Intrinsic specificity of a 'core' Tip60 acetyltransferase complex in Drosophila
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Abstract
The lysine acetyltransferase Tip60 (KAT5) regulates gene expression through acetylation of histone N- terminal ′tail′ domains. We determined the intrinsic substrate selectivity of a recombinant, 4-subunit TIP60 core module from Drosophila melanogaster with synthetic nucleosome arrays. We compared matched arrays of nucleosomes containing either the replication-dependent histone H2A, or the variant H2A.V (H2A.Z in mammals), a prominent substrate of Tip60. Targeted mass spectrometry allowed to quantify acetylation of individual lysines in histones H2A, H2A.V and H4. Overall, H4 and H2A/H2A.V were equally well acetylated. The analysis comprehensively identified selected sites of acetylation, their relative acetylation levels, diacetylation patterns and revealed surprisingly different acetylation rates of individual lysines. We also applied this defined acetylation system to evaluate the effectiveness and selectivity of a TIP60 inhibitor, NU9056. Remarkably, the inhibitor shows variable effectiveness at different acetylation sites. Knowledge about the intrinsic substrate selectivity of Tip60 is a prerequisite for a mechanistic understanding of the enzyme′s mode of action.
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Reply to the reviewers
Reviewer #1
(...) The study describes meticulously conducted and controlled experiments, showing the impressive biochemistry work consistently produced by this group. The statistical analysis and data presentation are appropriate, with the following major comments noted:
Response: We thank the reviewer for their thoughtful and constructive review of our manuscript. We appreciate the positive comments on our experimentation.
Major comments
Please clarify why K8ac/K12ac, K5ac/K16ac, K5ac/K12ac are not quantified (Figure 3). If undetected, state explicitly and annotate figures with "n.d." rather than leaving gaps. If detected but excluded, justify the …
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Reply to the reviewers
Reviewer #1
(...) The study describes meticulously conducted and controlled experiments, showing the impressive biochemistry work consistently produced by this group. The statistical analysis and data presentation are appropriate, with the following major comments noted:
Response: We thank the reviewer for their thoughtful and constructive review of our manuscript. We appreciate the positive comments on our experimentation.
Major comments
Please clarify why K8ac/K12ac, K5ac/K16ac, K5ac/K12ac are not quantified (Figure 3). If undetected, state explicitly and annotate figures with "n.d." rather than leaving gaps. If detected but excluded, justify the exclusion.
Response: We restricted ourselves to mapping those diacetylated motifs that can be readily identified by MS2. The characteristic ions of the d3-labeled and endogenous acetylated peptides in the MS2 spectra could not differentiate the diacetylated forms mentioned by the reviewer. Rather than expanding the figure with non-informative rows we amended the legend of figure 3 accordingly "Diacetylated forms K8-K12, K5-K16, K5-K12 could not be distinguished from each other by MS2 and were thus not included in the analysis".
The statement "Nevertheless, combinations of di- and triacetylation were much more frequent if K12ac was included, suggesting that K12 is the primary target." is under-supported because only two non-K12ac combinations are shown, and only one is lower than K12ac-containing combinations. Either soften the claim ("trend toward ... in our dataset") or expand the analysis to all observed di/tri combinations with effect sizes, n, and statistical tests.
Response: The reviewer is right our statement does properly reflect the data. It rather seems that combinations lacking K12ac are considerably less frequent (K5K8K16 tri-ac, K5K8 di-ac). We now modified the sentence as follows: "Peptides lacking K12ac were less frequent, suggesting that K12 is a primary target".
Please provide a more detailed discussion about the known nature of NU9056 inhibition and how it fits or doesn't fit with your data. Are there any structural studies on this?
Response: Unfortunately, NU9056 is very poorly described, neither the mode of interaction with Tip60 nor the mechanism of inhibition are known. The specificity of the chemical has not really been shown, but nevertheless it is used as a selective Tip60 inhibitor in several papers which is why we picked it in the first place. Our conclusions on the inhibitor are in the last paragraph of the discussion: "The fact that acetylation of individual lysines is inhibited with different kinetics argues against a mechanism involving competition with acetyl-CoA, but for an allosteric distortion of the catalytic center." We think that any further interpretation would likely be considered an overstatement.
