A Sterol-PI(4)P exchanger controls the Tel1/ATM axis of the DNA Damage Response
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Abstract
Upon DNA damage, cells activate the DNA Damage Response (DDR) to coordinate proliferation and DNA repair. Dietary, metabolic, and environmental inputs are emerging as modulators of how DNA surveillance and repair take place. Lipids hold potential to convey these cues, although little is known about how. We observed that lipid droplet (LD) number specifically increased in response to DNA breaks. We show that the selective storage of sterols into these LD concomitantly stabilizes phosphatidyl-4-inositol (PI(4)P) at the Golgi, where it binds the DDR kinase ATM. In turn, this titration attenuates the initial nuclear ATM-driven response to DNA breaks, thus allowing processive repair. Further, manipulating this loop impacts the kinetics of DNA damage signaling and repair in a predictable manner. Thus, our findings have major implications for tackling genetic instability pathologies through dietary and pharmacological interventions.
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Reply to the reviewers
We are very grateful about the thorough reading and deep understanding of the work that these 4 reviewers have provided. Although it is evident that they still request an improved profiling of some aspects, it is very encouraging that all four think the work is very interesting, original, insightful and adds a new layer of knowledge to the regulation of DNA damage sensing and repair. It is also very rewarding that the four reviewers estimate that this work will sew connections between different fields and interest a broad readership. This is why we have designed here a very deep revision, tailored to satisfy all the raised concerns except one, and this just …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
We are very grateful about the thorough reading and deep understanding of the work that these 4 reviewers have provided. Although it is evident that they still request an improved profiling of some aspects, it is very encouraging that all four think the work is very interesting, original, insightful and adds a new layer of knowledge to the regulation of DNA damage sensing and repair. It is also very rewarding that the four reviewers estimate that this work will sew connections between different fields and interest a broad readership. This is why we have designed here a very deep revision, tailored to satisfy all the raised concerns except one, and this just for technical reasons.
Please find below the original reviewers’ comments and our answers to them preceded by the symbol “>”:
Reviewer #1 (Evidence, reproducibility and clarity (Required)): Ovejero et al. report an increase in lipid droplet (LD) abundance after long (from 120' on) exposure of budding yeast cells to DNA damaging agents zeocin and camptothecin (CPT). Next, they analyze DNA damage signaling in yeast mutants that impair triacylglycerol (TAGs) or sterol (STEs) esterification. They observe a slight anticipation in Rad53/CHK2 phosphorylation (indicative of DDR signaling) in yeast stem mutants, as well as in yeast cells or human cells lines pre-treated with oleate upon zeocin treatment. Yeast stem mutants are sensitive to zeocin and captothecin, but only confer sensitivity to hydroxyurea upon combination with tagD mutations. Authors relate these phenotypes to a somewhat decreases DSB resection in yeh2D mutants (expected to have reduced steryl esters pools) and RPA-foci in steD yeast cells. Next, a reduction in single strand annealing recombination repair events upon zeocin treatment is reported using a genetic reporter in steD mutants and oleate-treated cells. From these data they conclude that inability to process sterols in response to DSBs leads to an exacerbated DDR and prevents DNA repair. Next, it is shown that Flag-tagged Tel1 distinctly interacts with mono-phosphate phosphoinositides, including PI(4)P. An interaction in vivo is also inferred through Proximity Ligation Assays (PLA) using anti-PI(4)P and anti-ATM antibodies in human cell lines, which was moderately downregulated upon treatment with MMS or zeocin. Over-expression of the Osh4/OSBP1 transporter, which consumes PI(4)P, increased the number of Tel1 (nuclear) foci upon zeocin treatment. Conversely Sac1 ablation, in which accumulation of PI(4)P is expected, abrogated nuclear Tel1 foci formation and reduced telomere length (a phenotype related to lack of Tel1 function). From these results authors conclude that Tel1 availability in the nucleus is influenced by PI(4)P availability. Lastly, treatment with an OSBP1 inhibitor led to a cell line and damaging agent -variable reduction of ATM phosphorylation and a mostly non-significant reduction of DNA resection, measured by native BrdU detection, in response to CPT treatment. Overall, authors conclude that i) biding of Tel1/ATM to PI(4)P modulates its functional availability in the nucleus, and that ii) DNA damage elicits the esterification and storage of sterols toward LDs, which contributes to tritate Tel1/ATM away from the nucleus dampening the DDR and affecting long-range resection.
Major comments: While the conclusion that Tel1/ATM binds PI(4)P and this interaction modulates Tel1/ATM functional availability at the nucleus is convincing, the conclusion that DSBs elicit a change in the metabolism of this lipid to "control" Tel1/ATM function is not demonstrated. The notion that sterol processing occurs in response to DSBs is not sufficiently supported by the data presented, as the increase in LD numbers is observed much after activation of the DDR (Rad53 phosphorylation) in Zeozin-treated yeast cells.
We are afraid that we have not been clear enough in explaining the kinetics giving rise to our model. As indicated by the reviewer, our work shows, through kinetic studies, that the storage of sterols within LD occurs at later stages than the activation of the DDR by Tel1 and Rad53 phosphorylation. Tel1 foci decline is necessary for subsequent engagement of downstream DNA long-range resection. Since we propose that sterol storage within LD is a means to attenuate Tel1 engagement at DSBs, it is thus logical (and thus compatible with the data we show) that LD number increase occurs simultaneously with Tel1 foci decrease, at late stages of the reactionWe will include this explanation and graph in the revised version of the work.
In addition, evidence is not provided on the mechanisms by which PI(4)P metabolism would be controlled, which would be expected to be DDR-independent as they are placed upstream of this signaling pathway in the author's model.
The key mechanism through which, in the end, PI(4)P metabolism will be controlled, is the esterification of sterols within LD. Given that, as clarified above, LD formation in response to DSBs occurs “late” (i.e., after 120 min), it is not excluded that the DDR itself can instruct, through phosphorylation of some effector(s), LD formation. In other words, by ordering LD formation, the DDR would be launching a self-limiting mechanism. In support, we now know, although we do not show in this work, that eliminating key DDR proteins prevents the formation of LD in response to DNA damage. Because of this, we have undertaken an educated-guess approach and chosen critical or rate-limiting enzymes in LD biology either possessing an S/T-Q cluster domain (predicted to be a phosphorylation substrate for the DNA Damage Response kinases (1), and/or retrieved in phospho-proteomic screens as specific DDR targets (2,3). This adds up to 28 proteins in S. cerevisiae and 45 proteins in Homo sapiens. Importantly, the emergent candidates fall into two identical categories in both organisms. To provide initial support for their pertinence, we have generated a point mutant in the putative S/T-Q cluster of one of the yeast candidates. Of high relevance, we find that the concerned mutant is impaired in correctly triggering LD formation in response to DNA damage, and we have now obtained a specific funding to pursue this characterization that, as such, constitutes a different work from the one presented in this manuscript. We hope that the reviewer is now convinced yet that she/he agrees in keeping this information for subsequent manuscript(s).
The damaging agents used have been suggested to alter the redox metabolism and even lipid peroxidation (Kitanovic 2009, Mizumoto 1993, Krol 2015, Todorova 2015, Ren 2019, Singh 2014). Hence it is possible that PI(4)P changes are not due to DSBs, but an indirect though relevant effect. In absence of direct evidence supporting an active regulation of PI(4)P dynamics in response to DNA breaks, this conclusion remains speculative and this should be noted in the manuscript.
