAs inducers, provokes a dynamic interaction of PML with the nucleolus, where the different phases linked to RNAPI inhibition can be discriminated into four basic structural subtypes of PNAs termed according to the 3D structures obtained by super-resolution microscopy as PML 'bowls', PML 'funnels', PML 'balloons' and PML nucleolus-derived structures (PML-NDS; (36)). The doxorubicin-induced inhibition of RNAPI leads to a nucleolar cap formation around which diffuse PML accumulates to form the PML bowl. Note that this event is rare as a minority of nucleolar caps are enveloped by PML (36). As the RNAPI inhibition continues, PML bowls protrude into PML funnels or transform into PML balloons wrapping the whole nucleolus. When the stress is relieved and RNAPI resumes activity, a PML funnel transforms into distinct compartments placed next to the non-segregated (i.e., reactivated) nucleoli, PML nucleolus-derived structures (PML-NDS). PML-NDSs contain nucleolar material, rDNA, and markers of DNA DSBs (36,37).

Cell cycle and cell division are not considered. Double-strand break repair is cell cycle dependent, and most experiments occur over days of treatment and recovery. It is unclear if the cultures are proliferating, or which cell cycle phase the cells are in at the time of analysis. It is also unclear if PNAs are repeatedly dissociating and reforming each cell division.

We agree this is an important point. In a complementary setting we previously published (Imrichova et al., doi: 10.18632/aging.102248. Epub 2019 Sep 7) that exposure of RPE-1 hTERT cells to doxorubicin caused cell cycle arrest and cellular senescence. Thus, most of such cells will not enter the cell cycle again. Regarding the I-PpoI-based model, we indeed did not show in the present manuscript how I-PpoI activation (rDNA damage) affects the cell cycle. In our preliminary experiments that address this issue, we saw that only about 1–3% of cells can recover from the stress and form colonies in a colony-forming assay. We will further repeat and corroborate these preliminary data and include these results in the revised manuscript, together with β-galactosidase staining to demonstrate the presence of senescent cells.

Furthermore, as suggested by this reviewer, we will assess the cell cycle phase/position of the cells in our experiments, to find out whether the cell cycle phase affects/correlates with the PNAs formation.

The relationship of PNA morphologies (bowl, funnel, balloon, and PML-NDS) also remains unclear. It is possible that PNAs mature/progress through the distinct morphologies, and that morphological presentation is a readout of repair or damage in the rDNA locus. However, this is not formally addressed.

This is partly explained by our response to Reviewer no 1, related to our previous live-cell imaging analyses. The 'bowl' emerges first and can be transformed into a 'funnel' or 'balloon'. All these PML structures are in contact with the nucleolar cap (the RNAPI is inhibited). Upon reactivation of RNAPI, the funnel can transform into the PML-NDS. At this moment, we cannot conclude to which precise process the individual structure is linked. However, we already know (Hornofova et al., DOI: 10.1016/j.dnarep.2022.103319) that the funnels colocalize with the highest portion of rDNA, which may reflect some process of concentration/clustering of rDNA. This observation is supported by results presented in this manuscript, which show that individual acrocentric chromosomes (NORs) also accumulate in one funnel. To summarize, the formation of the bowl reflects the aberration in rDNA. The funnel can accumulate rDNA and NORs in one site. The transition between the funnel and PML-NDS mirrors the changes after the reactivation of RNAPI and facilitates the sequestration of damaged rDNA/NORs outside of the active nucleolus. As the processes linked to the individual PNA are not solved yet, we will at least address this issue in a discussion.

An I-Ppol targeted sequence within the rDNA locus suggests 3D structural rearrangement following damage. An orthogonal approach measuring rDNA 3D architecture would benefit comprehension.

This is a very inspiring idea, although demanding and somewhat outside the focused scope of the present study. Our follow-up work will focus on the localization of individual NORs using immune-FISH after introducing the rDNA damage by I-PpoI. In the context of those studies, we also plan to analyze rDNA 3D architecture.

Following I-Ppol induction, it is possible that cells arrest in a G1 state. This may explain why targeting NHEJ has a greater impact on the number of 53BP1 foci and should be investigated.

We fully agree with this possibility and in response, we will perform a series of cell cycle analysis experiments to address this issue, during the revision phase of this manuscript. We will analyze whether I-Ppol-induced PNAs are linked to some cell cycle phase(s).

Conclusions: PNAs are a phenomenon of biological significance and understanding that significance is of value. More work is required to advance knowledge in this area. The authors may wish to examine the literature on APBs (Alt-associated PML-NBs), which are similar structures where telomeres associate with PML-NBs in a specific subset of cancers. It is possible that APBs and PNAs share similar biology, and prior efforts on APBs may help guide future PNA studies.

We will follow this recommendation by the reviewer. In ALT, PML is essential for clustering several (damaged) telomeres into APB. In PML-deficient cells, there is not only a defect in the formation of APB, but also the ALT telomeric DNA synthesis in G2 cells is blocked. As we already mentioned, funnel-like PNAs can accumulate several NORs. Thus, the recombination process between NORs might be facilitated. We will highlight this link and its relevance for cancer in our revised manuscript, thank you.

  1. Howard Hughes Medical Institute
  2. Wellcome Trust
  3. Max-Planck-Gesellschaft
  4. Knut and Alice Wallenberg Foundation