Why was the inhibitor experiment MS only performed for H2A.V and not H2A? Given the clear H2A vs H2A.V differences reported in Fig. 2, it would be useful to have the matched data for H2A.
Response: In these costly mass spec experiments we strive to balance limited resources and most informative output. Because H2A.V and H4 are the major functional targets of Tip60, we considered that documenting the effect of the inhibitor on these substrates would be most appropriate. In hindsight, including H2A would have been nice to have, but would not change our conclusions about the inhibitor.
The inhibitor observations are very interesting as they can highlight systems to study the loss of specific acetyl residues: can the authors perform WB/IF validation in treated cells? I understand it will not be possible with the H2A antibodies, but the difference in H4K5ac vs H4K12ac should be possible to validate in cells
Response: We attempted to monitor changes of histone modifications upon treatment of cells with NU9056 by immunoblotting. Probing H4K5 and K12, the results were variable. We also observed occasionally that acetylation of H4K5 and H4K12 was slightly diminished in whole cell extracts, but not in nuclear extracts. This reminded us that diacetylation of H4 at K5 and K12 is a feature of cytoplasmic H4 in complex with chaperones, a mark that is placed by HAT1 (Aguldo Garcia et al., DOI: 10.1021/acs.jproteome.9b00843; Varga et al., DOI: 10.1038/s41598-019-54497-0). The observed proliferation arrest by NU9056 may thus affect chromatin assembly and indirectly K5K12 acetylation. H4K12 is also acetylated by chameau (Chm).
We observed a reduction of acetylated H4K16 and H2A.V. H4K16 is not a preferred target of Tip60, but Tip60 acetylates MSL1 and MBDR2, two subunits of the NSL1 complex (Apostolou et al. DOI: 10.1101/2025.07.15.664872). We, therefore, consider that effects on H4 acetylation upon NU9056 treatment may at least partially be affected indirectly. Because we are not confident about the data and because our manuscript emphasizes the direct, intrinsic specificity of Tip60, we refrain from showing the corresponding Western blots.
You highlight that H2AK10 (a major TIP60 site here) is not conserved in human canonical H2A. Please expand the discussion of the potential function and physiological relevance. Maybe in relation to H2A.V being a fusion of different human variants?
Response: The reviewer noted an interesting aspect of the evolution of the histone H2A variants. It turns out that H2A.Z is the more ancient variant, from which H2A derived by mutation. H2A.Z/H2A.V sequences are more conserved than H2A sequences. We summarized these evolutionary notions in Baldi and Becker (DOI: 10.1007/s00412-013-0409-x). In the context of the question, this means that mammalian H2A.Z, Drosophila H2A.V and mammalian H2A still contain the ancient sequence (lacking K10), and Drosophila H2A acquired K10 by mutation. The evolutionary advantage associated with this mutation in unclear. We now added a small paragraph summarizing these ideas on page 13 of the (changes tracked in red).
To enable direct comparisons between variants and residues, please match y-axis scales where the biology invites comparison (e.g., H2A vs H2A.V; Figs. 2-3).
Response: We adjusted the Y-axes in Figure 2 and 3 to facilitate direct comparisons, where such comparison is informative.
Minor comments
Add 1-2 sentences in the abstract on the gap in the field being addressed by the study.
Response: We are grateful for this suggestion and have expanded the abstract accordingly (changes tracked in red).
Either in the introduction or discussion, comment on your prior Tip60 three-subunit data (Kiss et al.). The three-subunit complex was significantly less active on H4, as indicated in that publication, which is likely due to the absence of Eaf6.
Response: We thank the reviewer for the opportunity to emphasize this point. Motivated by findings in the yeast and mammalian systems that Eaf6 was important for acetylation, we added this subunit to our previously reconstituted 3-subunit 'piccolo' complex. As can be seen by the comparison of the older data (Kiss et al.) and the new data, the 4-subunit TIP60 core complex is a much more potent HAT. We amended the introduction (see marked text) accordingly. We also added a paragraph on what is known about the properties and function of Eaf6 to the discussion.
