KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Evaluated articles (Review Commons)
- Multiple peer reviews (damianpattinson)
Abstract
The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication.
Article activity feed
-
-
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on April 25, 2020, follows.
Summary
The manuscript by Fournier et al. highlights the importance of acetylation in the ChAM domain of PALB2 in regulating nucleosome binding and DNA repair. The text is well-written text and the experiments are well-designed. We read the manuscript, the reviews from Review Commons, as well as the rebuttal and plans for a revision. We believe the revision and the proposed added experiments will be needed to cement the conclusions.
Of the 5 experiments, #1 and #2 are critical, and we believe #4 and #5 are also important as BRCA1 is a key factor for PALB2 (#5) and the effect on HR (#4) should be documented experimentally. We do not consider experiment #3 (PALB2 foci) as critical. We encourage the authors to …
###This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on April 25, 2020, follows.
Summary
The manuscript by Fournier et al. highlights the importance of acetylation in the ChAM domain of PALB2 in regulating nucleosome binding and DNA repair. The text is well-written text and the experiments are well-designed. We read the manuscript, the reviews from Review Commons, as well as the rebuttal and plans for a revision. We believe the revision and the proposed added experiments will be needed to cement the conclusions.
Of the 5 experiments, #1 and #2 are critical, and we believe #4 and #5 are also important as BRCA1 is a key factor for PALB2 (#5) and the effect on HR (#4) should be documented experimentally. We do not consider experiment #3 (PALB2 foci) as critical. We encourage the authors to plan and execute this revision as they outlined with the above exception of experiment #3. You may want to consider combining KAT2A/B depletion and/or using KDAC inhibitors in the experiments with the 7R and 7K mutants, but we leave that suggestion to them.
-
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 thank all the Reviewers for taking the time to evaluate our manuscript and providing us with constructive feedback. We are pleased to hear that all Reviewers appreciate the importance and significance of our study, commenting that our conclusions are ‘convincing, are supported by the presented experimental results’ and that our study ‘will yield novel insights into the regulation and function of PALB2 in DNA repair’.
Please refer to our point-by-point response to the specific points raised, in which we highlight a couple of key experiments to be conducted to refine our study in bold. We are grateful for all the reviewers’ remarks and suggestions, which …
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 thank all the Reviewers for taking the time to evaluate our manuscript and providing us with constructive feedback. We are pleased to hear that all Reviewers appreciate the importance and significance of our study, commenting that our conclusions are ‘convincing, are supported by the presented experimental results’ and that our study ‘will yield novel insights into the regulation and function of PALB2 in DNA repair’.
Please refer to our point-by-point response to the specific points raised, in which we highlight a couple of key experiments to be conducted to refine our study in bold. We are grateful for all the reviewers’ remarks and suggestions, which will certainly lead to a substantial improvement in our manuscript.
Point-by-point response:
__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __
Fournier et al. detect acetylation within the chromatin association motif (ChAM) of PALB2 and demonstrate that KAT2 can acetylate these 7 lysine residues within this region. They then generate K to R mutations (7R) or K to Q mutations (7Q) at these sites and perform assays of fluorescence recovery after photobleaching (FRAP) to measure mobility as a measure of chromatin association, RAD51 foci, PALB2 recruitment at sites of laser-induced DNA damage, and sensitivity to olaparib. They find increased mobility of the 7Q mutant of PALB2 but not 7R in the absence of exogenous DNA damage, as well as defects in DNA damage-induced RAD51 foci and resistance to olaparib. On this basis, the authors conclude that acetylation is required for the association of PALB2 with undamaged chromatin and that deacetylation permits mobilization and association with BRCA1 to enable proper DNA repair. While the manuscript is generally well-written, many of the systems are rather elegant, and this study may yield novel insights into the regulation and function of PALB2 in DNA repair, there are some missing experiments to be added and important contradictions that should be resolved in order to fully establish the new model the authors propose.
**Major comments:**
1.There are some concerns about the interpretation of experiments with the 7R and 7Q mutants of PALB2. For example, in the description of results in Fig. S2C, the authors state "K to R substitutions maintain the charge yet are unable to accept acetylation and hence mimic constitutively non-acetyl lysine". However, in Fig. 4B the association of the 7R mutant with chromatin is similar to WT and in Fig. 7D,E the relative immobility of the 7R mutant is very similar to WT PALB2. Thus, the conclusion that acetylation is required for PALB2 association with damaged undamaged chromatin and for release of PALB2 upon DNA damage does not appear justified. Perhaps the authors need to better consider whether the 7R mutant mimics acetylation because of its charge. Even so, the mutant then maintains the charge normally associated with acetylated PALB2, calling into question whether deacetylation indeed "releases PALB2 from undamaged chromatin".
We agree with the Reviewer’s point that there is no or little difference between WT and the 7R mutant in regard to their enrichment on non-damaged chromatin, as detected by fractionation (Fig 4B), or their mobility, as detected by FRAP (Fig 4D and E). Note that Fig. 7D is our model and Fig. 7E does not exist. As the Reviewer suggests, it is possible that, in contrast to the 7Q mutant, which is defective in both nucleosome and DNA binding (Fig. 2E and F), the 7R mutant may maintain its electrostatic interaction with DNA, while lacking its acetylation-mediated nucleosome interaction, masking the impact of substitutions. This assumption is in line with our model in which ChAM DNA binding assists HR repair, which is supported by the 7R mutant but not by the 7Q mutant. To better dissect the question raised by the Reviewer, we will conduct biochemical analyses of the ChAM 7R mutant, testing its direct interaction with nucleosomes and DNA; the results will be included in the revised manuscript (Experiment 1).