We fully agree with the reviewer that the used genotoxins are triggering a myriad of effects which could elicit the same phenomenon by indirect means. Yet, we want to stress that the use of camptothecin, which elicits a very robust LD formation phenotype (Figure 1C), is very likely specific, as it is proven as a potent and direct trapper of Top1 onto DNA after having cleaved it. Nevertheless, we propose in the next paragraph two specific experiments to dismiss this problem, please see immediately below.
Authors conclude that LD is specific to DSB induction. This seems an overstatement as they just reported LD increases in response to two agents that also induce other kinds of DNA damage. To also strengthen the link between DSBs and PI(4)P modulation of Tel1 function, authors should analyze LD numbers, Rad53 phosphorylation and Tel1 nuclear re-localization in response to HO-induced DNA breaks (e.g., using the system employed in Figure 3C).
We humbly think that enzymatically-induced DNA breaks will both activate Rad53 phosphorylation and Tel1 nuclear concentration, as this has already been established, thus requiring no further exploration. Yet, it is very important to assess the reviewer’s suggestion concerning whether enzymatically-induced DNA breaks also trigger the formation of LD. To this end, we will perform two complementary studies in which, instead of using HO, which cuts only a few times in the genome, we will:
- a) exploit the naturally DSB-accumulating mutant rad3-102, which we previously characterized in the past (4), and which we already exploit in this work for recombination analyses (Figure S4A), to evaluate whether it endogenously harbors more LD in comparison with the WT.
- b) we have recently created a tool in which gRNAs targeted to different subsets of transposons in the genome can drive Cas9 to create DSB in a dose-dependent manner ((9), under revision in Genetics). We will use this system to monitor the LD formation in response to Cas9-triggered cuts. In addition, on figure 5A, significant differences in GFP-Tel1 foci abundance between WT and steD or yeh2D cells are only observed after 210', way after the slight effect on Rad53 phosphorylation is observed. This is at odds with the conclusion that Tel1 association to STEs modulates DDR signaling.
We are afraid that we have not been clear enough in explaining the kinetics giving rise to our model. As indicated by the reviewer, our work shows, through kinetic studies, that the storage of sterols within LD occurs at later stages than the activation of the DDR by Tel1 and Rad53 phosphorylation. Tel1 foci decline is necessary for subsequent engagement of downstream DNA long-range resection. Since we propose that sterol storage within LD is a means to attenuate Tel1 engagement at DSBs, it is thus logical (and thus compatible with the data we show) that LD number increase occurs simultaneously with Tel1 foci decrease, at late stages of the reactionWe will include this explanation and graph in the revised version of the work.
Minor comments:
Figure S1D and E, experiments should be carried out to include time points in which LD accumulation and cell cycle arrest are observed upon zeocin treatment (i.e., up to 210' as in Figure 1A)
We will provide cytometry profiles of cells at 210 min. These data exist already in our laboratory.
How do authors explain increased single strand annealing recombination frequencies in steD and oleate-treated wild type cells (Figure 4A). Should it not be expected that increased STEs also impair recombination induced by endogenous damage?
Only ste∆ (and not +oleate) indeed manifests an increase in basal recombination frequencies, likely arising from endogenous damage. Although the increase is observed, it is not significant. We agree anyway with the reviewer that, was the experiment to be repeated more times, the increase may be found significantly different. We do not have any honest proposal to explain this.
Data presented in figure 4B and 4C are not fully convincing. Performing time course experiments might help concluding if the differences observed represent a relevant defect in DSB processing.
We will perform a Pulsed Field Gel Electrophoresis (PFGE) kinetcis in response to zeocin with or without oleate pre-loading to reinforce the conclusion.
Is Figure 5B referring to Flag-tagged Tel1 or GFP-tagged Tel1 as stated in the figure legend?
There is a misunderstanding here, as the mentioned Figure 5B corresponds to P-ATM immunofluorescences in human cells, not to any tagged Tel1 experiment.
Treatment with the ATM inhibitor AZD0156 increased PI(4)P-ATM PLA signals. From these authors conclude that "association of ATM and PI(4)P inversely correlated with the need for ATM within the nucleus. Do they imply that treatment with ATM-inhibitors reduces the requirement for ATM function in the nucleus? The interpretation of this result should be further elaborated to sustain this conclusion.
We may have conveyed a wrong notion at this point. We do not imply at all that ATM inhibitors reduce the need for ATM in the nucleus. Instead, we imply that, by reinforcing ATM attachment to Golgi-resident PI(4)P, ATM inhibitors end up titrating ATM away from the nucleus. We will clarify our explanation to avoid misunderstandings.
An increase of GFP-Tel1 foci upon OSH4 overexpression is described on Figure 7B. These are described as nuclear in the results, but no reference is made in the figure or legend as to how nucleus positions are addressed in these experiments. This should be clarified.
We systematically combine the tagging of a nucleoplasmic protein (mCherry-Pus1) with the detection of GFP-Tel1 foci, as to unambiguously assess the nuclear position of Tel1 foci. We will include this explanation and the corresponding mCherry-Pus1 channel to clarify this.
Also, WT controls and quantifications should be included in the experiments shown on Figure 7C.
These experiments are quantified from the moment we did them, although we did not include such quantifications in the present version for the sake of space. We will do so in the revised version.
Reviewer #1 (Significance (Required)):
While the conclusion of lipid metabolism responding to DSBs is not convincing, the observation that Tel1/ATM function is modulated by PI(4)P biding is significant and advances the understanding on the function and regulation of this key kinase in promoting genome integrity maintenance. This is an unanticipated result which is highly novel and has implications for the modulation of Tel1/ATM function through pharmacological manipulation of lipid metabolism. This finding would be of broad interest for scientists working on the response to DNA damage and the maintenance of genome integrity. This reviewer belongs to that group and has limited expertise to evaluate the lipid metabolism genetic manipulation in the manuscript.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The authors show that cytoplasmic PI4P have a regulatory role on ATM response to DNA double strand breaks. The process involves a balance between exchange of PI4P between Golgi and ER in exchange of esterified sterols. The study is of interest, however provides indirect evidences to support their conclusions.
Major comments : 1). Since the major conclusion relates to PI4P association with ATM in basal conditions to keep ATM outside nucleus and known presence of PI4P, ATM in other organelles of a cell, further experiments such as cell fractionation experimental that show golgi specific interaction would support the main conclusion.
In continuation of 1st comment, since PI4P in substrate of PI4 phosphoinositol kinases, is there a competition between PI4kinases and ATM for PI4P binding should be addressed through immunoprecipitation studies.
First of all, we need to specify here that PI4kinases will phosphorylated PI4 to create PI(4)P. Thus, PI(4)P is the product, and not the substrate, of PI4kinases. We therefore do not expect any competition between such kinases and ATM.
Second, we take good note of the reviewer’s concern that the pool of PI(4)P at the Golgi may be shared, and also that it would be important to reinforce the notion of the relative subcellular localization of ATM under different treatments. To this end, we will perform the following integrative experiment:
Immunoprecipitation of PI(4)P could theoretically be done using our specific antibody, yet the IP efficiency of a lipid cannot be verified by western blot. Further, there are PI(4)P pools elsewhere in the cell that would mess up with interpretations. We therefore dismiss the use of anti-PI(4)P as a tool to perform immunoprecipitations.