3a. Text references Fig.1E before Fig.1C, please reorder
Response: We deleted the premature mentioning of Figure 1E and added the following explanation to the relevant panels in Figure 1: "The blot was reprobed with an antibody detecting H3 as an internal standard for nucleosome input."
3b. Fig.1B/C legend labels appear swapped.
Response: We thank the reviewer for spotting the swap. We corrected the figure legend.
3c. Fig.1E, 4A, 4B: add quantification
Response: We quantified each acetylation level, and added to the relevant panel of Figure 1 and 4 the following phrase: "The quantified levels of each acetylation mark over H3 are shown below each plot." Notably, the difference in acetylation signal strength between the two antibodies highlights the inherent variability of antibody-based detection.
3d. Fig.2A: Note explicitly that K5-K10 and K8-K10 are unresolvable pairs to explain the shading scheme used.
Response: The legend of Figure 2A now includes the following sentence. "Peptides that are diacetylated at either K5/K10 or K8/K10 cannot be resolved by MS2. The last row reminds of this fact by the patterning of boxes and displays the combined values."
Ensure consistent KAT5/TIP60 naming.
Response: Our naming follows this logic: We use 'Tip60' for the Drosophila protein and 'TIP60' for the Drosophila 'piccolo' or 'core' complexes. The mammalian protein is referred to by the capital acronym TIP60, as is established in the literature. We use KAT5/TIP60 according to the unified nomenclature in the introduction and parts of the discussion, when we refer to the enzymes in more general terms, independent of species. We scrutinized the manuscript again and made a few changes to adhere to the above scheme.
Consider moving the first two Discussion paragraphs (field context and challenges in antibody-based detection) into the Introduction to better frame the significance.
Response: We thank the reviewer for this suggestion that improved the manuscript a lot. We incorporated the first two paragraphs of the discussion into the introduction.
Significance
This is a valuable and timely study for the histone acetylation field. The substrate specificity of many individual HATs remains incompletely understood owing to (i) cross-reactivity and limited selectivity of many anti-acetyl-lysine antibodies, (ii) functional redundancy among KATs, (iii) variability across in-vitro assays (HAT domain vs full-length/complex; free histones vs oligonucleosomes), and (iv) incomplete translation of in-vitro specificity to in-vivo settings. These factors have produced conflicting reports in the literature. By combining quantitative mass spectrometry with carefully engineered oligonucleosomal arrays, the authors make a principal step toward deconvoluting TIP60 biology in a controlled yet close-to-physiologically relevant system. Conceptually, the work delineates intrinsic, site-specific preferences of the TIP60 core on variant versus canonical nucleosomes, consistent with largely distributive behaviour and site-dependent inhibitor sensitivity. The inhibitor-dependent shifts in acetylation patterns are particularly intriguing and could enable dissection of residue-specific functions, with potential translational implications for preclinical cancer research and biomarker development. Overall, this manuscript will be of interest to the chromatin community, and I am supportive of publication pending satisfactory resolution of the points raised above.
Response: Once more we thank the reviewer for their time and efforts devoted to help us improve the manuscript.
Reviewer #2
Major comments
(...) A central limitation of the study, noted by the authors, is the uncertainty regarding the biological relevance of the findings. While the in vitro system provides a controlled framework for analyzing residue specificity and kinetics, it does not address the functional significance of these results in a cellular or organismal context. This limitation is outside the scope of the current work but indicates potential directions for follow-up studies. Within its defined objectives, the study presents a methodological framework and dataset that contribute to understanding TIP60 activity in a biochemical setting.
Response: We agree with the referee.
Minor comments
While the manuscript is clearly presented overall, there are two minor issues that could be addressed:
In Figure 1, the panels are not ordered according to their appearance in the Results section. In addition, the legends for Figures 1B and 1C appear to be swapped.
Response: We thank the reviewer for spotting these oversights. We deleted the premature mentioning of Figure 1E and added the following explanation to the relevant panels in Figure 1: "The blot was reprobed with an antibody detecting H3 as an internal standard for nucleosome input." We also swapped the legends.