It is also worth noting that full-length PALB2 is enriched at a fraction of H3K36me3-marked exons (which comprise only 1-1.5 % of the whole genome), as shown in our previous genome-wide ChIP-seq analysis (Bleuyard et al., 2017, PNAS). Hence, it is also possible that bulk fractionation or FRAP analyses might not be sensitive enough to highlight the impact of the 7R mutation. Conversely, we foresee that the ChIP-qPCR method, detecting PALB2 association at defined genic regions as shown in Fig S5, will be more appropriate__. Thus, in the revised manuscript, we will expand our ChIP-qPCR analyses to further validate our proposed model (Experiment 2).__
2.Related to questions of interpreting results utilizing the 7R and 7Q mutants of PALB2, in Fig. 7B,C the 7R mutant but not 7Q supports RAD51 foci and resistance to olaparib similar to WT PALB2. The authors then state in the Discussion that "our work also suggests that caution should be exercised in the use of K to Q substitutions for functional studies of lysine acetylation". Thus, which mutant is giving the correct and reliable results?
We apologise for the miscommunication if this point was unclear. Using biochemical approaches, we established that ChAM acetylation, but not K to Q substitution, facilitates its association with nucleosomes (please compare Fig 2E and Fig 3B). This observation clearly demonstrates that K to Q substitution does not mimic acetylation at these residues, but instead renders PALB2 ChAM functionally null. The PALB2 7Q phenotypes therefore demonstrate the importance of the 7K patch for ChAM function in HR repair, rather than its acetylation status.
Perhaps even more importantly, if results with the 7Q mutant are suspect, the conclusion that deacetylation is required for HR (or DNA repair) is suspect because that is the only case where the authors see a defect in RAD51 foci and resistance to olaparib. Similarly, if the 7R mutant "mimics non-acetyl-lysine" then the fact that it has normal RAD51 foci and resistance to olaparib contradicts the conclusion that deacetylation is required for DNA repair.
Unfortunately, it is currently technically not possible to ‘lock’ the PALB2 7K patch in its acetylated status in vivo (i.e. preventing PALB2 dissociation from active genes). We thus agree with the Reviewer that it is difficult to draw definitive conclusions on the impact of constitutive PALB2 acetylation in HR, although the importance of the 7K-patch for the functionality of PALB2 is evidenced by the 7Q mutant phenotypes. Similarly, strictly speaking, our results using the 7R mutant support the notion that the ‘non-acetylated’ status of the 7K mutant, but not necessarily the dynamics of ‘de-acetylation’ events, can promote HR repair. In the revised manuscript, we will rephrase and clarify these points.
3.There are multiple concerns about Figs. 5 and S5. In Fig. 5A-C, difference in cell cycle progression after synchronization are relatively small and no rationale/interpretation is given for how this may be related to PALB2 function is given. In Fig. 5D,E differences in the levels of gamma-H2AX as a marker of DNA damage between different forms of PALB2 do not become readily apparent until about 6 or more days after addition of doxycycline. As such, it seems that these could be indirect effects and it is unclear how strongly this supports the importance of PALB2 acetylation in the DNA damage response.
We apologise for the miscommunication on these points. We have previously established that steady-state PALB2 chromatin association, jointly mediated by the ChAM and MRG15 interaction, protects a subset of active genes from DNA damage that may otherwise arise from replication-transcription conflicts (Bleuyard et al., PNAS 2017). The results presented in Fig 5 and S5 led us to propose that PALB2 chromatin association is, at least in part, mediated by the ChAM 7K patch, and its acetylation (hindered by 7Q and 7R substitutions, respectively) prevents DNA damage via a similar mechanism, i.e., protecting PALB2-bound genes during replication. This model nicely supports our observations that both 7Q/7R mutants exhibit slow S-phase progression and accumulation of gamma-H2AX over time. These points will be better articulated in the revised manuscript.
In Fig. S5, it is interesting that there are differences in the association of different forms of PALB2 with 3 distinct active loci, but no error bars or measures of statistical significance are given. Further at 2 of the 3 loci, the association of the 7Q mutant is closer to WT than the 7R mutant. Taken together, neither Fig. 5 nor Fig. S5 strongly support the key conclusion that acetylation regulates the association of PALB2 with actively transcribed genes to protect them.
We appreciate this constructive comment. The analysis was conducted once, albeit with three technical replicates, which explains why the results are presented without error bars. Nonetheless, we observe a consistent trend at three different loci, that both 7R and 7Q have chromatin association similar to the empty vector, which is background level (FLAG/IgG ChIP) and does not reflect real binding. __The revised manuscript will include the results from three biological replicates with statistical evaluation (Experiment 2). __
4.Figs. 6D-G and S6A-D conclude that "DNA damage triggers ChAM deacetylation and induces PALB2 mobilization" based upon FRAP experiments utilizing WT PALB2. But there is no control to demonstrate that this is a specific effect driven by the state of PALB2 acetylation. For example, DNA damage might cause global acetylation changes resulting in relaxed chromatin in which proteins that are not subject to acetylation-deacetylation also show increased mobility.
We thank the Reviewer for this valuable comment. It is true that we cannot formally exclude the possibility that changes in PALB2 mobility are indirect consequence of damage-induced chromatin reorganisation/increased chromatin mobility. However, our analyses clearly demonstrate that ChAM acetylation increases its association with nucleosomes (Fig. 3B), while non-nucleosome binding ChAM-null (7Q or deletion) increases PALB2 mobility (Fig. 2E, Fig. 4E and Fig. S4C). Further, WT PALB2 mobility increases after KAT2 depletion (i.e. reduction of chromatin acetylation of KAT2 targets, hence chromatin compaction) (Fig. 3F), but reduces upon KDAC inhibition (i.e. global increase in acetylation, hence chromatin relaxation) (Fig. 3G). Considering all these observations collectively, the increase in PALB2 mobility detectable upon DNA damage is unlikely to reflect global chromatin relaxation, and that PALB2 acetylation influences its mobility in both challenged and unchallenged cells. This point will be emphasised in the revised manuscript.
