Spatial Chromatin Organization Across the Cell Cycle: Insights from Auxin-Inducible Protein Depletion

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review by Artem Nurislamov and Anastasia Yunusova is thoughtfully written and commendably comprehensive. It should be of value to researchers studying chromatin architecture, particularly in mammalian cells, and of special interest to those studying CTCF and the SMC protein complexes condensin and cohesin. While the authors pitch their review as one that explains the pros and cons of auxin-induced degradation for chromatin studies (based on their title), the review provides a lot more. A title along the lines of “Insights into SMC complexes and CTCF in regulating chromatin structure and function as obtained by auxin-inducible protein depletion” would elicit more interest in the piece.

Main concern

The authors refer to “AID2.0 and AID2.1” (lines 688-690) without explaining exactly how these methods differ from the original technique (AID1.0). Given the emphasis of auxin-induced degradation throughout, I believe it is important to point out exactly what the improvements were in the upgraded versions. (Some was presented in the last paragraph (lines 805-848) but needs to be presented here.) In addition, the authors should discuss alternative conditional protein degradation strategies – PROTAC and dTAG – and how these techniques compare to the AID method – both their pluses and minuses.

Other concerns

1. In places the review was unnecessarily detailed; removal of such superfluous detail would make it more concise and readable. For example, the gist of lines 627-646 could be summarized in 2 short sentences.
2. Abstract and first paragraph contain many grammatical errors/vague phrasings:

• Line 13: “their function” not “its function”
• Lines 17, 18: “discuss studies” not “discuss the studies”
• Line 24: should be “lysosomal or proteasomal”
• Lines 31-32: should be “C-terminus of the target protein”
• Line 32: should be “the plant hormone (auxin) indole-3-acetic acid (IAA)”
• Line 37: “Drosophila” not “Drosophyla”
• Line 43: better would be “Recently an improved version has been presented…”

3. Table 1 is poorly organized. Top row should indicate AID version. 4^th row: cell line. 5^th row: citations.
4. Additional text issues:

• Line 287: Should be “chromosome redistribution”
• Line 313: Should be “TADs (topologically associating domains)”
• Lines 602-604: Better would be “for identifying cohesin-interacting motifs in CTCF N-terminal region [99] that allow CTCF to block cohesin translocation and protect cohesin from unloading by WAPL, thereby explaining why cohesin…”
• Figure 6 legend: define “LAD”
• Abbreviations Table: add “CBSs” (line 698). Other abbreviations were used in the text but not listed here.

5.   Finally, it is less awkward to say, “In a recent study by Yao et al [201], the authors aimed to address…”, rather than “In a recent study by [201], authors aimed to address…” (line 820). This awkward use of citations occurs multiple times throughout the manuscript.

Comments on the Quality of English Language

Grammar/syntax issues are discussed above.

Author Response

We thank the reviewers for their time spent and the insightful, constructive comments. We are pleased to take into account their recommendations in making the manuscript more accessible for a broader audience. The key revisions are summarized as follows:

We have incorporated the conclusion section. 

We have  shortened section 1 devoted to AID-technology.

We have included a summary of Ki67 functions based on the AID technology. 

Additionally, we have corrected the title, affiliations and the funding information.

Comments 1: This review by Artem Nurislamov and Anastasia Yunusova is thoughtfully written and commendably comprehensive. It should be of value to researchers studying chromatin architecture, particularly in mammalian cells, and of special interest to those studying CTCF and the SMC protein complexes condensin and cohesin. While the authors pitch their review as one that explains the pros and cons of auxin-induced degradation for chromatin studies (based on their title), the review provides a lot more. A title along the lines of “Insights into SMC complexes and CTCF in regulating chromatin structure and function as obtained by auxin-inducible protein depletion” would elicit more interest in the piece.