Instead, to explore the impact of PI(4)P levels on ATM both at the Golgi and within the nucleus, we will split our cultures in two to either immunoprecipitate specific cytoplasmic Trans-Golgi Network-associated proteins (we will test separately TGN46 and GOLPH3); or the nuclear ATM-interacting factor MRE11 from nuclei, then blot for co-immunoprecipitated ATM. The relative co-immunoprecipitated ATM is expected to vary under different treatments to which the cells will be exposed, namely:
- untreated
- zeocin, to trigger ATM need in the nucleus
- OSBP inhibition (+/- zeocin), to stabilize PI(4)P at the Golgi
- PIK93, an inhibitor of PI4 kinases that prevents PI(4)P synthesis
2). The authors claim that the ATM retention is the main function of PI4P in Golgi. The authors should rule out the possibility that the phenotype observed on DNA damage response is not due to non availability of PI4P substrate for PI4P kinases, that have recently been shown to participate in genome integrity maintenance.
We want to explain that we do not intend to say that PI(4)P main function at the Golgi is ATM retention, as PI(4)P is a molecule binding and modulating multiple proteins, as for example the aforementioned GOLPH3. We will first revise our text to correct it, in case we have conveyed this incorrect notion, as it stems from the reviewer’s comment.
Second, the reviewer evokes the notion that PI(4)P can be the substrate of a second phosphorylation, which could give rise to PI(3,4)P or to PI(4,5)P, which could still undergo remodeling into PI(3)P, for example. Recent work by Dr Michael Sheetz’s lab demonstrated that this set of phosphoinositides serves to drive the nucleation and activation of the ATR-Chk1 branch of the DNA Damage Response upon genotoxic stress, yet was completely inert with respect to the ATM-Chk2 branch (5). To rule out the possibility, as evoked by the reviewer, that the oleate-induced DDR phenomena we describe relate to these other events, we have now explored the response of the ATR-Chk1 branch when comparing the response of zeocin-treated cells that have been pre-loaded or not with oleate. We observe that the ATR-Chk1 branch is unaltered by oleate loading. Thus, we can now propose that the PI(4)P branch exclusively modulates the ATM-Chk2 axis.
3). Does Oleate treatment influences Rad53 protein levels in addition to its phosphorylation that affect DNA damage response may be addressed.
Exponential cultures from three different WT, three different ste∆ and three different yeh2∆ strains have now been taken and pre-loaded for 2 hours with 0.05% oleate, then total levels of Rad53 (without induction of DNA damage) assessed. We can now formally say that basal levels of Rad53 protein are not altered by this incubation. We will include this control in the revised manuscript.
4). Does Yeh2 deletion reduces LDS should be checked.
We frequently use yeh2∆ cells in our studies. In particular, we have recently published work characterizing the phenotype of this strain with respect to the formation of lipid droplets in the nucleus (6). We are currently exploiting those same sets of data to quantify the total number of LD in order to satisfy the reviewer’s concern.
5). Figure 4D representation should show % of phospho reduction of initial activation and a better western blot image should be shown that show equal loading of samples.
We are currently repeating these gels and blots for the sake of clarity, as requested.
6). In immunoprecipitation experiments, kindly include isotypee IgG controls as well to rule out non-specificity.
Of course, this important control will be included every time.
Minor points: 1). Figure S1F do not show oleate treatment as presented in results section.
We will revise the accurate naming.
2). A better gel for S4B should be presented with ponceau of the same gel.
We are currently repeating this gel and associated blot for the sake of clarity, as requested.
3). Nuclear PI4Ps has also been previously reported, an explanation to the specific interaction of ATM and PI4P in the Golgi should be addressed/discussed.
We take it that the reviewer is referring here to the recent work by Fáberová et al (7) in which PI(4)P and PI(4,5)P were described as very dynamic in the nucleus, and mostly related then to mRNA transcription, splicing and export. We will reinforce the connection of our phenomenon to the Golgi-associated pool of PI(4)P thanks to the co-immunoprecipitation experiments proposed above, and will timely contextualize these in light of the paper by Fáberová and co-workers in the revised version. Thank you for reminding us of this work.
Reviewer #2 (Significance (Required)):
The current work definitely adds a layer in our understanding to ATM regulation and cross-talk between different PIKK family of kinases. ATM localisation in extra nuclear regions of a cell has been described earlier with significant impact on cell physiology such as mitochondria etc., ATM retention at golgi and limiting nuclear ATM levels is significant advance at ATM activity regulation, while signifying non canonical function of PI4P.
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
Summary:
In this manuscript, the authors propose that ATM/Tel1 signaling is regulated in a spatiotemporal manner during genotoxic stress both in yeast and mammalian cells. They show that Lipid droplets accumulate in response to genotoxic stress. As a consequence, there is a decrease of exchange of PI4P from the Golgi to ER, thus dampening ATM/Tel1 signaling by sequestering this kinase into the Golgi. The authors combined findings in yeast and mammals showing that this mechanism is conserved throughout eukaryotes. For this purpose, they use a vast number of techniques that support their proposed model.
Major comments:
The conclusions were made based on evidence combining yeast genetics, immunofluorescence, DNA end resection analysis and pharmacological interventions. The hypothesis that ATM is kept away from the nucleus by physically interacting with PI4P at the Golgi, thus allowing processive repair is bold and contributes for a better understanding of the choreography of the DDR kinases during DSB repair. However, many of the experiments in yeast and mammals show only mild phenotypes and there is no evidence that this mode of ATM dampening impact cell viability in mammals.
We agree with the reviewer that the effects associated to the reported phenomenon are indeed mild. This is a fact. We would like to remind that the metabolism of sterols is finely controlled, and at many different levels, in a very complex manner. For example, sterol increases in the cell will immediately be compensated by reduced synthesis, while synthesis inhibition will immediately promote uptake from the medium, and/or release from stores (for example, see (8)). As a natural consequence, the window of manipulation and, more importantly, the strength of the phenotypes we can uncover are small.
Therefore, I have some comments and suggestions of experiments that I think could improve the quality of the manuscript. I believe that most of these new experiments does not require much time and resources.
- Does oleate treatment in RPE-1/Huh-7 cells induce loss of viability? An experiment showing loss of viability like MT-assay or decreased cell proliferation would reinforce the importance of the mechanism proposed.
This experiment was already included in the previous version, yet it may have escaped the attention of the reviewer. We show in Figure S2E that oleate treatment restricts viability in Huh-7 cells alone, and also worsens their tolerance to zeocin. Perhaps we should reconsider moving this result to the main figures so that it does not go unnoticed.
- In yeast there is evidence that a ste delta strain show sensitivity to zeocin/CPT, but there is no experiment showing the same effect on cells lacking Yeh2. Since both strains share similar phenotypes, it would be interesting to show that increased kinetics of Rad53 signaling leads to sensitivity to genotoxins.
We have now performed this experiment, we will include the matching information for yeh2∆ cells, which agrees with the predictions.
- The conclusion that ste delta cells exposed to zeocin leads to unproductive events due to defects in DNA-end resection could be reinforced by a decrease in Rad52 foci. It has been previously shown by the group of Dr. Marcus Smolka, that inhibition of DNA-end resection decreases Rad52 foci (https://doi.org/10.1083/jcb.201607031). Since the authors were able to monitor Rad52-YFP (Figure S1A), it shouldn't consume time and resources.
The reviewer is right that this experiment should not be time- or resources-consuming. We will evaluate the accumulation of Rad52 foci in response to the concerned genotoxin in ste∆ cells.