5.Fig. 7B shows that the 7Q mutant has diminished RAD51 foci while Fig. S7C,D suggests based upon a different methodology (laser-induced damage) that the 7Q mutant does not affect PALB2 recruitment. Since the issue of recruitment is key to the mechanism proposed, the authors should examine PALB2 foci instead as this may be a more sensitive assay of PALB2 recruitment.
We appreciate the Reviewer’s point. We would like to highlight, however, the well-documented role of BRCA1 in PALB2 recruitment to sites of DNA damage. This supports our notion that the 7Q mutant is recruited to sites of DNA damage, likely mediated via its interaction with BRCA1. As depicted in Fig. 7D, we propose that the 7K patch-mediated PALB2 engagement with damaged DNA, which is disrupted by the K to Q substitutions, is essential for proper RAD51 loading onto DNA, hence RAD51 foci formation and HR repair. This is in line with our observation that PALB2 ChAM deletion, similarly to the 7Q mutant, perturbs damage-induced RAD51 foci formation (Bleuyard et al., EMBO Rep. 2012). We believe that the laser-induced experiments provide high sensitivity and resolution for PALB2 recruitment kinetics, as the data were obtained with real-time live-cell imaging.
6.The authors state in the last sentence of the Results section that "lysine residues within the ChAM 7K-patch are indispensable for PALB2 function in HR" but never test the mutants for HR using reporter assays. The manuscript would be strengthened by performing such assays.
RAD51 foci formation and sensitivity to PARP inhibition are well-accepted readouts for HR repair. Conversely, we have been cautious about existing HR reporter systems, which evaluate gene-conversion or targeting events triggered by a ‘clean’ enzyme-induced DSB, but not an authentic repair of ‘dirty’ DSB induced by IR or olaparib.
7.The model for the role of ChAM acetylation in regulating PALB2 function presented in Fig. 7D is not fully supported by the data presented. Critically, while association with RAD51 and BRCA2 is tested in Fig. S7B, the authors hypothesize that deacetylation is required to release PALB2 to enable association with BRCA1 but this is not tested utilizing the mutants.
We appreciate the Reviewer’s point. It has been demonstrated that PALB2 interaction with BRCA1 is triggered by damage-induced PALB2 phosphorylation (Ahlskog et al., EMBO Reports, 2016), as well as removal of KEAP1-mediated ubiquitylation in S and G2 (Orthwein et al., Nature 2015). Our preliminary analyses further suggest that BRCA1-PALB2 interaction is highly dynamic, and we propose that damage-induced PALB2 modification and its mobilisation jointly facilitate this interaction.
Also, there are some specific points that should be considered in the context of the model. This includes how DNA damage may trigger deacetylation, and whether it is the deacetylated state or the process of deacetylation of ChAM that is critical. Also, if acetylation is important for protecting active genes in the absence of DNA damage, is deacetylation necessary to release PALB2 local or global. This is important, because if it is local there needs to be a specific mechanism for local deacetylation, while if deacetylation is global that could result in transcriptionally active genes becoming unprotected.
We thank this Reviewer for this valuable comment. We agree that, while this study establishes that ChAM is deacetylated upon DNA damage, it remains unclear whether the dynamic ‘de-acetylation’ of PALB2, rather than the ‘non-acetylated status’ of PALB2, is important for HR repair, and whether or not this is a local event. However, we would like to highlight that PALB2-bound genes are mostly periodic, e.g. those required for cell cycle progression (Bleuyard et al., 2017, PNAS). It would therefore be reasonable to speculate that DNA damage triggers the suppression of periodic gene expression as a part of DNA damage checkpoint signalling, possibly in a KDAC-dependent manner, which then allows release of PALB2 without risking DNA damage that could otherwise be caused by replication-transcription conflict. Mobilised PALB2 might then be recruited to sites of DNA damage for HR repair. Further study will be required to fully evaluate this model, for example by identifying the specific KDAC involved in ChAM deacetylation and tracking individual PALB2 molecules, which we consider to be beyond the scope of the present study. In the revised manuscript, we will better describe our model, and further detail the arising questions to be addressed in future studies.
**Minor Comments:**
a.Some parts of the Materials and Methods are overly long (such as the subsection on "Protein purification" and "immunofluorescence microscopy") and could be shortened by consolidating experimental details that are largely the same for related processes.
We propose to move these experimental sections to the supplementary information.
b.In the description of Fig. 1D, the statement "7K-patch, which is common to PALB2 orthologs" is misleading since there is not complete conservation of each lysine residue across each ortholog.
We agree with this Reviewer’s comment and will amend the description in the revised manuscript accordingly.
c.Figs. 3E,F and S3B,C perform FRAP in cells with knockdown of KAT2A/B as a surrogate for chromatin association. The authors note that this global reduction in acetylation increases PALB2 diffusion, but there is concern that this experiment is not very informative because the increased mobility may have nothing to do acetylation of PALB2.
Please refer to our answer in response to the Reviewer’s point 4.
__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __
This manuscript reports the control of PALB2 - chromatin interaction by the acetylation of a particular lysine-rich domain of the protein called ChAM. This acetylation is shown to be mediated by the acetyltransferases KAT2A/B. Following these investigations, the authors made an effort to place their findings in the context of DNA replication and DNA repair. The proposed model is that the acetylation-dependent interaction of PALB2 with chromatin could ensure the protection of the genome during DNA replication and control DNA repair.
**Specific remarks**
1 - Based on different experiments, essentially the one shown in Fig. 3B, the authors conclude that the acetylation of the ChAM domain enhances its association with nucleosomes. However, taking into account the experimental setting, this conclusion should be largely tuned down. Indeed, this enhanced acetylation-dependent nucleosome binding was observed when the experiment was carried out in the presence of excess of free naked DNA. Under these conditions, the non-acetylated ChAM fragments became mostly trapped by DNA (clearly shown in Fig. 3C/D), and hence would not be available for nucleosome binding, while the acetylated ChAM fragments would remain available for nucleosome association because of their reduced DNA-binding ability.