Response 1:  We thank the Reviewer for the evaluation of our manuscript. We support the reviewer's assertion that the title “Auxin-inducible protein depletion for chromatin studies: advantages and limitations” does not fulfill the contents of the review. To accurately reflect the paper's content we propose the revised title: “Spatial chromatin organization across the cell cycle: insights from auxin-inducible protein depletion”

Comments 2: The authors refer to “AID2.0 and AID2.1” (lines 688-690) without explaining exactly how these methods differ from the original technique (AID1.0). Given the emphasis of auxin-induced degradation throughout, I believe it is important to point out exactly what the improvements were in the upgraded versions. (Some was presented in the last paragraph (lines 805-848) but needs to be presented here.) In addition, the authors should discuss alternative conditional protein degradation strategies – PROTAC and dTAG – and how these techniques compare to the AID method – both their pluses and minuses.

Response 2: We thank the Reviewer for the suggestions. The brief description of AID 2.0 and AID 2.1 systems is mentioned in Section 1 “Auxin-inducible degron system”. Since we focus mainly on chromatin studies we have decided to confine the introduction to the AID technology and to limit only the AID system. We believe it may help to keep attention on the main biological premise. 

Comments 3: In places the review was unnecessarily detailed; removal of such superfluous detail would make it more concise and readable. For example, the gist of lines 627-646 could be summarized in 2 short sentences.

Response 3:

Thank you for your suggestion. Indeed, the paragraph in lines 627-646 was too detailed. Thus, we present a shorter version (starting in line 622).

Comments 4: Abstract and first paragraph contain many grammatical errors/vague phrasings:

Line 13: “their function” not “its function”

Lines 17, 18: “discuss studies” not “discuss the studies”

Line 24: should be “lysosomal or proteasomal”

Lines 31-32: should be “C-terminus of the target protein”

Line 32: should be “the plant hormone (auxin) indole-3-acetic acid (IAA)”

Line 37: “Drosophila” not “Drosophyla”

Line 43: better would be “Recently an improved version has been presented…”

Response 4: Thank you for pointing this out. We have corrected as advised. 

Comments 5: Table 1 is poorly organized. Top row should indicate AID version. 4th row: cell line. 5th row: citations.

Response 5: 

As suggested by another reviewer, we decided to remove Table 1 because it does not provide any real comparison with pros and cons of each approach. 

Comments 6: Additional text issues:

• Line 287: Should be “chromosome redistribution”
• Line 313: Should be “TADs (topologically associating domains)”
• Lines 602-604: Better would be “for identifying cohesin-interacting motifs in CTCF N-terminal region [99] that allow CTCF to block cohesin translocation and protect cohesin from unloading by WAPL, thereby explaining why cohesin…”
• Figure 6 legend: define “LAD”
• Abbreviations Table: add “CBSs” (line 698). Other abbreviations were used in the text but not listed here.

Response 6: All the above comments have been addressed as suggested. Thank you.

 

Comments 7: Finally, it is less awkward to say, “In a recent study by Yao et al [201], the authors aimed to address…”, rather than “In a recent study by [201], authors aimed to address…” (line 820). This awkward use of citations occurs multiple times throughout the manuscript.
Response 7:

Thank you for your observation. We refined these references according to your recommendations. E.g. “Nora et al. [150] reported that AID-tagged CTCF in mESCs…”, “In a recent study by Yao et al. [201], the authors aimed to address…”

Reviewer 2 Report

Comments and Suggestions for Authors

This is a highly densed review of the AID technology for protein depletion and on the current knowledge of how chromosomal conformations are regulated. Although informative, this reviewer feels that the review article could improve on the focus of the manuscript. It is helpful to know about the state-of-the-field for the major chromosomal regulators such as condensins. However, the extensive discussion about the AID technology comes across as less important and often distracting from the main biological premise. Below are comments to help improve this manuscript:

