An MNase-ChIP-Seq Protocol to Profile Histone Modifications at a DNA Break in Yeast

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors in the reviewed article entitled "An MNase-ChIP-seq protocol to profile histone modifications at a DNA break in yeast" presented the complete experimental procedure. The protocol is important for scientists, not only beginners, to elevate histone post-transcriptional modification. The above is important from the point of gene expression, especially from the DNA repair point of view. The authors correctly described induced histone methylation after double-strand break induction.

The article is well written with details in procedure, readable and easy to follow. The critical points have been also described. The graphics are in good shape and resolution, additionally the references were well settled.

I have some relevant remarks:

- How many DSB are critical for bacterial, yeast, and mammalian cells?

- In the article, I have discovered many typing mistakes and a lack of cohesion – it must be clarified before publication.

- The description of the OD should be given

- the overnight is not a precise description – should be given in hours

- what is the meaning of 1.66*10, TBE buffer 1x, DB 3x, 18’000

In conclusion, after correction, I can recommend the article for publication.

Author Response

Response to reviewer 1

1-How many DSB are critical for bacterial, yeast, and mammalian cells?

We have now added a general statement in the Introduction noting that cells across diverse organisms exhibit a very limited tolerance to DNA DSBs, as even a small number of unrepaired DSBs can severely compromise viability.        

 

2-In the article, I have discovered many typing mistakes and a lack of cohesion – it must be clarified before publication.

We thank the reviewer for pointing this out. We sincerely apologize for the typographical errors and for the sections that lacked cohesion. We have now thoroughly revised the entire manuscript and corrected mistakes. We believe the revised version is substantially clearer and more coherent.

 

3-The description of the OD should be given

We have specified the exact optical density values used for each step (OD₆₀₀), replacing the previous generic wording.

 

4-The overnight is not a precise description – should be given in hours

We agree that “overnight” is imprecise. We have now replaced it with the exact incubation time (reported in hours).

 

5-What is the meaning of 1.66*10, TBE buffer 1x, DB 3x, 18’000

The meaning of 1.66*10, TBE buffer 1×, DB 3×, and 18’000 has been clarified in the revised text.

Reviewer 2 Report

Comments and Suggestions for Authors

Elena Di Nisio and colleagues describe a chromatin immunoprecipitation protocol  based on the use of micrococcal nuclease digestion combined with immunoprecipitation followed by massive parallel sequencing. The protocol is described around the study of inducible double-strand breaks in yeast and the characterization of histone modifications that are present around such event.

The presented protocol is well structured and quite detailed notably for scientists that would be interested in studying the same system. To gain on relevance, it would be important to provide a broader application view, for instance the use of Mnase-ChIP-seq assays for other type of assays. Potentially this could be extended in the discussion part of the document, which is currently missing (no discussion part described in the current version).

As following, you can find some major and minor comments that could help to improve the content of the document:

Main comments

• While the introduction properly highlights the positive points in using the enzymatic cleavage of the chromatin by the micrococcal nuclease; the authors forgot to mention that enzymatic chromatin fragmentation presents per see a sequence bias. In the particular case of micrococcal nuclease, cleavage preferences have been observed at sites rich in adenylate, deoxyadenylate or thymidylate. Indeed, the way in which the introduction is written let believe that the enzymatic chromatin fragmentation is better than the mechanical fragmentation by sonication…or this last approach is the most even way to fragment chromatin, devoid of sequence bias and by consequence more appropriate for ChIP assays. This being said, the reason why the enzymatic strategies (Cut&tag, or Cut&run) gained major use in these last years is because these methods allow to dramatically decrease the amounts of required material.

 

• In line 72 is written: “…In this assay, crosslinking is not required, and DNA does not need to be fragmented before immunoprecipitation”. This paragraph is not clear to me since the chromatin is well fragmented by the Mnase that is part of the protein A-Mnase complex…why do you indicate that the DNA does not need to be fragmented?

 

• In line 75 the authors indicate that they describe a ChIP protocol fragmented by Mnase digestion. To be fare with previous efforts, it is essential to indicate that this strategy has been developed previously (e.g. Lorzadeh et al; Cell Reports 2016), including efforts performed in yeast (e.g. Chereji et al; Mol. Cell 2018).