Consequently, the acetylation of the ChAM domain would only play a role on the availability of PALB2 for chromatin/nucleosome binding and not directly stimulate nucleosome binding. Therefore, the nucleosome-binding capacity of ChAM by itself should not be dependent on ChAM domain acetylation.
If true, this hypothesis could also be relevant in vivo since the poly-K in the ChAM domain could also non-specifically interact with nuclear RNAs and hence its acetylation, by releasing it from nuclear RNAs, would make it available for chromatin-binding. The importance of RNAs in the regulation of PALB2 nucleosome-binding could be tested in the experiments shown in Fig. 2C and 2E by adding RNase to the pull-down medium (WT +/-RNase or addition of increasing exogenous RNAs).
We are grateful for the Reviewer’s detailed comments and find the potential involvement of RNA very intriguing. Indeed, transcriptionally active loci, which are bound by PALB2, are enriched in nascent RNA, and such local RNA may play an important role in promoting the association of acetylated PALB2 with nucleosomes. However, we believe that investigating the role of RNAs in PALB2 nucleosome binding is beyond the scope of this study. As discussed extensively in response to this Reviewer’s point 2 below, we believe the mode of interaction of ChAM with nucleosomes to be highly complex, being jointly mediated by the N-terminal conserved region and the C-terminal lysine cluster. We will discuss these issues more extensively in the revised manuscript.
2 - The real question is as follows. While acetylation makes the protein available for nucleosome binding, which part of the ChAM domain is actually mediating nucleosome binding and whether lysine acetylation could be directly involved in this binding. Another question would be to identify the elements in the nucleosome mediating this interaction, histones (core domain, tails, post-translational modifications, specific histone types), histone-DNA, etc...
We entirely agree with the Reviewer’s question – despite the increasing recognition of the physiological importance of the PALB2 ChAM and our efforts in understanding the mode of association of ChAM with nucleosomes (including the potential involvement of histone tail modifications), this specific question remains enigmatic.
Explicitly, our previous work demonstrated that substitutions of residues within the evolutionarily highly conserved N-terminal part of the ChAM perturb its association with nucleosomes (Bleuyard et al., 2017, PNAS; Bleuyard et al., 2017, Wellcome Open Research). A recent study by the laboratory of Prof Jackson proposed that basic residues across the ChAM are part of a binding interface with an acidic patch of histone H2A in its nucleosomal context (Belotserkovskaya et al., Nat Comm. 2020). Our results presented in this study introduced an additional complexity, showing that the C-terminal 7K basic patch is essential for ChAM-nucleosome interaction. Intriguingly, our study also suggests that the regions flanking ChAM, which are phosphorylated at multiple residues, play roles in regulating ChAM binding to nucleosomes (Fig 2B and C; please refer to our answer to the Reviewer’s minor point 6 too).
We are currently working towards solving the structure of ChAM in complex with a nucleosome, which may help to clarify this very important question. At this point, we think that the question about complete elements for the ChAM interaction with nucleosome is out of the scope of this manuscript, and should be addressed in future work. To make this point clear, we will provide an updated overview of the ChAM elements affecting nucleosome interaction in the revised manuscript.
3 - Taking into account the authors conclusions on the role of ChAM domain acetylation and its impact on PALB2 mobility, in Figure 4D/E, one should expect a difference of t1/2 when wild-type and 7R mutant are assayed by FRAP. At least the measures of t1/2 in the wild-type should have been more heterogeneous compared to the 7R mutant due to the acetylation of the wild-type PALB2 by the endogenous HATs (the impact of endogenous HATs on the wild-type sequence is shown in Fig. 3F). Could the authors comment on this?
We appreciate this Reviewer’s point. As mentioned in our responses to Reviewer 1’s points 1 and 3, we are unable to exclude the possibility that the 7R mutant still maintains its DNA-binding capacity, masking detectable change in its chromatin enrichment and mobility. Also, PALB2 in vivo chromatin association is limited to a small fraction of periodic genes, hence FRAP assay may not be sensitive enough to detect minute but critical differences. We will conduct biochemical assessment of the ChAM 7R mutant and ChIP-qPCR analyses to assess PALB2 binding to specific genes, results of which will be included in the revised manuscript (Experiments 1 and 2).
4 - It would be better to remove the data presented in Fig. 5 since, as currently presented, these investigations remain shallow and do not bring much information on what is happening. The presented data are rather confusing since, in the absence of further investigations, it is not clear which one(s) of the mechanisms involved in the control of DNA replication is controlled by PALB2 and many explanations, including artefacts, remain possible.
The manuscript would gain in interest if the authors would devote the functional studies only to the repair part (Fig.6 and 7).
We feel it is important to show Fig 5, as although the results may appear confusing, they highlight the importance of the acetylation of the 7K patch at the cellular level. Namely, the non-acetylatable 7R mutant fails to support normal cellular growth, likely due to its impaired association with active genes (Fig S5), which might provide in vivo evidence that non-acetylation of the 7K patch promotes PALB2 release from chromatin (please refer to our response to Reviewer 1’s point 3). We are confident that we will be able to clarify this point with our additional ChIP-qPCR analyses (Experiment 2).
**Minor points**
5 - High background of non-enzymatic acetylation of PALB2 fragments makes the identification of KAT2A/B specific acetylation not very convincing. The immunoblot detection of acetylation fragments shown in Figure S1 is much more convincing. Therefore, the authors may consider to present Fig S1 as a main Figure and Fig.1B as a supplementary one.
We will swap or add Fig S1B with or to Fig 1B in the revised manuscript.
6 - It would be interesting if the authors would comment on why the presence of regions flanking the ChAM domain (Fig. 1A, construct #5) significantly reduces chromatin (Fig. 1B) and nucleosome binding (Fig. 1C).