1. Since this is not a technology-centric paper, the authors should not have the first section be about the AID technology. The technology section should either come after the chromatin architecture section or, better yet, trimmed and integrated into the other sections. It feels as though the manuscript is conflicted between a focus on the AID technology or the biology of chromatin.
2. In line 30, the authors refer to 'Mini-AID-tag' but continue to refer to simply 'AID' in the rest of the tex and Figures. There should be a logical consistency to the terminology.
3. In line 37, Drosophila was mis-spelled.
4. Starting in line 39, it is unclear whether it is the combination of the OsTIR1 mutations and the 5-Ph-IAA are needed for enhanced protein depletion, or it is one or the other.
5. There should be a distinct section dedicated to Cohesins since it plays a major role in addition to Condensins.
6. It is unclear what are some of the major knowledge gaps that are still remaining. It would be very helpful to clearly define these at the end of the manuscript.

Comments on the Quality of English Language

Could use minor English editing.

Author Response

We thank the reviewers for their time spent and the insightful, constructive comments. We are pleased to take into account their recommendations in making the manuscript more accessible for a broader audience. The key revisions are summarized as follows:

We have incorporated the conclusion section. 

We have  shortened section 1 devoted to AID-technology.

We have included a summary of Ki67 functions based on the AID technology. 

Additionally, we have corrected the title, affiliations and the funding information. 

 

Comments 1: This is a highly densed review of the AID technology for protein depletion and on the current knowledge of how chromosomal conformations are regulated. Although informative, this reviewer feels that the review article could improve on the focus of the manuscript. It is helpful to know about the state-of-the-field for the major chromosomal regulators such as condensins. However, the extensive discussion about the AID technology comes across as less important and often distracting from the main biological premise. Below are comments to help improve this manuscript:

Since this is not a technology-centric paper, the authors should not have the first section be about the AID technology. The technology section should either come after the chromatin architecture section or, better yet, trimmed and integrated into the other sections. It feels as though the manuscript is conflicted between a focus on the AID technology or the biology of chromatin.

 We thank the Reviewer for the valuable suggestions. We also agree that discussion of the AID technology might distract attention from our primary focus on chromatin organization. However, since the discussed studies were mostly conducted using the AID technology, we believe that a brief excursion into the AID technology, its pros and cons, is worthwhile if only to be presented to an audience unfamiliar with this technique. In accordance with the reviewer’s suggestion we have reduced  the section on AID technology.. 

Comments 2: In line 30, the authors refer to 'Mini-AID-tag' but continue to refer to simply 'AID' in the rest of the text and Figures. There should be a logical consistency to the terminology.

Response 2: We thank the Reviewer for this comment, we replaced “Mini-AID-tag, a domain” by “AID-tag, a short degron domain”.

Comments 3: In line 37, Drosophila was mis-spelled.

Response 3: Thanks, we fixed this annoying misspell. 

Comments 4: Starting in line 39, it is unclear whether it is the combination of the OsTIR1 mutations and the 5-Ph-IAA are needed for enhanced protein depletion, or it is one or the other.

Response 4: OsTIR1(F74G) mutant in combination with a 5-Ph-IAA ligand provided sharp protein degradation and no detectable leaky degradation. This information has also been added in text. 

Comments 5: There should be a distinct section dedicated to Cohesins since it plays a major role in addition to Condensins.

Response 5: Since cohesin is a key determinant of interphase chromosome architecture, its role and the effects of its depletion with AID-system were described in section 4.3 “Cohesin and its cofactors: effects on interphase chromatin topology and gene expression upon depletion“ (Starting in line 675). 

Comments 6: It is unclear what are some of the major knowledge gaps that are still remaining. It would be very helpful to clearly define these at the end of the manuscript.