- Since the authors propose that there is a DNA repair defect due to inhibition of long-range DNA-end resection, it would be important to monitor gamma-H2A(X) signal either in yeast or mammals.
Taking into consideration the reviewer’s suggestion, we have now performed anti-yH2AX immunofluorescence of all the implied conditions (genotoxins +/- oleate pre-load) and will quantify them to answer the concern.
- How do the authors exclude the possibility that yeast mutants or oleate treatment in yeast/mammalian cells change membrane permeability allowing an increase in genotoxin concentration?
Although this is a very reasonable criticism, we want to remind the data we present in Figure S4A in which we use the naturally DSB-bearing rad3-102 cells for recombination analyses, showing that, in the absence of any genotoxin, the same phenotype also applies. Yet, we want to reinforce the notion that LD formation in response to DSB can also occur when the breaks are not chemically, but physically, induced. To this end, and also to match a related request by Reviewer 1, we will:
- a) exploit the naturally DSB-accumulating mutant rad3-102 (4) to evaluate whether it endogenously harbors more LD in comparison with the WT.
- b) we have recently created a tool in which gRNAs targeted to different subsets of transposons in the genome can drive Cas9 to create DSB in a dose-dependent manner ((9), under revision in Genetics). We will use this system to monitor the LD formation in response to Cas9-triggered cuts. In addition, on figure 5A, significant differences in GFP-Tel1 foci abundance between WT and steD or yeh2D cells are only observed after 210', way after the slight effect on Rad53 phosphorylation is observed. This is at odds with the conclusion that Tel1 association to STEs modulates DDR signaling.
We are afraid that we have not been clear enough in explaining the kinetics giving rise to our model. As indicated by the reviewer, our work shows, through kinetic studies, that the storage of sterols within LD occurs at later stages than the activation of the DDR by Tel1 and Rad53 phosphorylation. Tel1 foci decline is necessary for subsequent engagement of downstream DNA long-range resection. Since we propose that sterol storage within LD is a means to attenuate Tel1 engagement at DSBs, it is thus logical (and thus compatible with the data we show) that LD number increase occurs simultaneously with Tel1 foci decrease, at late stages of the reactionWe will include this explanation and graph in the revised version of the work.
- It would be interesting to investigate genetic interactions between ste delta (or yeh2delta) and yeast mutants with DNA-end resection problems (exo1delta; sae2delta). For instance, it has been shown that Sae2 antagonizes checkpoint signaling by competing with Rad9 to DSB sites (https://doi.org/10.1073/pnas.1816539115). Also, cells lacking Sae2 show an increase in Rad53 signaling due to increased Tel1 Signaling. Therefore, an epistatic effect between these two pathways would reinforce the hypothesis of the manuscript.
we will build the double mutant sae2∆ yeh2∆ and assess the potential epistatic behavior they may display with respect to some key phenotypes (Tel1 foci formation, Rad53 phosphorylation…).
- The authors showed that Tel1-GFP does not accumulate in the nucleus in cells lacking Sac1 (Figure 7C). Tel1 is important to cope with increased DSBs in the absence of Mec1, thus avoiding genomic instability. Cells lacking both Mec1 and Tel1 show a sick phenotype with an exponential increase in gross chromosomal rearrangements and sensitivity to genotoxins. Therefore, does deletion of Mec1 (and Sml1) in sac1 delta phenocopies a mec1tel1 delta? Alternatively, does pharmacological inhibition of ATR in the presence of the OSBP1 inhibitor causes loss of viability or chromosomal aberrations?
We will delete SAC1 in mec1∆ sml1∆ and compare the fitness, through growth drop assays, with respect to the mutant tel1∆ mec1∆ sml1∆.
We will expose cells either to OSBP1 inhibitor, ATR inhibitor, or both, and assess the phosphorylation of their downstream common effector H2AX. Additionally, we will assess the effect on cell growth of the combination of ATRi and OSBP1i using synergy matrices. We will determine if the combination of both drugs synergizes or not to impair cell proliferation and reduce cell viability.
- Finally, it seems strange to me that ATR/Mec1 signaling is not mentioned throughout the entire manuscript. Does PI4P pathway affect only ATM/Tel1? In Figure 2D, an antibody against phospho-CHK1 could be used to monitor ATR signaling. In line with that, I would like to see in the discussion how these new findings are in line with evidence from a 2019 paper showing that phophoinositides PIP2 and PIP3, but not PI4P are important for ATR signaling (DOI: 10.1038/s41467-017-01805-9). They showed that a nuclear pool of PIP2 increases upon DNA damage induction and rapidly accumulates at DNA lesions. This event is important for the recruitment of ATR. Since PI4P is substrate for PIP2 synthesis and there is a nuclear pool of PI4P and PIP2, I think it is important to discuss if the results presented here are in line with these previous findings.
The reviewer evokes recent work by Dr Michael Sheetz’s lab demonstrating that a different set of phosphoinositides serves to drive the nucleation and activation of the ATR-Chk1 branch of the DNA Damage Response upon genotoxic stress, yet was completely inert with respect to the ATM-Chk2 branch (5). We have now explored, also to satisfy a similar concerned raised by Reviewer 2, the response of the ATR-Chk1 branch when comparing the response of zeocin-treated cells that have been pre-loaded or not with oleate. We observe that the ATR-Chk1 branch is unaltered by oleate loading. Thus, we can now propose that the PI(4)P branch exclusively modulates the ATM-Chk2 axis.
We will of course give the needed credit to this work and contextualize our findings accordingly.
Minor comments:
- Line 124: The correct is Figure S1E, lower panel and not Figure S1F -Lines 127-128: Figure S2A does not show zeocin treatment
Both minor mistakes will be corrected.
Reviewer #3 (Significance (Required)):
Together, these new findings, if corroborated by others, might be important to open new lines of investigation in basic and translational research regarding human diseases as explored in the discussion section. I believe this paper will attract attention not only from the DDR field but also from other areas of research such as nutrient and lipid signaling both in yeast and mammals. I hope I was able to collaborate in this review, since my main expertise is in the area of DNA damage signaling using budding yeast as an organism model.
Reviewer #4 (Evidence, reproducibility and clarity (Required)):
This is a very interesting study where Sara et al. demonstrated a link between lipid metabolism with DNA repair response (DDR). In this study, they have proposed ATM as a novel PI4P-effector. The sterol deposition into lipid droplets impacts the Golgi PI4P level due to lipid exchange machinery facilitated by OSBP1, therefore regulating the cytosolic retention of ATM due to PI4P binding. Although how lipid droplets in the cytosol sense the DNA damage and control the initiation of DDR by regulating ATM is still unclear, this study linked lipid biology/PI signaling to DNA damage repair and showed the evolutionary conservation of PI signaling and DNA repair machinery from yeast to humans. The experiments are well designed, nicely controlled, with a high quality of data presentation. With some improvements, this work could be a very interesting study attracting a broad readership.
In their model, ATM is PI4P-bound and sequestered inside the cytosol under basal conditions. Upon genotoxic stress, activation of OSBP1 removes PI4P and free PI4P-bound ATM for nuclear translocation of DNA repair. This could also be interpreted as genotoxic stress-induced PIP-kinase activity, where PI4P is processed into PIP2 or PIP3, somehow redirecting ATM into the nucleus to initiate its activation for DDR. Those aspects should be discussed and improved.