We are grateful for this Reviewer’s comment. Indeed, we noticed that the inclusion of the ChAM C-terminal flanking region perturbs its chromatin association. This region is highly enriched with serine and threonine residues which could be targeted for phosphorylation by cell cycle regulators (CDKs and PLK1) and DNA damage-responsive kinases (ATM and ATR). It is therefore tempting to speculate that, when phosphorylated, this flanking region could mask the basic patch of the ChAM, hence facilitating the release of PALB2 from undamaged chromatin region and its recruitment to sites of DNA damage. In the revised manuscript, we will provide the complete list of PTMs and discuss this point.
__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __
KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity
The authors describe a series of experiments examining the consequence of acetylation, within a defined motif (Chromatin Association Motif; ChAM), on the cellular roles of the protein PALB2 (Partner and Localizer of BRCA2).
The key conclusions drawn by the authors are generally convincing and are supported by the presented experimental results, which indicate that acetylation of PALB2 by KAT2A/KAT2B modulates its cellular behaviour and response to DNA damage. However please see specific comments below:
**Major Comments**
Expression of full-length PALB2 in the heterologous host E. coli is highly problematic, as the WD40 domain is generally not correctly folded. The authors use the ArticExpress strain to try and solve/alleviate this problem - but it is clear from the materials and methods section that an ATP-wash step has had to be introduced in order to release the recombinant protein from the chaperone system encoded by the ArticExpress system; i.e. indicating poor / mis-folding. Whilst this does not strictly have an effect on the results presented in Figure 1 (detection of in vitro acetylation sites), they have implications for the wider scientific community, as this may lead to the erroneous assumption that is possible to produce functional / folded full-length PALB2 in this way.
We apologise if the manuscript conveyed the message that we are able to produce functionally active, full-length PALB2 in bacteria, which was clearly not our intention. Our aim was to test whether KAT2A was able to acetylate PALB2 in vitro. We agree that the folding and the biochemical properties (e.g. WD40-mediated BRCA2 binding) of the bacterially produced full length PALB2 were not fully assessed. We believe that this does not affect the overall conclusions of this study. In the revised manuscript, we will correct this error to make this point clear.
In vitro modification assays are prone to producing post-translational modifications that are not fully reflective of those observed in vivo, and therefore need to be treated with some caution. This is highlighted by the relatively low modification of K438 in vitro by KAT2A; esp. as this is an acetylation site that has been previously mapped in vivo (by the authors). It would have been useful to include / see the effects on PALB2 function in vivo by modification / alteration of this single site.
We appreciate the Reviewer’s constructive comment. Redundancy of acetylation acceptor residues within a lysine cluster is common, as is also the case for many ubiquitination events, hence we analysed the 7K patch mutant for phenotypic studies. For the same reason, we trust that the outcome of the characterisation of a K438 mutant would not significantly change our conclusions.
Figure 3C and Figure 3D do not fully support or reflect the conclusions drawn by the authors - any peptide containing a cluster of positive charged residues are likely to interact with DNA through charge neutralisation of the phosphodiester backbone, concomitantly any alteration to this region of charge (i.e. via acetylation) will perturb this interaction.
We totally agree with the Reviewer’s view and state, in the main text referring to the results shown in Fig. 3C and D, that “As anticipated, lysine acetylation, which neutralises the positive charge on the lysine side chain, conferred reduced affinity for negatively charged DNA”. In the revised manuscript, we will make this point clearer.
Furthermore, experiments performed with the synthetic acetylated peptides do not agree with those carried out with the GST-ChAM constructs - GST-ChAM interacts with the nicked and linear forms of the pBS plasmid (Figure 2F) but does not interact with the supercoiled form. The WT synthetic ChAM peptide, in contrast, interacts with all three plasmid states at high concentrations. It is suggested that these two figures are removed.
It is true that we cannot exclude the potential difference between GST-ChAM and synthetic ChAM peptide: for example, 26 kDa of GST, which can form a dimer, might mask the full biochemical properties of ChAM in DNA binding. However, we believe that the difference is more likely caused by the concentration of ChAM used. While we used the synthetic ChAM peptides at concentrations of 2.97, 5.94, 29.3 µM for Fig. 3C, we used 5.94 µM of GST-ChAM for Fig. 2F, for which we apologise for the omission of the experimental conditions used. This notion is supported by the side-by-side experiment, which was not shown in the original manuscript. In the revised manuscript, we will make these points clear.
p. 18 : the authors used a PALB2 variant, where the lysines in the 7K patch are mutated to arginine - but don't fully characterise the effects of introducing these particular mutations on the ability of the ChAM fragment to bind to DNA, or indeed to nucleosomes; this is an important control.
We appreciate the Reviewer’s comment. Biochemical analyses of the 7R mutant were not conducted, as ChAM produced in bacteria is not expected to be acetylated. Nonetheless, as also linked with the concerns of Reviewers 1 and 2, we recognise the importance of 7R biochemical characterisation for accurate interpretation of in vivo phenotypes. We will assess the DNA and nucleosome binding of the ChAM 7R mutant, which will be included in the revised manuscript (Experiment 1).
Figure 6 : it would be good to show a second supporting example for deacetylation of PALB2 in response to DNA damage - perhaps treatment with MMC?
We appreciate the Reviewer’s comment. Indeed, we have conducted the analysis upon MMC and Olaparib exposure. Curiously, however, no clear change of ChAM acetylation was detectable. Note that, for this experiment, we assessed the acetylation level of a GFP-fusion of ChAM, exogenously expressed in HEK293, along with endogenous gamma-H2AX as a readout of DNA damage signalling. Unlike ionising radiation, which triggered strong induction of gamma-H2AX (Fig. 6), no clear increase of gamma-H2AX was detectable upon MMC/Olaparib exposure. Hence, we propose that the reduction of ChAM acetylation reflects the cellular response to DNA damage. We will make these points clear in the revised manuscript.