Response 6: We sincerely thank the Reviewer for the important observation. We agree that a conclusion section highlighting important unresolved issues and suggesting several future research directions would be very helpful. We have extended our review by including conclusion section (Starting in line 837)

 

Reviewer 3 Report

Comments and Suggestions for Authors

The MS. by A. Nurislamov and A. Yunusova entitled “Auxin-inducible protein depletion for chromatin studies: advantages and limitations“ reviews the experimental works employing the auxin-inducible degradation (AID) of proteins for the study of large-scale chromatin structure in the course of cell cycle, especially for revealing the role of condensins I and II, cohesin, and CTCF. Advantages of the AID method include its fast depletion kinetics and reversibility. In combination with other methods, such as Hi-C, it can provide valuable information about the formation of mitotic chromosomes and organization of the interphase chromatin.

 

In my opinion the MS. may be published after a minor revision.

 

My specific remarks.

 

--The Title does not quite reflect the contents of the review. Although advantages and limitations of the AID are mentioned in the MS., it seems chiefly focused on the results of experiments. Besides, other methods, such as Hi-C, are discussed as well. It would be more proper to put the subject of chromatin organization in the title, but relocate “the auxin-inducible protein depletion“ to a subtitle.   

 

--Fig 1: The ubiquitin conjugation enzyme (E2) is marked on the Figure, but not defined in the legend, and only the bridging ubiquitin ligase (E3) is mentioned in the text.

I also think that the adaptor, which is one of the main components of AID, should be indicated on the Figure.

 

--Lines 43-44:  “Recently it has been presented an improved version of AID 2 - 43 AID 2.1 system“

 

My suggestion is: “An improved version of AID 2 - 43 AID 2.1 system has been presented recently“

 

--Lines 66-67: “Even in the absence of condensins, chromatin was still condensed albeit

no distinct rod-shaped chromosomes were observed.“

 

Since the sentence is in the past tense: “after degradation of condensin…“ would be better than “…in the absence of condensins.“

 

--Lines 67: altering in the molecular density around mitotic chromosomes after nuclear envelope breakdown

 

I would suggest “changes in the molecular density“ or “altering the molecular density“

 

--Section 2. Key regulators of mitotic chromatin architecture.

 

Other regulators, which are no less important, deserve to be mentioned in the section. For one thing, mutual isolation of chromosomes in prophase and their clustering in telophase cannot be achieved without Ki67 (this remarks goes with the suggestion about editing the Title).

 

--Fig 2:  The figure at rhe right seems superfluous, for the mini-auxin tag could be shown on the left figure, and the peculiar colouring hardly adds any significant information.

But indication of the whole motor domains (in addition to ATPase heads) would be useful.

 

--Lines 86-88: “After multiple cyclic switching the conformation between O and B shapes the size of the DNA  loop significantly increases.“

 

I would suggest: “After multiple cycles of transition (or switching) between O and B conformations“

 

-- Fig 3A: The legend says: “…~400-kb outer loops mediated by condensin II (red).“ One may think that the red helix represents condensin II. Apparently, the word “red“ should be after “loops.“

 

--Lines 141-143: “The question of how mitotic chromosomes are folded has long intrigued researchers and mutually with the development of novel techniques continues to attract intense research efforts.“

 

The word “mutually“ does not fit here.

 

--Lines 184-186: In barley mitotic chromosomes, which are much larger (522–675 Mb), the second diagonal band is positioned at a larger distance ~30 Mb with a predicted helical turn to be ~400 nm.

 

The size of chromosomes depends not only on the size of genome but also on the cell/spindle size. Although this phenomenon is mentioned below (in lines 205-212), it probably should be presented here to avoid misunderstanding.

 

--Lines 201-202: Plant metaphase chromosome spreads exhibit clearly visible helical structure, in contrast to vertebrates.

 

The expression “clearly visible helical structure“ is somewhat mysterious. For all I know, neither the peculiar holes in electronograms of plant chromosomes, nor banding observed in light microscopy reveal distinct helical structures. I would be glad to learn more about it but, unfortunately, the sentence lacks reference.