Both Reviewers 2 and 3 have somehow evoked a similar concern. More precisely, the work by Dr Michael Sheetz’s lab demonstrating that a different set of phosphoinositides serves to drive the nucleation and activation of the ATR-Chk1 branch of the DNA Damage Response upon genotoxic stress, yet was completely inert with respect to the ATM-Chk2 branch (5). We have now explored, to satisfy all reviewers’ concerns, the response of the ATR-Chk1 branch when comparing the response of zeocin-treated cells that have been pre-loaded or not with oleate. We observe that the ATR-Chk1 branch is unaltered by oleate loading. Thus, we can now propose that the PI(4)P branch exclusively modulates the ATM-Chk2 axis.
Additionally, we will of course give the needed credit to this work and contextualize our findings accordingly.
Upon stress, there is nuclear activation of p53-phosphoinositide (PI) signalosomes and PIP-kinases. Also, there is a significant PIP2 pool inside the nucleus with an involvement in DNA damage repair. Those papers and their relevance to the current study need to be discussed. If ATM is a novel PI4P-effector, there is also nuclear PI4P formation or nuclear PI4P accumulation upon stresses based on recent studies; how the ATM interacts with PIPn in the nucleus upon translocation? A know ATM substrate p53 is PIP2/PIP3 bound in the nucleus based on recent studies. Will ATM prefer to interact with other PIPn-bound proteins in the nucleus or PIPn regulate their interaction needs to be discussed.
These additional notions are in line with the previous paragraph presented by the reviewer, and our answers too. We will provide a constructive overview of all these ideas in the revised version of the manuscript.
Major points:
- The PI4P-ATM complex is supported only by PLA and PIP strips. Need more robust biochemical characterization of the interaction: co-IP, lipid binding, and/or in vitro constitution.
We agree with the need to perform assays in which PI(4)P is embedded in a bilayer, as to confidently assess whether Tel1 can bind it in that context. We have now performed a pilot experiment in which we have confronted purified FLAG-Tel1 to liposomes harboring PI(4)P. Western blot analysis using anti-FLAG antibody shows the encouraging result that FLAG-Tel1 can be found there. As a control, we have performed the same process but in the absence of any liposomes. We observe that a residual fraction of FLAG-Tel1 can nevertheless be found in this control, most probably because the buffer used during the liposome assay makes part of FLAG-Tel1 precipitate.To avoid this type of background and to increase our trust in the results, we propose to perform the liposome assay but on a discontinuous density gradient, so that liposomes will be retrieved in the top layer (and bound FLAG-Tel1 with them, if that is the case), while any precipitated FLAG-Tel1 will be in the bottom fraction (liposome floatation assay). As a further control, we will include the same liposomes but lacking PI(4)P. We expect to be successful in the floatation assays. If we are not, we will repeat the experiment presented above to be confident that the observed increase is reproducible.
- The use of drug inhibitors only in the final figure is problematic. KD or KO experiments should be performed to confirm that ATM and the exchanger are the relevant targets.
We have now used siRNAs against the exchanger protein, OSBP1, with a very high silencing rate success. We have next monitored the activation status of the chromatin-associated ATM target KAP1, in order to monitor the predicted decrease of ATM activity specifically inside the nucleus. Our results confirm the role of OSBP1, by KD experiments as requested by the reviewer, in attenuating ATM nuclear participation.
- Poor quality of some WBs (e.g Fig. S1F).
We have now repeated the Western Blot to detect Rad53-P in response to 20 mM HU in WT versus ste∆cells.
- Lack of statistical analyses for some data (e.g. Fig. 1B-E)
We had already included, in the previous version, the complete statistical analyses corresponding to Figures 1B to E and evoked here by the reviewer. They were indeed included in Figure S1C, and our brief reference to them in the text may have escaped her/his attention. We will make a clear reference to this in the revised version.
Additional clarification points:
Figure 1: No representative images were shown for quantifications in Figure 1C, D, E.
If the reviewer / editor estimates it pertinent, we can of course include them. Yet, they will be very redundant with the images displayed in Figure 1A.
Line 121: Should be Figure S1E, upper panel. Line 124: Should be Figure S1E, lower panel. Figure 2D-E, please show the quantification of the ratio of pCHK2/CHK2 with an N=3
We will correct / include the requested changes.
Figure S2B: needs quantification of NileRed staining to conclude induction in LD formation
We will quantify the LD as requested.
Figure 3C, to show the selectivity of ATM-binding toward PI4P, PLA of ATM with other PIPn species should be assessed, such as PI3P, PI4,5P2, and PI3,4,5P3.
We have provided an overview of the binding preferences of ATM with respect to the full battery of phosphoinositides in the strip-binding assay shown in Figures S5C and 6B. Other than that, we are afraid that PLA studies as the ones we develop in the current manuscript for PI(4)P are not feasible, since no reliable antibodies exist for most of the phosphoinositide species evoked by the reviewer.
Figure S6A, PI4P level could be assessed by IF staining using PI4P antibody besides using PI4P sensor.
We will use our PI(4)P antibody to monitor by immunofluorescence the behavior of this molecule in response to either MMS or zeocin, as suggested.
References
- Cheung HC, San Lucas FA, Hicks S, Chang K, Bertuch AA, Ribes-Zamora A. An S/T-Q cluster domain census unveils new putative targets under Tel1/Mec1 control. BMC Genomics. 2012;
- Bensimon A, Schmidt A, Ziv Y, Elkon R, Wang SY, Chen DJ, et al. ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage. Sci Signal. 2010;
- BastosdeOliveira FM, Kim D, Cussiol JR, Das J, Jeong MC, Doerfler L, et al. Phosphoproteomics Reveals Distinct Modes of Mec1/ATR Signaling during DNA Replication. Mol Cell. 2015;
- Moriel-Carretero M, Aguilera A. A Postincision-Deficient TFIIH Causes Replication Fork Breakage and Uncovers Alternative Rad51- or Pol32-Mediated Restart Mechanisms. Mol Cell. 2010;37(5):690–701.
- Wang YH, Hariharan A, Bastianello G, Toyama Y, Shivashankar G V., Foiani M, et al. DNA damage causes rapid accumulation of phosphoinositides for ATR signaling. Nat Commun. 2017;
- Kumanski S, Forey R, Cazevieille C, Moriel-Carretero M. Nuclear Lipid Droplet Birth during Replicative Stress. Cells. 2022;11(1390).
- Fáberová V, Kalasová I, Krausová A, Hozák P. Super-Resolution Localisation of Nuclear PI(4)P and Identification of Its Interacting Proteome. Cells. 2020;9(5):1–17.
- Luo J, Yang H, Song BL. Mechanisms and regulation of cholesterol homeostasis. Nat Rev Mol Cell Biol [Internet]. 2020;21(4):225–45. Available from: http://dx.doi.org/10.1038/s41580-019-0190-7
- Coiffard J, Santt O, Kumanski S, Pardo B, Moriel-Carretero M. A CRISPR-Cas9-based system for the dose-dependent study of 4 DNA double strand breaks sensing and repair 5 6. bioRxiv [Internet]. 2021;1–37. Available from: https://doi.org/10.1101/2021.10.21.465387.