**Minor Comments**
p. 16 : 'Our MS analysis of the chromatin-associated GFP-ChAM fragment identified actelyation of all seven lysines within the 7K-patch (Fig. 3A, marked with arrows).
This part of the manuscript is potentially a little confusing, as Fig. 3A references a series of synthetic peptides rather than the GFP-ChAM fragments themselves.
We appreciate the Reviewer’s point. Indeed, Fig. 3A shows 1) MS of the chromatin-associated fraction of GFP-ChAM (the top part with arrows) and 2) a schematic diagram of synthetic peptides that we used for biochemical analyses (the bottom part). In the revised manuscript, we will clarify this point and indicate the MS result and the schematic of synthetic peptides in two separate panels and refer each of them appropriately.
p. 20 : Furthermore, using the FRAP approach, we observed clear differences in diffusion rates of FE-PALB2 following damage by IR, MMC, or olaparib treatment...
FE-PALB2 = FL-PALB2?
We apologise for the confusion. In our study, FE-PALB2 refers to Flag-EGFP tagged PALB2 (full-length). This is defined in the text “To this end, a tandem FLAG- and EGFP-tagged full-length wild-type (WT) PALB2 (FE-PALB2)” (p. 17).
__ __
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity
The authors describe a series of experiments examining the consequence of acetylation, within a defined motif (Chromatin Association Motif; ChAM), on the cellular roles of the protein PALB2 (Partner and Localizer of BRCA2).
The key conclusions drawn by the authors are generally convincing and are supported by the presented experimental results, which indicate that acetylation of PALB2 by KAT2A/KAT2B modulates its cellular behaviour and response to DNA damage. However please see specific comments below:
Major Comments
Expression …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
KAT2-mediated acetylation switches the mode of PALB2 chromatin association to safeguard genome integrity
The authors describe a series of experiments examining the consequence of acetylation, within a defined motif (Chromatin Association Motif; ChAM), on the cellular roles of the protein PALB2 (Partner and Localizer of BRCA2).
The key conclusions drawn by the authors are generally convincing and are supported by the presented experimental results, which indicate that acetylation of PALB2 by KAT2A/KAT2B modulates its cellular behaviour and response to DNA damage. However please see specific comments below:
Major Comments
Expression of full-length PALB2 in the heterologous host E. coli is highly problematic, as the WD40 domain is generally not correctly folded. The authors use the ArticExpress strain to try and solve/alleviate this problem - but it is clear from the materials and methods section that an ATP-wash step has had to be introduced in order to release the recombinant protein from the chaperone system encoded by the ArticExpress system; i.e. indicating poor / mis-folding. Whilst this does not strictly have an effect on the results presented in Figure 1 (detection of in vitro acetylation sites), they have implications for the wider scientific community, as this may lead to the erroneous assumption that is possible to produce functional / folded full-length PALB2 in this way.
In vitro modification assays are prone to producing post-translational modifications that are not fully reflective of those observed in vivo, and therefore need to be treated with some caution. This is highlighted by the relatively low modification of K438 in vitro by KAT2A; esp. as this is an acetylation site that has been previously mapped in vivo (by the authors). It would have been useful to include / see the effects on PALB2 function in vivo by modification / alteration of this single site.
Figure 3C and Figure 3D do not fully support or reflect the conclusions drawn by the authors - any peptide containing a cluster of positive charged residues are likely to interact with DNA through charge neutralisation of the phosphodiester backbone, concomitantly any alteration to this region of charge (i.e. via acetylation) will perturb this interaction.
Furthermore, experiments performed with the synthetic acetylated peptides do not agree with those carried out with the GST-ChAM constructs - GST-ChAM interacts with the nicked and linear forms of the pBS plasmid (Figure 2F) but does not interact with the supercoiled form. The WT synthetic ChAM peptide, in contrast, interacts with all three plasmid states at high concentrations. It is suggested that these two figures are removed.p. 18 : the authors used a PALB2 variant, where the lysines in the 7K patch are mutated to arginine - but don't fully characterise the effects of introducing these particular mutations on the ability of the ChAM fragment to bind to DNA, or indeed to nucleosomes; this is an important control.
Figure 6 : it would be good to show a second supporting example for deacetylation of PALB2 in response to DNA damage - perhaps treatment with MMC?
Minor Comments
p. 16 : 'Our MS analysis of the chromatin-associated GFP-ChAM fragment identified actelyation of all seven lysines within the 7K-patch (Fig. 3A, marked with arrows).
This part of the manuscript is potentially a little confusing, as Fig. 3A references a series of synthetic peptides rather than the GFP-ChAM fragments themselves.
p. 20 : Furthermore, using the FRAP approach, we observed clear differences in diffusion rates of FE-PALB2 following damage by IR, MMC, or olaparib treatment...
FE-PALB2 = FL-PALB2?
Significance
The paper is generally incremental in nature - offering additional information about the effects of acetylation within the ChAM region of PALB2 on its interaction with chromatin, and on the cellular response to DNA damage.
The reported findings would be of interest to scientists working in the area of DNA damage repair and DNA damage signalling, esp. those with a keen interest in the regulation and control of homologous recombination.
Field of expertise: Biochemistry / Biophysics / DNA damage response / Structural Biology
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This manuscript reports the control of PALB2 - chromatin interaction by the acetylation of a particular lysine-rich domain of the protein called ChAM. This acetylation is shown to be mediated by the acetyltransferases KAT2A/B. Following these investigations, the authors made an effort to place their findings in the context of DNA replication and DNA repair. The proposed model is that the acetylation-dependent interaction of PALB2 with chromatin could ensure the protection of the genome during DNA replication and control DNA repair.