 

--Line 213:  Section 2 is supposed to be about “Key regulators of mitotic chromatin architecture.“ But subsection 2.1.2. is named “Condensins, cohesin, and the configuration of interphase chromosomes.“ Perhaps, instead of mitotic chromatin architecture, “chromatin structure in the cell cycle“ would be better.

 

Fig 4A:  I would suggest replacing one of the icons (mosquitoe or fly) with an image of budding yeast to emphasize the presence of the Rabl configuration in all biological kingdoms.

 

--Lines 535-536: “Supercoiled regions are maintained by topoisomerases“

 

“Restrained“ or “regulated“ would be more correct than “maintained“ (the latter term suggests preservation).

 

--Fig 6A: The picture seems incomplete without the nucleolus, the most prominent structure of the nucleus, crucial for chromatin organization.

 

-- Fig 6A: Compartments A and B are not separated in the left picture; both are connected to the same square.

 

--Lines 602-605: “This strategy was successfully employed for identifying a cohesin-interacting motifs in CTCF N-terminal region [99], allowing CTCF to block cohesin translocation and protect from unloading by WAPL and explaining why cohesin stalls at CTCF sites in orientation specific manner.“

 

The sentence will need editing. It is not clear what is protected.  Probably the authors meant to say “…prevent unloading of cohesin from DNA.“ As for the the last clause: “and explaining why cohesin stalls…,“ I would suggest to make a separate sentence of it.

 

--Line 615: “though with considerations“

 

The authors probably meant “with reservations.“

 

Table 1. “Comparison of AID-based CTCF complementation systems.“

 

The table is not very informative. There is no actual comparison (as to results or drawbacks), only a list of publications and methods used.

 

--Lines 691-692: “Cohesin and its cofactors: effects on interphase chromatin topology and gene expression upon  depletion“

 

This may be better: “Effects of depletion on interphase chromatin topology and gene expression.“

 

--Line 768: “Acute RNAPII loss differentially affects CRE-anchored interactions“

 

Perhaps the review should question conclusions of the cited work. One would expect multiple effects after the depletion (even brief) of RNAPII. E.g. are the effects on the enhancer-anchored loops specific?

 

--Line 776: “Transcription can also generate DNA supercoiling“

 

Rather: “Transcription does generate supercoiling“

 

--Lines 780-781: Notably, most type II topoisomerase inhibitors target both TOP2A and TOP2B, an issue that can be resolved by using degron-tagged proteins.

 

The sentence is vague. The authors probably meant that the effects of the two enzymes may be distiguished by the AID method.

 

--Lines 798-799: “…reduced negative super-coiling upon topoisomerase or RNAPII depletion may interfere cohesin extrusion activity“

 

Rather: “…interfere with cohesin extrusion activity“ or “disrupt cohesin extrusion activity.“

 

--Line 805: Current limitations of the AID system

 

Two problems are discussed in this section, basal degradation and direct effects of auxin. Perhaps it should be mentioned, that the method requires introduction of three different components (ligase complex, tag, and auxin) in the cell, which greatly complicates the procedure.

 

--Lines 836-838: “Moreover, one of the upregulated AHR pathway genes was found to change its position within the nucleus upon lamin B depletion [204], finding it difficult to distinguish  between lamin depletion effect and effect of auxin addition.“

 

I would suggest: “…which made it difficult to distinguish…“ instead of “finding it difficult to distinguish…“

 

--The MS. has no Conclusion; instead there is only a section about the method limitations.

But adding general conclusive remarks would make the MS. more interesting. The section may include hypotheses about other proteins which may be treated by AID to investigate the processes of chromatin maintenance and transformation. For example, could this method be useful in the study of inactive X-chromosome folding? Or nucleolus assembly after mitosis?  

 

 

 

Author Response

We thank the reviewers for their time spent and the insightful, constructive comments. We are pleased to take into account their recommendations in making the manuscript more accessible for a broader audience. The key revisions are summarized as follows:

We have incorporated the conclusion section. 