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Referee #4
Evidence, reproducibility and clarity
This is a very interesting study where Sara et al. demonstrated a link between lipid metabolism with DNA repair response (DDR). In this study, they have proposed ATM as a novel PI4P-effector. The sterol deposition into lipid droplets impacts the Golgi PI4P level due to lipid exchange machinery facilitated by OSBP1, therefore regulating the cytosolic retention of ATM due to PI4P binding. Although how lipid droplets in the cytosol sense the DNA damage and control the initiation of DDR by regulating ATM is still unclear, this study linked lipid biology/PI signaling to DNA damage repair and showed the evolutionary conservation of PI …
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Referee #4
Evidence, reproducibility and clarity
This is a very interesting study where Sara et al. demonstrated a link between lipid metabolism with DNA repair response (DDR). In this study, they have proposed ATM as a novel PI4P-effector. The sterol deposition into lipid droplets impacts the Golgi PI4P level due to lipid exchange machinery facilitated by OSBP1, therefore regulating the cytosolic retention of ATM due to PI4P binding. Although how lipid droplets in the cytosol sense the DNA damage and control the initiation of DDR by regulating ATM is still unclear, this study linked lipid biology/PI signaling to DNA damage repair and showed the evolutionary conservation of PI signaling and DNA repair machinery from yeast to humans. The experiments are well designed, nicely controlled, with a high quality of data presentation. With some improvements, this work could be a very interesting study attracting a broad readership.
In their model, ATM is PI4P-bound and sequestered inside the cytosol under basal conditions. Upon genotoxic stress, activation of OSBP1 removes PI4P and free PI4P-bound ATM for nuclear translocation of DNA repair. This could also be interpreted as genotoxic stress-induced PIP-kinase activity, where PI4P is processed into PIP2 or PIP3, somehow redirecting ATM into the nucleus to initiate its activation for DDR. Those aspects should be discussed and improved.
Upon stress, there is nuclear activation of p53-phosphoinositide (PI) signalosomes and PIP-kinases. Also, there is a significant PIP2 pool inside the nucleus with an involvement in DNA damage repair. Those papers and their relevance to the current study need to be discussed. If ATM is a novel PI4P-effector, there is also nuclear PI4P formation or nuclear PI4P accumulation upon stresses based on recent studies; how the ATM interacts with PIPn in the nucleus upon translocation? A know ATM substrate p53 is PIP2/PIP3 bound in the nucleus based on recent studies. Will ATM prefer to interact with other PIPn-bound proteins in the nucleus or PIPn regulate their interaction needs to be discussed.
Major points:
- The PI4P-ATM complex is supported only by PLA and PIP strips. Need more robust biochemical characterization of the interaction: co-IP, lipid binding, and/or in vitro constitution.
- The use of drug inhibitors only in the final figure is problematic. KD or KO experiments should be performed to confirm that ATM and the exchanger are the relevant targets.
- Poor quality of some WBs (e.g Fig. S1F).
- Lack of statistical analyses for some data (e.g. Fig. 1B-E)
Additional clarification points:
Figure 1: No representative images were shown for quantifications in Figure 1C, D, E.
Line 121: Should be Figure S1E, upper panel.
Line 124: Should be Figure S1E, lower panel.
Figure 2D-E, please show the quantification of the ratio of pCHK2/CHK2 with an N=3
Figure S2B: needs quantification of NileRed staining to conclude induction in LD formation.
Figure 3C, to show the selectivity of ATM-binding toward PI4P, PLA of ATM with other PIPn species should be assessed, such as PI3P, PI4,5P2, and PI3,4,5P3.
Figure S6A, PI4P level could be assessed by IF staining using PI4P antibody besides using PI4P sensor.
Significance
This is a very interesting study where Sara et al. demonstrated a link between lipid metabolism with DNA repair response (DDR). In this study, they have proposed ATM as a novel PI4P-effector. The sterol deposition into lipid droplets impacts the Golgi PI4P level due to lipid exchange machinery facilitated by OSBP1, therefore regulating the cytosolic retention of ATM due to PI4P binding. Although how lipid droplets in the cytosol sense the DNA damage and control the initiation of DDR by regulating ATM is still unclear, this study linked lipid biology/PI signaling to DNA damage repair and showed the evolutionary conservation of PI signaling and DNA repair machinery from yeast to humans. The experiments are well designed, nicely controlled, with a high quality of data presentation. With some improvements, this work could be a very interesting study attracting a broad readership.
In their model, ATM is PI4P-bound and sequestered inside the cytosol under basal conditions. Upon genotoxic stress, activation of OSBP1 removes PI4P and free PI4P-bound ATM for nuclear translocation of DNA repair. This could also be interpreted as genotoxic stress-induced PIP-kinase activity, where PI4P is processed into PIP2 or PIP3, somehow redirecting ATM into the nucleus to initiate its activation for DDR. Those aspects should be discussed and improved.
Upon stress, there is nuclear activation of p53-phosphoinositide (PI) signalosomes and PIP-kinases. Also, there is a significant PIP2 pool inside the nucleus with an involvement in DNA damage repair. Those papers and their relevance to the current study need to be discussed. If ATM is a novel PI4P-effector, there is also nuclear PI4P formation or nuclear PI4P accumulation upon stresses based on recent studies; how the ATM interacts with PIPn in the nucleus upon translocation? A know ATM substrate p53 is PIP2/PIP3 bound in the nucleus based on recent studies. Will ATM prefer to interact with other PIPn-bound proteins in the nucleus or PIPn regulate their interaction needs to be discussed.
Major points:
- The PI4P-ATM complex is supported only by PLA and PIP strips. Need more robust biochemical characterization of the interaction: co-IP, lipid binding, and/or in vitro constitution.
- The use of drug inhibitors only in the final figure is problematic. KD or KO experiments should be performed to confirm that ATM and the exchanger are the relevant targets.
- Poor quality of some WBs (e.g Fig. S1F).
- Lack of statistical analyses for some data (e.g. Fig. 1B-E)
Additional clarification points:
Figure 1: No representative images were shown for quantifications in Figure 1C, D, E.
Line 121: Should be Figure S1E, upper panel.
Line 124: Should be Figure S1E, lower panel.
Figure 2D-E, please show the quantification of the ratio of pCHK2/CHK2 with an N=3
Figure S2B: needs quantification of NileRed staining to conclude induction in LD formation.
Figure 3C, to show the selectivity of ATM-binding toward PI4P, PLA of ATM with other PIPn species should be assessed, such as PI3P, PI4,5P2, and PI3,4,5P3.
Figure S6A, PI4P level could be assessed by IF staining using PI4P antibody besides using PI4P sensor.
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Referee #3
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors propose that ATM/Tel1 signaling is regulated in a spatiotemporal manner during genotoxic stress both in yeast and mammalian cells. They show that Lipid droplets accumulate in response to genotoxic stress. As a consequence, there is a decrease of exchange of PI4P from the Golgi to ER, thus dampening ATM/Tel1 signaling by sequestering this kinase into the Golgi. The authors combined findings in yeast and mammals showing that this mechanism is conserved throughout eukaryotes. For this purpose, they use a vast number of techniques that support their proposed model.
Major comments:
The conclusions …
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Referee #3
Evidence, reproducibility and clarity
Summary:
In this manuscript, the authors propose that ATM/Tel1 signaling is regulated in a spatiotemporal manner during genotoxic stress both in yeast and mammalian cells. They show that Lipid droplets accumulate in response to genotoxic stress. As a consequence, there is a decrease of exchange of PI4P from the Golgi to ER, thus dampening ATM/Tel1 signaling by sequestering this kinase into the Golgi. The authors combined findings in yeast and mammals showing that this mechanism is conserved throughout eukaryotes. For this purpose, they use a vast number of techniques that support their proposed model.