Specific remarks
1 - Based on different experiments, essentially the one shown in Fig. 3B, the authors …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
This manuscript reports the control of PALB2 - chromatin interaction by the acetylation of a particular lysine-rich domain of the protein called ChAM. This acetylation is shown to be mediated by the acetyltransferases KAT2A/B. Following these investigations, the authors made an effort to place their findings in the context of DNA replication and DNA repair. The proposed model is that the acetylation-dependent interaction of PALB2 with chromatin could ensure the protection of the genome during DNA replication and control DNA repair.
Specific remarks
1 - Based on different experiments, essentially the one shown in Fig. 3B, the authors conclude that the acetylation of the ChAM domain enhances its association with nucleosomes. However, taking into account the experimental setting, this conclusion should be largely tuned down. Indeed, this enhanced acetylation-dependent nucleosome binding was observed when the experiment was carried out in the presence of excess of free naked DNA. Under these conditions, the non-acetylated ChAM fragments became mostly trapped by DNA (clearly shown in Fig. 3C/D), and hence would not be available for nucleosome binding, while the acetylated ChAM fragments would remain available for nucleosome association because of their reduced DNA-binding ability.
Consequently, the acetylation of the ChAM domain would only play a role on the availability of PALB2 for chromatin/nucleosome binding and not directly stimulate nucleosome binding. Therefore, the nucleosome-binding capacity of ChAM by itself should not be dependent on ChAM domain acetylation.
If true, this hypothesis could also be relevant in vivo since the poly-K in the ChAM domain could also non-specifically interact with nuclear RNAs and hence its acetylation, by releasing it from nuclear RNAs, would make it available for chromatin-binding. The importance of RNAs in the regulation of PALB2 nucleosome-binding could be tested in the experiments shown in Fig. 2C and 2E by adding RNase to the pull-down medium (WT +/-RNase or addition of increasing exogenous RNAs).
2 - The real question is as follows. While acetylation makes the protein available for nucleosome binding, which part of the ChAM domain is actually mediating nucleosome binding and whether lysine acetylation could be directly involved in this binding. Another question would be to identify the elements in the nucleosome mediating this interaction, histones (core domain, tails, post-translational modifications, specific histone types), histone-DNA, etc...
3 - Taking into account the authors conclusions on the role of ChAM domain acetylation and its impact on PALB2 mobility, in Figure 4D/E, one should expect a difference of t1/2 when wild-type and 7R mutant are assayed by FRAP. At least the measures of t1/2 in the wild-type should have been more heterogeneous compared to the 7R mutant due to the acetylation of the wild-type PALB2 by the endogenous HATs (the impact of endogenous HATs on the wild-type sequence is shown in Fig. 3F). Could the authors comment on this?
4 - It would be better to remove the data presented in Fig. 5 since, as currently presented, these investigations remain shallow and do not bring much information on what is happening. The presented data are rather confusing since, in the absence of further investigations, it is not clear which one(s) of the mechanisms involved in the control of DNA replication is controlled by PALB2 and many explanations, including artefacts, remain possible.
The manuscript would gain in interest if the authors would devote the functional studies only to the repair part (Fig.6 and 7).
Minor points
5 - High background of non-enzymatic acetylation of PALB2 fragments makes the identification of KAT2A/B specific acetylation not very convincing. The immunoblot detection of acetylation fragments shown in Figure S1 is much more convincing. Therefore, the authors may consider to present Fig S1 as a main Figure and Fig.1B as a supplementary one.
6 - It would be interesting if the authors would comment on why the presence of regions flanking the ChAM domain (Fig. 1A, construct #5) significantly reduces chromatin (Fig. 1B) and nucleosome binding (Fig. 1C).
Significance
This manuscript brings interesting information on the control of the activity of PALB2 by acetylation which depends on specific cellular HATs. By doing so, it also opens the door to envision a role for a metabolic reprogramming of protein acetylation in the control of PALB2 activity, as discussed by the authors.
This is an interesting report but the functional part is rather weak, decreasing the interest of the manuscript as a whole. This report would gain in interest if the authors would develop the first part (Figs 1- 4), while reducing the second part (Figs 5 -7) to the presentation and discussion of the strongest functional data (see my comments).
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Fournier et al. detect acetylation within the chromatin association motif (ChAM) of PALB2 and demonstrate that KAT2 can acetylate these 7 lysine residues within this region. They then generate K to R mutations (7R) or K to Q mutations (7Q) at these sites and perform assays of fluorescence recovery after photobleaching (FRAP) to measure mobility as a measure of chromatin association, RAD51 foci, PALB2 recruitment at sites of laser-induced DNA damage, and sensitivity to olaparib. They find increased mobility of the 7Q mutant of PALB2 but not 7R in the absence of exogenous DNA damage, as well as defects in DNA damage-induced RAD51 foci …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Fournier et al. detect acetylation within the chromatin association motif (ChAM) of PALB2 and demonstrate that KAT2 can acetylate these 7 lysine residues within this region. They then generate K to R mutations (7R) or K to Q mutations (7Q) at these sites and perform assays of fluorescence recovery after photobleaching (FRAP) to measure mobility as a measure of chromatin association, RAD51 foci, PALB2 recruitment at sites of laser-induced DNA damage, and sensitivity to olaparib. They find increased mobility of the 7Q mutant of PALB2 but not 7R in the absence of exogenous DNA damage, as well as defects in DNA damage-induced RAD51 foci and resistance to olaparib. On this basis, the authors conclude that acetylation is required for the association of PALB2 with undamaged chromatin and that deacetylation permits mobilization and association with BRCA1 to enable proper DNA repair. While the manuscript is generally well-written, many of the systems are rather elegant, and this study may yield novel insights into the regulation and function of PALB2 in DNA repair, there are some missing experiments to be added and important contradictions that should be resolved in order to fully establish the new model the authors propose.