For the quantitative MS data (N = 2 biological replicates), the phrasing "Error bars represent the two replicate values" could be refined. With N = 2, showing individual data points or the range may convey the information more transparently than conventional error bars, which are typically associated with statistical measures (e.g., SEM) from larger sample sizes. Alternatively, a brief note explaining the choice to use two replicates and represent them with error bars could be added.
Response: We appreciate the reviewer's comment and have revised the figure to display individual data points for the two biological replicates instead of error bars, providing a clearer representation of the data distribution. We changed the phrasing 'Error bars represent...' to "Bars represent the mean of two biological replicates (each consisting of two TIP60 core complexes and two nucleosome arrays - each analyzed with two technical replicates), with individual replicate values shown as open circles." and hope that this describes the data better.
Significance
Krause and colleagues, using a clean in vitro system, define the substrate specificity of the Drosophila TIP60 core complex. They identify the main acetylation sites and their kinetic dynamics on H2A, H2A.V, and H4 tails, and further characterize the inhibitory activity of NU9056. This work addresses a longstanding question in the field and provides compelling evidence to support its conclusions. Future studies will be needed to establish the biological relevance of these findings.
Response: We thank the reviewer for a thoughtful and constructive review of our manuscript. We appreciate the suggestions that helped to improve the manuscript.
Reviewer #3
(...) However, the authors should revisit some additional points:
Major comments:
The Tip60 core complex is usually described as containing three subunits: Tip60, Ing3 and E(Pc). The authors also included Eaf6 in their analysis, however, their motivation to include Eaf6 specifically remains unclear. They should explain in the manuscript why Eaf6 was included and how this could affect the observed acetylation pattern.
Response: We thank the reviewer for the opportunity to emphasize this point. Motivated by findings in the yeast and mammalian systems that Eaf6 was important for acetylation, we added this subunit to our previously reconstituted 3-subunit piccolo complex. As can be seen by the comparison of the older data (ref Kiss) and the new data, the 4-subunit Tip60 core complex is a much more potent HAT. We amended the introduction accordingly. We also added a paragraph on what is known about the properties and function of Eaf6 to the discussion. Please see the amended text marked in red.
The authors investigated the effectiveness of two Tip60 inhibitors by testing their effects on H4K12ac using an antibody. They state that "TH1834 had no detectable effect on either complex [Tip60 or Msl], even at very high concentrations." However, the initial publication describing TH1834 also stated that this inhibitor particularly affected H2AX with not direct effect on H4 acetylation. The authors should revisit TH1834 and specifically investigate its effect on H2A and, in particular, on H2Av as H2Av is the corresponding ortholog of H2AX.
Response: The case of TH1834 is not very strong in the literature, which is why we discontinued the line of experimentation when we did not see any effect of TH1834 (2 different batches) on the preferred substrate. The reviewer's suggestion is very good, but given our limited resources we decided to remove the data and discussion of TH1834 from the manuscript (old Figure 4A). The deletion of these very minor data does not diminish the overall conclusion and significance of the manuscript.
The authors performed a detailed analysis of NU9056 effects. However, they did not include effects on H2A. H2A is distinct from H4 and H2Av as it is the only one containing K10 and this lysine also showed high levels of acetylation by Tip60. Therefore, a comprehensive analysis of Nu9056 effects should include analyzing its effects on H2A acetylation.
Response: In these costly mass spec experiments, we strive to balance limited resources and most informative output. Because H2A.V and H4 are the major functional targets of Tip60, we considered that documenting the effect of the inhibitor on these substrates would be most appropriate. In hindsight, including H2A would have been nice to have, but would not change our conclusions about the inhibitor.
The authors have previously reported non-histone substrates of Tip60. It would be interesting to test whether the two investigated Tip60 inhibitors affect acetylation of non-histone substrates of Tip60. This analysis would greatly increase the understanding of how selective these inhibitors are. (OPTIONAL)
Response: We agree with the reviewer that the proposed experiments may be an interesting extension of our current work. However, the Becker lab will be closed down by the end of this year due to retirement, precluding major follow-up studies at this point.