We have  shortened section 1 devoted to AID-technology.

We have included a summary of Ki67 functions based on the AID technology. 

Additionally, we have corrected the title, affiliations and the funding information. 

 

Comments 1: The MS. by A. Nurislamov and A. Yunusova entitled “Auxin-inducible protein depletion for chromatin studies: advantages and limitations“ reviews the experimental works employing the auxin-inducible degradation (AID) of proteins for the study of large-scale chromatin structure in the course of cell cycle, especially for revealing the role of condensins I and II, cohesin, and CTCF. Advantages of the AID method include its fast depletion kinetics and reversibility. In combination with other methods, such as Hi-C, it can provide valuable information about the formation of mitotic chromosomes and organization of the interphase chromatin.

In my opinion the MS. may be published after a minor revision. 

My specific remarks.

--The Title does not quite reflect the contents of the review. Although advantages and limitations of the AID are mentioned in the MS., it seems chiefly focused on the results of experiments. Besides, other methods, such as Hi-C, are discussed as well. It would be more proper to put the subject of chromatin organization in the title, but relocate “the auxin-inducible protein depletion“ to a subtitle.   

Response 1: We thank the Reviewer for the evaluation of our manuscript and considering it for publication. We completely agree that the title should clearly define the focus on the chromatin organization studies. To accurately reflect the paper's content we propose the revised title: “Spatial chromatin organization across the cell cycle: insights from auxin-inducible protein depletion”

Comments 2: Fig 1: The ubiquitin conjugation enzyme (E2) is marked on the Figure, but not defined in the legend, and only the bridging ubiquitin ligase (E3) is mentioned in the text.

Response 2: We have extended the legends of Figure 1 and mentioned the ubiquitin conjugation enzyme (E2 ligase). 

Comments 3: I also think that the adaptor, which is one of the main components of AID, should be indicated on the Figure.

Response 3: Thanks for the suggestion, however, in all honesty, we could not understand the meaning of the adaptor. Is it Skp1 that binds the F-box protein (OsTIR1), a scaffold protein Cullin and an Rbx? In that case, Skp1 is indicated on Figure 1. Otherwise, we’d ask the Reviewer to explain the task in more detail. 

Comments 4: --Lines 43-44:  “Recently it has been presented an improved version of AID 2 - 43 AID 2.1 system“ My suggestion is: “An improved version of AID 2 - 43 AID 2.1 system has been presented recently“

Response 4: Thanks for the suggestion, we fixed it.

Comments 5: --Lines 66-67: “Even in the absence of condensins, chromatin was still condensed albeit

no distinct rod-shaped chromosomes were observed.“ Since the sentence is in the past tense: “after degradation of condensin…“ would be better than “…in the absence of condensins.“

Response 5: We have corrected as advised.Thank you.

Comments 6:--Lines 67: altering in the molecular density around mitotic chromosomes after nuclear envelope breakdown  I would suggest “changes in the molecular density“ or “altering the molecular density“

Response 6: Thanks for the comments, we have amended as suggested.

Comments 7:--Section 2. Key regulators of mitotic chromatin architecture.

Other regulators, which are no less important, deserve to be mentioned in the section. For one thing, mutual isolation of chromosomes in prophase and their clustering in telophase cannot be achieved without Ki67 (this remarks goes with the suggestion about editing the Title).

Response 7: We thank the Reviewer for this valuable observation. In response, we have extended Section 2 by reviewing studies using AID-technologies to elucidate the role of Ki67 (Starting in line 410). 

Comments 8:--Fig 2:  The figure at rhe right seems superfluous, for the mini-auxin tag could be shown on the left figure, and the peculiar colouring hardly adds any significant information.

But indication of the whole motor domains (in addition to ATPase heads) would be useful.

Response 8: We recognize and share the reviewer's concern regarding Figure 2. Due to its dispensability we have decided to exclude it from the review. Accordingly, the numbering of the following figures is shifted.