Major comments:
1.There are some concerns about the interpretation of experiments with the 7R and 7Q mutants of PALB2. For example, in the description of results in Fig. S2C, the authors state "K to R substitutions maintain the charge yet are unable to accept acetylation and hence mimic constitutively non-acetyl lysine". However, in Fig. 4B the association of the 7R mutant with chromatin is similar to WT and in Fig. 7D,E the relative immobility of the 7R mutant is very similar to WT PALB2. Thus, the conclusion that acetylation is required for PALB2 association with damaged undamaged chromatin and for release of PALB2 upon DNA damage does not appear justified. Perhaps the authors need to better consider whether the 7R mutant mimics acetylation because of its charge. Even so, the mutant then maintains the charge normally associated with acetylated PALB2, calling into question whether deacetylation indeed "releases PALB2 from undamaged chromatin".
2.Related to questions of interpreting results utilizing the 7R and 7Q mutants of PALB2, in Fig. 7B,C the 7R mutant but not 7Q supports RAD51 foci and resistance to olaparib similar to WT PALB2. The authors then state in the Discussion that "our work also suggests that caution should be exercised in the use of K to Q substitutions for functional studies of lysine acetylation". Thus, which mutant is giving the correct and reliable results? Perhaps even more importantly, if results with the 7Q mutant are suspect, the conclusion that deacetylation is required for HR (or DNA repair) is suspect because that is the only case where the authors see a defect in RAD51 foci and resistance to olaparib. Similarly, if the 7R mutant "mimics non-acetyl-lysine" then the fact that it has normal RAD51 foci and resistance to olaparib contradicts the conclusion that deacetylation is required for DNA repair.
3.There are multiple concerns about Figs. 5 and S5. In Fig. 5A-C, difference in cell cycle progression after synchronization are relatively small and no rationale/interpretation is given for how this may be related to PALB2 function is given. In Fig. 5D,E differences in the levels of gamma-H2AX as a marker of DNA damage between different forms of PALB2 do not become readily apparent until about 6 or more days after addition of doxycycline. As such, it seems that these could be indirect effects and it is unclear how strongly this supports the importance of PALB2 acetylation in the DNA damage response. In Fig. S5, it is interesting that there are difference in the association of different forms of PALB2 with 3 distinct active loci, but no error bars or measures of statistical significance are given. Further at 2 of the 3 loci, the association of the 7Q mutant is closer to WT than the 7R mutant. Taken together, neither Fig. 5 nor Fig. S5 strongly support the key conclusion that acetylation regulates the association of PALB2 with actively transcribed genes to protect them.
4.Figs. 6D-G and S6A-D conclude that "DNA damage triggers ChAM deacetylation and induces PALB2 mobilization" based upon FRAP experiments utilizing WT PALB2. But there is no control to demonstrate that this is a specific effect driven by the state of PALB2 acetylation. For example, DNA damage mmight cause global acetylation changes resulting in relaxed chromatin in which proteins that are not subject to acetylation-deacetylation also show increased mobility.
5.Fig. 7B shows that the 7Q mutant has diminished RAD51 foci while Fig. S7C,D suggests based upon a different methodology (laser-induced damage) that the 7Q mutant does not affect PALB2 recruitment. Since the issue of recruitment is key to the mechanism proposed, the authors should examine PALB2 foci instead as this may be a more sensitive assay of PALB2 recruitment.
6.The authors state in the last sentence of the Results section that "lysine residues within the ChAM 7K-patch are indispensable for PALB2 function in HR" but never test the mutants for HR using reporter assays. The manuscript would be strengthened by performing such assays.
7.The model for the role of ChAM acetylation in regulating PALB2 function presented in Fig. 7D is not fully supported by the data presented. Critically, while association with RAD51 and BRCA2 is tested in Fig. S7B, the authors hypothesize that deacetylation is required to release PALB2 to enable association with BRCA1 but this is not tested utilizing the mutants. Also, there are some specific points that should be considered in the context of the model. This include how DNA damage may trigger deacetylation, and whether it is the deacetylated state or the process of deacetylation of ChAM that is critical. Also, if acetylation is important for protecting active genes in the absence of DNA damage, is deacetylation necessary to release PALB2 local or global. This is important, because if it is local there needs to be a specific mechanism for local deacetylation, while if deacetylation is global that could result in transcriptionally active genes becoming unprotected.
Minor Comments:
a.Some parts of the Materials and Methods are overly long (such as the subsection on "Protein purification" and "immunofluorescence microscopy") and could be shortened by consolidating experimental details that are largely the same for related processes.
b.In the description of Fig. 1D, the statement "7K-patch, which is common to PALB2 orthologs" is misleading since there is not complete conservation of each lysine residue across each ortholog.
c.Figs. 3E,F and S3B,C perform FRAP in cells with knockdown of KAT2A/B as a surrogate for chromatin association. The authors note that this global reduction in acetylation increases PALB2 diffusion, but there is concern that this experiment is not very informative because the increased mobility may have nothing to do acetylation of PALB2.
Significance
The role of acetylation of PALB2 in the regulation and function of PALB2 was previously unknown. In this context, this is potentially an important manuscript in the DNA damage response and DNA repair field. Notably, these authors previously identified the ChAM motif which promotes chromatin association of PALB2 (JY Bleuyard et al. PMID:22193777) and demonstrated that PALB2 may associate with actively replicating genes aside of its general role in DNA repair (JY Bleuyard et al. PMID:28673974). In this context, the finding that acetylation of the ChAM motif appears to be associated with undamaged chromatin leads to the conclusion that acetylation may specifically regulate chromatin association of PALB2 and represent a switch for association with undamaged chromatin and damaged chromatin.
This work should be of specific interest to those working in the fields of DNA repair and DNA damage responses, as well as those with interests in chromatin biology. This study should be of general interest to individuals interested in cell biology, transcription, and cancer biology and therapeutics.
This reviewer's expertise is in the area of DNA damage responses and DNA repair.
-
-