Major comments:
The conclusions were made based on evidence combining yeast genetics, immunofluorescence, DNA end resection analysis and pharmacological interventions. The hypothesis that ATM is kept away from the nucleus by physically interacting with PI4P at the Golgi, thus allowing processive repair is bold and contributes for a better understanding of the choreography of the DDR kinases during DSB repair. However, many of the experiments in yeast and mammals show only mild phenotypes and there is no evidence that this mode of ATM dampening impact cell viability in mammals. Therefore, I have some comments and suggestions of experiments that I think could improve the quality of the manuscript. I believe that most of these new experiments does not require much time and resources.
- Does oleate treatment in RPE-1/Huh-7 cells induce loss of viability? An experiment showing loss of viability like MT-assay or decreased cell proliferation would reinforce the importance of the mechanism proposed.
- In yeast there is evidence that a ste delta strain show sensitivity to zeocin/CPT, but there is no experiment showing the same effect on cells lacking Yeh2. Since both strains share similar phenotypes, it would be interesting to show that increased kinetics of Rad53 signaling leads to sensitivity to genotoxins.
- The conclusion that ste delta cells exposed to zeocin leads to unproductive events due to defects in DNA-end resection could be reinforced by a decrease in Rad52 foci. It has been previously shown by the group of Dr. Marcus Smolka, that inhibition of DNA-end resection decreases Rad52 foci (https://doi.org/10.1083/jcb.201607031). Since the authors were able to monitor Rad52-YFP (Figure S1A), it shouldn't consume time and resources.
- Since the authors propose that there is a DNA repair defect due to inhibition of long-range DNA-end resection, it would be important to monitor gamma-H2A(X) signal either in yeast or mammals.
- How do the authors exclude the possibility that yeast mutants or oleate treatment in yeast/mammalian cells change membrane permeability allowing an increase in genotoxin concentration?
- It would be interesting to investigate genetic interactions between ste delta (or yeh2delta) and yeast mutants with DNA-end resection problems (exo1delta; sae2delta). For instance, it has been shown that Sae2 antagonizes checkpoint signaling by competing with Rad9 to DSB sites (https://doi.org/10.1073/pnas.1816539115). Also, cells lacking Sae2 show an increase in Rad53 signaling due to increased Tel1 Signaling. Therefore, an epistatic effect between these two pathways would reinforce the hypothesis of the manuscript.
- The authors showed that Tel1-GFP does not accumulate in the nucleus in cells lacking Sac1 (Figure 7C). Tel1 is important to cope with increased DSBs in the absence of Mec1, thus avoiding genomic instability. Cells lacking both Mec1 and Tel1 show a sick phenotype with an exponential increase in gross chromosomal rearrangements and sensitivity to genotoxins. Therefore, does deletion of Mec1 (and Sml1) in sac1 delta phenocopies a mec1tel1 delta? Alternatively, does pharmacological inhibition of ATR in the presence of the OSBP1 inhibitor causes loss of viability or chromosomal aberrations?
- Finally, it seems strange to me that ATR/Mec1 signaling is not mentioned throughout the entire manuscript. Does PI4P pathway affect only ATM/Tel1? In Figure 2D, an antibody against phospho-CHK1 could be used to monitor ATR signaling. In line with that, I would like to see in the discussion how these new findings are in line with evidence from a 2019 paper showing that phophoinositides PIP2 and PIP3, but not PI4P are important for ATR signaling (DOI: 10.1038/s41467-017-01805-9). They showed that a nuclear pool of PIP2 increases upon DNA damage induction and rapidly accumulates at DNA lesions. This event is important for the recruitment of ATR. Since PI4P is substrate for PIP2 synthesis and there is a nuclear pool of PI4P and PIP2, I think it is important to discuss if the results presented here are in line with these previous findings.
Minor comments:
- Line 124: The correct is Figure S1E, lower panel and not Figure S1F
- Lines 127-128: Figure S2A does not show zeocin treatment
Significance
Together, these new findings, if corroborated by others, might be important to open new lines of investigation in basic and translational research regarding human diseases as explored in the discussion section. I believe this paper will attract attention not only from the DDR field but also from other areas of research such as nutrient and lipid signaling both in yeast and mammals. I hope I was able to collaborate in this review, since my main expertise is in the area of DNA damage signaling using budding yeast as an organism model.
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Referee #2
Evidence, reproducibility and clarity
The authors show that cytoplasmic PI4P have a regulatory role on ATM response to DNA double strand breaks. The process involves a balance between exchange of PI4P between Golgi and ER in exchange of esterified sterols. The study is of interest, however provides indirect evidences to support their conclusions.
Major comments:
1). Since the major conclusion relates to PI4P association with ATM in basal conditions to keep ATM outside nucleus and known presence of PI4P, ATM in other organelles of a cell, further experiments such as cell fractionation experimental that show golgi specific interaction would support the main conclusion.
2).…
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Referee #2
Evidence, reproducibility and clarity
The authors show that cytoplasmic PI4P have a regulatory role on ATM response to DNA double strand breaks. The process involves a balance between exchange of PI4P between Golgi and ER in exchange of esterified sterols. The study is of interest, however provides indirect evidences to support their conclusions.
Major comments:
1). Since the major conclusion relates to PI4P association with ATM in basal conditions to keep ATM outside nucleus and known presence of PI4P, ATM in other organelles of a cell, further experiments such as cell fractionation experimental that show golgi specific interaction would support the main conclusion.
2). In continuation of 1st comment, since PI4P in substrate of PI4 phosphoinositol kinases, is there a competition between PI4kinases and ATM for PI4P binding should be addressed through immunoprecipitation studies.
3). The authors claim that the ATM retention is the main function of PI4P in Golgi. The authors should rule out the possibility that the phenotype observed on DNA damage response is not due to non availability of PI4P substrate for PI4P kinases, that have recently been shown to participate in genome integrity maintenance.
4). Does Oleate treatment influences Rad53 protein levels in addition to its phosphorylation that affect DNA damage response may be addressed.
5). Does Yeh2 deletion reduces LDS should be checked.
6). Figure 4D representation should show % of phospho reduction of initial activation and a bettier western blot image should be shown that show equal loading of samples.
7). In ammunoprecipitation experiments, kindly include isotypee IgG controls as well to rule out non-specificity.
Minor points:
1). Figure S1F do not show oleate treatment as presented in results section.
2). A better gel for S4B should be presented with ponceau of the same gel.
3). Nuclear PI4Ps has also been previously reported, an explanation to the specific interaction of ATM and PI4P in the Golgi should be addressed/discussed.
Significance
The current work definitely adds a layer in our understanding to ATM regulation and cross-talk between different PIKK family of kinases. ATM localisation in extra nuclear regions of a cell has been described earlier with significant impact on cell physiology such as mitochondria etc., ATM retention at golgi and limiting nuclear ATM levels is significant advance at ATM activity regulation, while signifying non canonical function of PI4P.