Comments 9: --Lines 86-88: “After multiple cyclic switching the conformation between O and B shapes the size of the DNA  loop significantly increases.“

I would suggest: “After multiple cycles of transition (or switching) between O and B conformations“

Response 9: Thanks for the suggestion, we made corrections as advised. 

Comments 10:-- Fig 3A: The legend says: “…~400-kb outer loops mediated by condensin II (red).“ One may think that the red helix represents condensin II. Apparently, the word “red“ should be after “loops.“

Response 10: Thanks for the observation, we have added additional information into the legend of Figure 3 to make this clearer. 

Comments 11: --Lines 141-143: “The question of how mitotic chromosomes are folded has long intrigued researchers and mutually with the development of novel techniques continues to attract intense research efforts.“ The word “mutually“ does not fit here.

Response 11:The word “mutually” was deleted from the text. Thank you for the suggestion. 

Comments 12:--Lines 184-186: In barley mitotic chromosomes, which are much larger (522–675 Mb), the second diagonal band is positioned at a larger distance ~30 Mb with a predicted helical turn to be ~400 nm.

The size of chromosomes depends not only on the size of genome but also on the cell/spindle size. Although this phenomenon is mentioned below (in lines 205-212), it probably should be presented here to avoid misunderstanding.

Response 12: Thanks for the suggestion. We have modified the manuscript by shortening and moving a part of text to the conclusion section. For now the mention that mitotic chromosome length does not depend only on the size of the genome but also scales with cell/spindle size is followed immediately after the statement of correlation between chromosome length and size of condensin-mediated loops line 181). We hope it can dispel some misunderstanding. 

Comments 13:--Lines 201-202: Plant metaphase chromosome spreads exhibit clearly visible helical structure, in contrast to vertebrates.

 The expression “clearly visible helical structure“ is somewhat mysterious. For all I know, neither the peculiar holes in electronograms of plant chromosomes, nor banding observed in light microscopy reveal distinct helical structures. I would be glad to learn more about it but, unfortunately, the sentence lacks reference.

Response 13: Thanks for the observation. We share the reviewer's concern about the absence of references. Writing this sentence we have referred to the early works on the structure of metaphase chromosomes and chromonema. Unfortunately, we were unable to find any good evidence describing the ‘clearly visible’ helical pattern of mitotic chromosomes in plants in the modern literature. More recent studies describing the helical organization for chromosomes relied on genome-wide chromatin contact profiles with Hi-C data not microscopical observation. So we decided to revise this paragraph and eliminate this vague phrasing. 

Comments 14 --Line 213:  Section 2 is supposed to be about “Key regulators of mitotic chromatin architecture.“ But subsection 2.1.2. is named “Condensins, cohesin, and the configuration of interphase chromosomes.“ Perhaps, instead of mitotic chromatin architecture, “chromatin structure in the cell cycle“ would be better.

Response 14 Thanks for the suggestion. Since section 2 is entirely devoted to regulators of mitotic chromatin architecture we would like to retain the title of section 2. However, to eliminate the aforementioned discrepancy we have changed the title of section 2.1.2 t from “Condensins, cohesin, and the configuration of interphase chromosomes.“ to “Condensins, cohesin and the transition of mitotic architectural features in the subsequent interphase”  

Comments 15  Fig 4A:  I would suggest replacing one of the icons (mosquitoe or fly) with an image of budding yeast to emphasize the presence of the Rabl configuration in all biological kingdoms.

Response 15:Thanks for the suggestion. We have changed the mosquito icon to budding yeast as advised. 

Comments 16: --Lines 535-536: “Supercoiled regions are maintained by topoisomerases“

 “Restrained“ or “regulated“ would be more correct than “maintained“ (the latter term suggests preservation).