__ Minor comments: __
Fig. 1 a: instead of "blue residues", would be more accurate to refer to "blue arrows"?
Response: Yes of course - the text has been revised accordingly.
Fig.1 b-c: it would be helpful to include which staining (silver/Ponceau?) was performed here.
Response: The legends now contain the relevant information.
Fig. 2a: I did not understand the shading for the K5/K8-K10ac panel from the figure legend. The explanation is present in the main text but would be helpful in the figure legend to allow easy access for readers.
Response: We agree and revised text accordingly.
Fig. 4 c: bar graphs on the top: the X-values are missing.
Response: The figure has been revised accordingly.
This sentence in the discussion seems to require revision: "Whereas the replication-dependent H2A resides in most nucleosomes in the genome, H2A.V, the only H2A variant histone in Drosophila, is incorporated by exchange of H2A, independent of replication."
Response: We revised the sentence as follows to improve clarity. "While the replication-dependent H2A is present in most nucleosomes across the genome, H2A.V, the only H2A variant in Drosophila, is incorporated through replication-independent exchange of H2A."
In this sentence: "A comparison with the TIP60 core complex is instructive since both enzymes are MYST acetyltransferases and bear significant similarity in their catalytic center." do the authors mean "informative" rather than "instructive"?
Response: We replaced 'instructive' by 'informative.
Significance
The findings are novel and expand our knowledge of Tip60 histone tail acetylation dynamics and specificity. The manuscript does not address the biological relevance of distinct acetylation marks, which is clearly beyond the scope of the study, but discuss their relevance where possible. The analysis of NU9056 is informative and relevant in a broad context. Optionally, the authors could expand their analysis of NU9056 on its effects on non-histone Tip60 targets to increase impact further. Their analysis of TH1834, however, is currently insufficient as they focused on H4 acetylation alone, which has already been reported to not be affected by TH1834. The authors should include an analysis of TH1834 effects on H2A and H2A.V acetylation. The manuscript is well written, easy to follow and of appropriate length. The methods are elegant and the findings of the study are novel. The manuscripts targets researchers specifically interested in chromatin remodeling as well as a broader audience using the Tip60 inhibitor NU9056.
Response: We thank the reviewer for their profound assessment and the general appreciation of our work. We agree that the analysis of the TH1834 is not satisfactory at this point and have removed the corresponding data and description from figure 4. The deletion of these very minor data does not diminish the overall conclusion and significance of the manuscript.
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Referee #3
Evidence, reproducibility and clarity
In their manuscript Krause et al investigate Tip60 selectivity on histone tail acetylation. They use elegant mass spectrometry analysis to analyze lysine acetylation marks and combination of acetylation marks of histone tails of the Tip60 targets H2A, H2A.V and H4. They further consider distinct dynamics by performing a time course experiment and compare Tip60 to MOF. Using these methods, the authors describe interesting and previously undescribed selectivity, dynamics and di-acetylation patterns of Tip60 that will be the starting point of follow-up studies diving into the biological relevance of these findings. Lastly, they …
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Referee #3
Evidence, reproducibility and clarity
In their manuscript Krause et al investigate Tip60 selectivity on histone tail acetylation. They use elegant mass spectrometry analysis to analyze lysine acetylation marks and combination of acetylation marks of histone tails of the Tip60 targets H2A, H2A.V and H4. They further consider distinct dynamics by performing a time course experiment and compare Tip60 to MOF. Using these methods, the authors describe interesting and previously undescribed selectivity, dynamics and di-acetylation patterns of Tip60 that will be the starting point of follow-up studies diving into the biological relevance of these findings. Lastly, they investigate the effects of two Tip60 inhibitors and characterize the effects of NU9056 on Tip60 histone tail acetylation in detail. These studies showed that NU9056 has selective effects, impacting some lysine acetylations with greater efficiency than others. As antibodies available to investigate histone acetylations affected by NU9056 are not selective enough, these findings are relevant for any applicant of NU9056.