 

• In line 88 the authors indicate that ChIP-based approach is likely more suitable than Cut&Run…I guess they mean that sonication is better than an enzymatic strategy…so what is not clear is why then the authors used an Mnase approach if a classical ChIP-seq method is better…notably because in yeast assays the number of cells is not a real limitation?

 

• While the protocol appears quite clear and well structured, a “trouble-shooting” section might be of interest. For instance, it would be important to stratify this section in three main points; (i) observation; (ii) potential explanation; (iii) potential solution.

 

• In figure 7 I have failed to see the region where the DSB took place and notably where the H3K79me1 modification should appear enriched. In Figure 7 I can see from the H3K79me1/H3 ratio several regions appearing as peaks, but they coincidentally appear in areas where H3 appears rather depleted. It would be essential to display a “zoom-out” view of the region such that we could appreciate the accumulation of H3K79me1 beyond the region where the DSB took place…in such manner we could see the background levels associated to the assay.

 

Minor comments

• In figure 3 replace “Galacose” by “Galactose”

 

Author Response

Response to Reviewer 2

1-To gain on relevance, it would be important to provide a broader application view, for instance the use of Mnase-ChIP-seq assays for other type of assays. Potentially this could be extended in the discussion part of the document, which is currently missing (no discussion part described in the current version).

In the revised version of the manuscript, we have added a dedicated Discussion section. In this section, we provide a broader overview of the potential applications of the MNase‑ChIP‑seq protocol, extending its relevance beyond the specific assay described. In particular, we highlight how the same approach can be adapted to investigate additional chromatin‑associated processes and protein–DNA interactions, thereby generalizing the applicability of the method.

 

2-While the introduction properly highlights the positive points in using the enzymatic cleavage of the chromatin by the micrococcal nuclease; the authors forgot to mention that enzymatic chromatin fragmentation presents per see a sequence bias. In the particular case of micrococcal nuclease, cleavage preferences have been observed at sites rich in adenylate, deoxyadenylate or thymidylate. Indeed, the way in which the introduction is written let believe that the enzymatic chromatin fragmentation is better than the mechanical fragmentation by sonication…or this last approach is the most even way to fragment chromatin, devoid of sequence bias and by consequence more appropriate for ChIP assays. This being said, the reason why the enzymatic strategies (Cut&tag, or Cut&run) gained major use in these last years is because these methods allow to dramatically decrease the amounts of required material.

We thank the reviewer for pointing this out. MNase has long been recognized for its intrinsic sequence preferences during DNA cleavage, reflecting the sequence‑dependent energetics of base‑pair destabilization and the structural accommodation of DNA within its catalytic binding pocket. However, numerous studies have clearly demonstrated that protein occupancy effectively shields DNA from MNase digestion, independently of the underlying DNA sequence (Sulkowski & Laskowski, Biochim. Biophys. Acta, 1968; Zhang & Gralla, Nucleic Acids Res., 1989; Di Luo et al., Nucleic Acids Res., 2018). In addition, it is well established that MNase cleaves not only its preferred DNA motifs but also a broader range of sequences, albeit with reduced efficiency (Horz & Altenburger, Nucleic Acids Res., 1981; Sulkowski & Laskowski, Biochim. Biophys. Acta, 1970; Mikulski et al., J. Biol. Chem., 1969; Dingwall et al., Nucleic Acids Res., 1981). More recently, Luo and colleagues analyzed MNase cleavage of nucleosomal DNA at base‑pair resolution, showing that MNase sequence preference is strongly constrained by DNA accessibility. Notably, they demonstrated that cleavage at inward‑facing nucleosomal sites reflects accessibility rather than intrinsic MNase sequence preference, indicating that protein binding can override even strong sequence biases (Luo et al, Nucleic Acids Res., 2018). These key findings are summarized in the Introduction section of the revised manuscript.

Furthermore, as suggested, we have added a statement noting that CUT&Tag and CUT&RUN have gained widespread use in recent years because they significantly reduce the amount of material required for chromatin profiling. However, we point out that this advantage is less critical when working with yeast, where biological material is readily available.

 

3-In line 72 is written: “…In this assay, crosslinking is not required, and DNA does not need to be fragmented before immunoprecipitation”. This paragraph is not clear to me since the chromatin is well fragmented by the Mnase that is part of the protein A-Mnase complex…why do you indicate that the DNA does not need to be fragmented?