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Referee #1
Evidence, reproducibility and clarity
Ovejero et al. report an increase in lipid droplet (LD) abundance after long (from 120' on) exposure of budding yeast cells to DNA damaging agents zeocin and camptothecin (CPT). Next, they analyze DNA damage signaling in yeast mutants that impair triacylglycerol (TAGs) or sterol (STEs) esterification. They observe a slight anticipation in Rad53/CHK2 phosphorylation (indicative of DDR signaling) in yeast stem mutants, as well as in yeast cells or human cells lines pre-treated with oleate upon zeocin treatment. Yeast stem mutants are sensitive to zeocin and captothecin, but only confer sensitivity to hydroxyurea upon combination with …
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Referee #1
Evidence, reproducibility and clarity
Ovejero et al. report an increase in lipid droplet (LD) abundance after long (from 120' on) exposure of budding yeast cells to DNA damaging agents zeocin and camptothecin (CPT). Next, they analyze DNA damage signaling in yeast mutants that impair triacylglycerol (TAGs) or sterol (STEs) esterification. They observe a slight anticipation in Rad53/CHK2 phosphorylation (indicative of DDR signaling) in yeast stem mutants, as well as in yeast cells or human cells lines pre-treated with oleate upon zeocin treatment. Yeast stem mutants are sensitive to zeocin and captothecin, but only confer sensitivity to hydroxyurea upon combination with tagD mutations. Authors relate these phenotypes to a somewhat decreases DSB resection in yeh2D mutants (expected to have reduced steryl esters pools) and RPA-foci in steD yeast cells. Next, a reduction in single strand annealing recombination repair events upon zeocin treatment is reported using a genetic reporter in steD mutants and oleate-treated cells. From these data they conclude that inability to process sterols in response to DSBs leads to an exacerbated DDR and prevents DNA repair. Next, it is shown that Flag-tagged Tel1 distinctly interacts with mono-phosphate phosphoinositides, including PI(4)P. An interaction in vivo is also inferred through Proximity Ligation Assays (PLA) using anti-PI(4)P and anti-ATM antibodies in human cell lines, which was moderately downregulated upon treatment with MMS or zeocin. Over-expression of the Osh4/OSBP1 transporter, which consumes PI(4)P, increased the number of Tel1 (nuclear) foci upon zeocin treatment. Conversely Sac1 ablation, in which accumulation of PI(4)P is expected, abrogated nuclear Tel1 foci formation and reduced telomere length (a phenotype related to lack of Tel1 function). From these results authors conclude that Tel1 availability in the nucleus is influenced by PI(4)P availability. Lastly, treatment with an OSBP1 inhibitor led to a cell line and damaging agent -variable reduction of ATM phosphorylation and a mostly non-significant reduction of DNA resection, measured by native BrdU detection, in response to CPT treatment. Overall, authors conclude that i) biding of Tel1/ATM to PI(4)P modulates its functional availability in the nucleus, and that ii) DNA damage elicits the esterification and storage of sterols toward LDs, which contributes to tritate Tel1/ATM away from the nucleus dampening the DDR and affecting long-range resection.
Minor comments:
Figure S1D and E, experiments should be carried out to include time points in which LD accumulation and cell cycle arrest are observed upon zeocin treatment (i.e., up to 210' as in Figure 1A)
How do authors explain increased single strand annealing recombination frequencies in steD and oleate-treated wild type cells (Figure 4A). Should it not be expected that increased STEs also impair recombination induced by endogenous damage?
Data presented in figure 4B and 4C are not fully convincing. Performing time course experiments might help concluding if the differences observed represent a relevant defect in DSB processing.
Is Figure 5B referring to Flag-tagged Tel1 or GFP-tagged Tel1 as stated in the figure legend?
Treatment with the ATM inhibitor AZD0156 increased PI(4)P-ATM PLA signals. From these authors conclude that "association of ATM and PI(4)P inversely correlated with the need for ATM within the nucleus. Do they imply that treatment with ATM-inhibitors reduces the requirement for ATM function in the nucleus? The interpretation of this result should be further elaborated to sustain this conclusion.
An increase of GFP-Tel1 foci upon OSH4 overexpression is described on Figure 7B. These are described as nuclear in the results, but no reference is made in the figure or legend as to how nucleus positions are addressed in these experiments. This should be clarified. Also, WT controls and quantifications should be included in the experiments shown on Figure 7C.
Major comments:
While the conclusion that Tel1/ATM binds PI(4)P and this interaction modulates Tel1/ATM functional availability at the nucleus is convincing, the conclusion that DSBs elicit a change in the metabolism of this lipid to "control" Tel1/ATM function is not demonstrated.
The notion that sterol processing occurs in response to DSBs is not sufficiently supported by the data presented, as the increase in LD numbers is observed much after activation of the DDR (Rad53 phosphorylation) in Zeozin-treated yeast cells. In addition, evidence is not provided on the mechanisms by which PI(4)P metabolism would be controlled, which would be expected to be DDR-independent as they are placed upstream of this signaling pathway in the author's model. The damaging agents used have been suggested to alter the redox metabolism and even lipid peroxidation (Kitanovic 2009, Mizumoto 1993, Krol 2015, Todorova 2015, Ren 2019, Singh 2014). Hence it is possible that PI(4)P changes are not due to DSBs, but an indirect though relevant effect.
In absence of direct evidence supporting an active regulation of PI(4)P dynamics in response to DNA breaks, this conclusion remains speculative and this should be noted in the manuscript.
Authors conclude that LD is specific to DSB induction. This seems an overstatement as they just reported LD increases in response to two agents that also induce other kinds of DNA damage. To also strengthen the link between DSBs and PI(4)P modulation of Tel1 function, authors should analyze LD numbers, Rad53 phosphorylation and Tel1 nuclear re-localization in response to HO-induced DNA breaks (e.g., using the system employed in Figure 3C)
In addition, on figure 5A, significant differences in GFP-Tel1 foci abundance between WT and steD or yeh2D cells are only observed after 210', way after the slight effect on Rad53 phosphorylation is observed. This is at odds with the conclusion that Tel1 association to STEs modulates DDR signaling.
Minor comments:
Figure S1D and E, experiments should be carried out to include time points in which LD accumulation and cell cycle arrest are observed upon zeocin treatment (i.e., up to 210' as in Figure 1A)
How do authors explain increased single strand annealing recombination frequencies in steD and oleate-treated wild type cells (Figure 4A). Should it not be expected that increased STEs also impair recombination induced by endogenous damage?
Data presented in figure 4B and 4C are not fully convincing. Performing time course experiments might help concluding if the differences observed represent a relevant defect in DSB processing.
Is Figure 5B referring to Flag-tagged Tel1 or GFP-tagged Tel1 as stated in the figure legend?
Treatment with the ATM inhibitor AZD0156 increased PI(4)P-ATM PLA signals. From these authors conclude that "association of ATM and PI(4)P inversely correlated with the need for ATM within the nucleus. Do they imply that treatment with ATM-inhibitors reduces the requirement for ATM function in the nucleus? The interpretation of this result should be further elaborated to sustain this conclusion.
An increase of GFP-Tel1 foci upon OSH4 overexpression is described on Figure 7B. These are described as nuclear in the results, but no reference is made in the figure or legend as to how nucleus positions are addressed in these experiments. This should be clarified. Also, WT controls and quantifications should be included in the experiments shown on Figure 7C.
Significance
While the conclusion of lipid metabolism responding to DSBs is not convincing, the observation that Tel1/ATM function is modulated by PI(4)P biding is significant and advances the understanding on the function and regulation of this key kinase in promoting genome integrity maintenance. This is an unanticipated result which is highly novel and has implications for the modulation of Tel1/ATM function through pharmacological manipulation of lipid metabolism. This finding would be of broad interest for scientists working on the response to DNA damage and the maintenance of genome integrity. This reviewer belongs to that group and has limited expertise to evaluate the lipid metabolism genetic manipulation in the manuscript.
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