However, the authors should revisit some additional points:
Major comments:
- The Tip60 core complex is usually described as containing three subunits: Tip60, Ing3 and E(Pc). The authors also included Eaf6 in their analysis, however, their motivation to include Eaf6 specifically remains unclear. They should explain in the manuscript why Eaf6 was included and how this could affect the observed acetylation pattern
- The authors investigated the effectiveness of two Tip60 inhibitors by testing their effects on H4K12ac using an antibody. They state that "TH1834 had no detectable effect on either complex [Tip60 or Msl], even at very high concentrations." However, the initial publication describing TH1834 also stated that this inhibitor particularly affected H2AX with not direct effect on H4 acetylation. The authors should revisit TH1834 and specifically investigate its effect on H2A and, in particular, on H2Av as H2Av is the corresponding ortholog of H2AX.
- The authors performed a detailed analysis of NU9056 effects. However, they did not include effects on H2A. H2A is distinct from H4 and H2Av as it is the only one containing K10 and this lysine also showed high levels of acetylation by Tip60. Therefore, a comprehensive analysis of Nu9056 effects should include analyzing its effects on H2A acetylation.
- The authors have previously reported non-histone substrates of Tip60. It would be interesting to test whether the two investigated Tip60 inhibitors affect acetylation of non-histone substrates of Tip60. This analysis would greatly increase the understanding of how selective these inhibitors are. (OPTIONAL)
Minor comments:
- Fig. 1 a): instead of "blue residues", would be more accurate to refer to "blue arrows"?
- Fig.1 b-c): it would be helpful to include which staining (silver/Ponceau?) was performed here
- Fig. 2a): I did not understand the shading for the K5/K8-K10ac panel from the figure legend. The explanation is present in the main text but would be helpful in the figure legend to allow easy access for readers.
- Fig. 4 c) bar graphs on the top: the X-values are missing.
- This sentence in the discussion seems to require revision: "Whereas the replication-dependent H2A resides in most nucleosomes in the genome, H2A.V, the only H2A variant histone in Drosophila, is incorporated by exchange of H2A, independent of replication."
- In this sentence: "A comparison with the TIP60 core complex is instructive since both enzymes are MYST acetyltransferases and bear significant similarity in their catalytic center." do the authors mean "informative" rather than "instructive"?
Significance
The findings are novel and expand our knowledge of Tip60 histone tail acetylation dynamics and specificity. The manuscript does not address the biological relevance of distinct acetylation marks, which is clearly beyond the scope of the study, but discuss their relevance where possible. The analysis of NU9056 is informative and relevant in a broad context. Optionally, the authors could expand their analysis of NU9056 on its effects on non-histone Tip60 targets to increase impact further. Their analysis of TH1834, however, is currently insufficient as they focused on H4 acetylation alone, which has already been reported to not be affected by TH1834. The authors should include an analysis of TH1834 effects on H2A and H2A.V acetylation.
The manuscript is well written, easy to follow and of appropriate length. The methods are elegant and the findings of the study are novel. The manuscripts targets researchers specifically interested in chromatin remodeling as well as a broader audience using the Tip60 inhibitor NU9056.
My expertise: I am a researcher working with Drosophila melanogaster and have published on the functions of the Tip60-p400 complex. I do not have extensive expertise in nucleosome arrays, the major method applied in this manuscript.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this study, Krause and colleagues investigate the intrinsic substrate selectivity of the four-subunit TIP60 core module from Drosophila melanogaster using synthetic nucleosome arrays. To quantitatively assess acetylation at individual lysines on histones H2A, the variant H2A.V, and H4, the authors employ targeted mass spectrometry, thereby overcoming the limitations of antibody-based approaches. Contrary to earlier reports, their results reveal that the TIP60 core complex displays a selective lysine acetylation pattern, with distinct kinetics toward specific residues on each histone tail. For example, H2A lysines K5, K8, …
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Referee #2
Evidence, reproducibility and clarity
Summary:
In this study, Krause and colleagues investigate the intrinsic substrate selectivity of the four-subunit TIP60 core module from Drosophila melanogaster using synthetic nucleosome arrays. To quantitatively assess acetylation at individual lysines on histones H2A, the variant H2A.V, and H4, the authors employ targeted mass spectrometry, thereby overcoming the limitations of antibody-based approaches. Contrary to earlier reports, their results reveal that the TIP60 core complex displays a selective lysine acetylation pattern, with distinct kinetics toward specific residues on each histone tail. For example, H2A lysines K5, K8, and K10 were acetylated, with K10 exhibiting the highest modification levels. On H2A.V, K4 and K7 were modified, with K7 showing greater initial efficiency. For H4, K12 was identified as the primary target, and its acetylation was further enhanced in the presence of H2A.V. The study also examined the activity of the KAT5 inhibitor NU9056, uncovering variable inhibition across different acetylation sites. Overall, the authors conclude that intrinsic substrate selectivity is central to understanding the mechanism of Tip60 activity and that the presence of H2A variants can modulate both the efficiency and specificity of acetylation.