With “DNA does not need to be fragmented before immunoprecipitation,” we intended to emphasize that, in CUT&RUN, no preliminary chromatin fragmentation step (such as sonication or MNase digestion performed before the immunoprecipitation step) is required. Instead, fragmentation is carried out only after antibody binding, directly by the pA‑MNase fusion protein. We have revised the text accordingly to avoid ambiguity.

 

4-In line 75 the authors indicate that they describe a ChIP protocol fragmented by Mnase digestion. To be fare with previous efforts, it is essential to indicate that this strategy has been developed previously (e.g. Lorzadeh et al; Cell Reports 2016), including efforts performed in yeast (e.g. Chereji et al; Mol. Cell 2018).

We modified the text according to this suggestion.

 

5-In line 88 the authors indicate that ChIP-based approach is likely more suitable than Cut&Run…I guess they mean that sonication is better than an enzymatic strategy…so what is not clear is why then the authors used an Mnase approach if a classical ChIP-seq method is better…notably because in yeast assays the number of cells is not a real limitation?

By stating that “a ChIP‑based approach is more suitable than CUT&RUN,” we are indeed referring to the comparison between ChIP workflows and CUT&RUN. CUT&RUN relies on chromatin cleavage after antibody binding in permeabilized cells, and in yeast this step is technically challenging because cell permeabilization requires enzymatic digestion of the cell wall (e.g., Zymolyase). This treatment becomes only partially effective after crosslinking and can increase background levels. Moreover, Zymolyase preparations often contain protease activities, which may further compromise CUT&RUN performance and data quality. For these reasons, CUT&RUN is less reliable in yeast compared with other organisms. By contrast, in ChIP‑based approaches, crosslinking is followed by cell lysis, so chromatin is extracted after the cell wall has been physically disrupted. Subsequent steps—including MNase digestion—occur on isolated chromatin and do not depend on permeabilization efficiency. We have now revised the manuscript to clarify this distinction.

 

6-While the protocol appears quite clear and well structured, a “trouble-shooting” section might be of interest. For instance, it would be important to stratify this section in three main points; (i) observation; (ii) potential explanation; (iii) potential solution.

We thank the reviewer for this useful suggestion. In the revised version of the manuscript, we have added a dedicated troubleshooting section. As recommended, we structured it in a clear and practical format. Specifically, we included a table listing the main issues that may arise during the procedure, organized into four columns: Issue, Observation, Potential explanation, and Potential solutions. We believe this structured presentation improves the clarity and usefulness of the protocol.

 

7-In figure 7 I have failed to see the region where the DSB took place and notably where the H3K79me1 modification should appear enriched. In Figure 7 I can see from the H3K79me1/H3 ratio several regions appearing as peaks, but they coincidentally appear in areas where H3 appears rather depleted. It would be essential to display a “zoom-out” view of the region such that we could appreciate the accumulation of H3K79me1 beyond the region where the DSB took place…in such manner we could see the background levels associated to the assay.

We thank the reviewer for this constructive comment. In the revised version of Figure 7, we have added a schematic representation indicating the position of the HO‑induced DSB on chromosome III, together with the genomic coordinates of the region analyzed for H3K79me1 enrichment. This addition clarifies the location of the break relative to the plotted ChIP‑seq signals. The chromatin surrounding the break is known to undergo histone depletion shortly after DSB induction, a phenomenon that prevents meaningful analysis of histone PTMs in the immediate vicinity of the break. For this reason, our analysis focuses on flanking regions where nucleosomes are retained.

To further demonstrate that H3K79me1 enrichment is specific to the region surrounding the DSB, we now include the same analysis performed on an unrelated locus on chromosome VIII. In this control region, the H3K79me1/H3 ratio is comparable between the wild‑type and the control mutant strain, confirming that the observed enrichment on chromosome III is localized and DSB‑dependent.

Regarding the “zoom‑out” view suggested by the reviewer: we have not included a broader genomic window for chromosome III because additional analyses to precisely define the full extent of H3K79me1 spreading are still ongoing and will be presented in a separate study. At this stage, our aim was to provide a focused demonstration of the local enrichment around the DSB, supported by the chromosome VIII control. We hope that these additions and clarifications improve the interpretability of Figure 7.

 

8-In figure 3 replace “Galacose” by “Galactose”

We modified the figure according to this suggestion.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have replied to all my comments.