Major comments:
The study by Krause et al. examines the in vitro substrate selectivity of the Drosophila TIP60 core complex and the lysine-specific effects of the inhibitor NU9056. The authors use a defined in vitro system with recombinant proteins and nucleosome arrays, together with targeted mass spectrometry, to assess intrinsic enzyme activity while avoiding potential issues of antibody specificity and avidity. Heatmaps and bar plots derived from the MS data show site-specific acetylation patterns and the effects of the inhibitor. A comparative analysis with the MSL core complex, which has a well-characterized selectivity, is used as a reference point for interpreting the specificity of TIP60. The observation that NU9056 exhibits different levels of effectiveness on individual lysines, including residues within the same histone tail, is supported by the quantitative MS measurements. A central limitation of the study, noted by the authors, is the uncertainty regarding the biological relevance of the findings. While the in vitro system provides a controlled framework for analyzing residue specificity and kinetics, it does not address the functional significance of these results in a cellular or organismal context. This limitation is outside the scope of the current work but indicates potential directions for follow-up studies. Within its defined objectives, the study presents a methodological framework and dataset that contribute to understanding TIP60 activity in a biochemical setting.
Minor comments:
While the manuscript is clearly presented overall, there are two minor issues that could be addressed:
- In Figure 1, the panels are not ordered according to their appearance in the Results section. In addition, the legends for Figures 1B and 1C appear to be swapped.
- For the quantitative MS data (N = 2 biological replicates), the phrasing "Error bars represent the two replicate values" could be refined. With N = 2, showing individual data points or the range may convey the information more transparently than conventional error bars, which are typically associated with statistical measures (e.g., SEM) from larger sample sizes. Alternatively, a brief note explaining the choice to use two replicates and represent them with error bars could be added.
Significance
Krause and colleagues, using a clean in vitro system, define the substrate specificity of the Drosophila TIP60 core complex. They identify the main acetylation sites and their kinetic dynamics on H2A, H2A.V, and H4 tails, and further characterize the inhibitory activity of NU9056. This work addresses a longstanding question in the field and provides compelling evidence to support its conclusions. Future studies will be needed to establish the biological relevance of these findings.
-
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Referee #1
Evidence, reproducibility and clarity
Summary
This study uses defined, reconstituted nucleosome arrays (H2A- or H2A.V-containing) and the four-subunit Drosophila TIP60 core complex to map intrinsic substrate selectivity across time courses and in the presence of reported TIP60 inhibitors (NU9056, TH1834). Key findings are: (i) selective H2A-tail acetylation (K10 > K8 > K5) with negligible K12/K14; (ii) preferential H2A.V K4 and K7 acetylation with distinct kinetics and low co-occurrence on a single tail; (iii) H4K12 is strongly favoured over other H4 sites; (iv) acetylation patterns are consistent with a more distributive (non-processive) mechanism relative to MOF/MSL; …
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Referee #1
Evidence, reproducibility and clarity
Summary
This study uses defined, reconstituted nucleosome arrays (H2A- or H2A.V-containing) and the four-subunit Drosophila TIP60 core complex to map intrinsic substrate selectivity across time courses and in the presence of reported TIP60 inhibitors (NU9056, TH1834). Key findings are: (i) selective H2A-tail acetylation (K10 > K8 > K5) with negligible K12/K14; (ii) preferential H2A.V K4 and K7 acetylation with distinct kinetics and low co-occurrence on a single tail; (iii) H4K12 is strongly favoured over other H4 sites; (iv) acetylation patterns are consistent with a more distributive (non-processive) mechanism relative to MOF/MSL; (v) NU9056 inhibits TIP60 activity with site-specific differences suggestive of a non-competitive/allosteric component, whereas TH1834 shows no effect in this Drosophila system.
Major comments
The study describes meticulously conducted and controlled experiments, showing the impressive biochemistry work consistently produced by this group. The statistical analysis and data presentation are appropriate, with the following major comments noted:
- Please clarify why K8ac/K12ac, K5ac/K16ac, K5ac/K12ac are not quantified (Figure 3). If undetected, state explicitly and annotate figures with "n.d." rather than leaving gaps. If detected but excluded, justify the exclusion.
- The statement "Nevertheless, combinations of di- and triacetylation were much more frequent if K12ac was included, suggesting that K12 is the primary target." is under-supported because only two non-K12ac combinations are shown, and only one is lower than K12ac-containing combinations. Either soften the claim ("trend toward ... in our dataset") or expand the analysis to all observed di/tri combinations with effect sizes, n, and statistical tests.
- Please provide a more detailed discussion about the known nature of NU9056 inhibition and how it fits or doesn't fit with your data. Are there any structural studies on this?
- Why was the inhibitor experiment MS only performed for H2A.V and not H2A? Given the clear H2A vs H2A.V differences reported in Figure 2, it would be useful to have the matched data for H2A.
- The inhibitor observations are very interesting as they can highlight systems to study the loss of specific acetyl residues: can the authors perform WB/IF validation in treated cells? I understand it will not be possible with the H2A antibodies, but the difference in H4K5ac vs H4K12ac should be possible to validate in cells.
- You highlight that H2A K10 (a major TIP60 site here) is not conserved in human canonical H2A. Please expand the discussion of the potential function and physiological relevance. Maybe in relation to H2A.V being a fusion of different human variants?
- To enable direct comparisons between variants and residues, please match y-axis scales where the biology invites comparison (e.g., H2A vs H2A.V; Figs. 2-3).
Minor comments
- Add 1-2 sentences in the abstract on the gap in the field being addressed by the study.
- Either in the introduction or discussion, comment on your prior Tip60 three-subunit data (Kiss et al.). The three-subunit complex was significantly less active on H4, as indicated in that publication, which is likely due to the absence of Eaf6.
- Figure order/legends:
a. Text references Fig.1E before Fig.1C, please reorder
b. Fig.1B/C legend labels appear swapped.
c. Fig.1E, 4A, 4B: add quantification
d. Fig.2A: Note explicitly that K5-K10 and K8-K10 are unresolvable pairs to explain the shading scheme used
- Ensure consistent KAT5/TIP60 naming.
- Consider moving the first two Discussion paragraphs (field context and challenges in antibody-based detection) into the Introduction to better frame the significance.
Significance
This is a valuable and timely study for the histone acetylation field. The substrate specificity of many individual HATs remains incompletely understood owing to (i) cross-reactivity and limited selectivity of many anti-acetyl-lysine antibodies, (ii) functional redundancy among KATs, (iii) variability across in-vitro assays (HAT domain vs full-length/complex; free histones vs oligonucleosomes), and (iv) incomplete translation of in-vitro specificity to in-vivo settings. These factors have produced conflicting reports in the literature. By combining quantitative mass spectrometry with carefully engineered oligonucleosomal arrays, the authors make a principal step toward deconvoluting TIP60 biology in a controlled yet close-to-physiologically relevant system. Conceptually, the work delineates intrinsic, site-specific preferences of the TIP60 core on variant versus canonical nucleosomes, consistent with largely distributive behaviour and site-dependent inhibitor sensitivity. The inhibitor-dependent shifts in acetylation patterns are particularly intriguing and could enable dissection of residue-specific functions, with potential translational implications for preclinical cancer research and biomarker development. Overall, this manuscript will be of interest to the chromatin community, and I am supportive of publication pending satisfactory resolution of the points raised above.
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