Response 16: Thank you for your observation. We updated this sentence: “Supercoiled regions are regulated by topoisomerases…”

Comments 17: --Fig 6A: The picture seems incomplete without the nucleolus, the most prominent structure of the nucleus, crucial for chromatin organization.

-- Fig 6A: Compartments A and B are not separated in the left picture; both are connected to the same square.

Response 17: We updated the Figure 6 according to your suggestions. Now Fig.6A presents a more detailed schematic representation of interphase nucleus.

Comments 18: --Lines 602-605: “This strategy was successfully employed for identifying a cohesin-interacting motifs in CTCF N-terminal region [99], allowing CTCF to block cohesin translocation and protect from unloading by WAPL and explaining why cohesin stalls at CTCF sites in orientation specific manner.“

The sentence will need editing. It is not clear what is protected.  Probably the authors meant to say “…prevent unloading of cohesin from DNA.“ As for the the last clause: “and explaining why cohesin stalls…,“ I would suggest to make a separate sentence of it.

Response 18: We updated this sentence according to your comments: “This strategy was successfully employed for identifying a cohesin-interacting motifs in CTCF N-terminal region [99] that allow CTCF to block cohesin translocation and protect cohesin from unloading by WAPL, thereby explaining why cohesin stalls at CTCF sites in orientation specific manner.”

Comments 19:--Line 615: “though with considerations“

The authors probably meant “with reservations.“

Response 19:Thank you for your observation. We updated this sentence: “This approach revealed mutation-specific effects on proliferation and binding, with potential caveats, including competition between endogenous and mutant CTCF and increased protein dosage due to coexpression”

Comments 20:Table 1. “Comparison of AID-based CTCF complementation systems.“

The table is not very informative. There is no actual comparison (as to results or drawbacks), only a list of publications and methods used.

Response 20:Thank you for your comment. Indeed, this table doesn’t provide any pros and cons in comparison. Since this information is already mentioned in the text, we decided to remove Table 1.

Comments 21:--Lines 691-692: “Cohesin and its cofactors: effects on interphase chromatin topology and gene expression upon  depletion“

This may be better: “Effects of depletion on interphase chromatin topology and gene expression.“

Response 21: According to your suggestion, we changed this subtitle to “Cohesin and its cofactors: effects of depletion on interphase chromatin topology and gene expression”

Comments 22:- --Line 768: “Acute RNAPII loss differentially affects CRE-anchored interactions“

Perhaps the review should question conclusions of the cited work. One would expect multiple effects after the depletion (even brief) of RNAPII. E.g. are the effects on the enhancer-anchored loops specific?

Response 22: We thank the reviewer for the suggestion. We extended the sentence with hypothesis mentioned in referred article:
“Acute RNAPII loss differentially affects CRE-anchored interactions: although enhancers and promoters share similar epigenetic features, promoter–promoter loops remain largely unaffected, whereas enhancer-anchored loops are markedly reduced [122], suggesting that distinct, yet poorly defined mechanisms may contribute to spatial communication between promoters.”

Given the limited data on RNAPII depletion and promoter–enhancer communication, we are currently unable to draw firm conclusions from these results. We have instead highlighted this knowledge gap as a topic for future investigation in the Conclusion section.

Comments 23:--Line 776: “Transcription can also generate DNA supercoiling“

Rather: “Transcription does generate supercoiling“

Response 23:Thank you for your observation. We refined this sentence to “Transcription also generates DNA supercoiling”

--Lines 780-781: Notably, most type II topoisomerase inhibitors target both TOP2A and TOP2B, an issue that can be resolved by using degron-tagged proteins.

Comments 24:The sentence is vague. The authors probably meant that the effects of the two enzymes may be distiguished by the AID method.

Response 24:Thank you for your comment. We refined this part for clarity  “Notably, most type II topoisomerase inhibitors affect both TOP2A and TOP2B, making it difficult to separate their individual functions. This limitation can be addressed by using degron-tagged TOP2A or TOP2B, which allows selective depletion of each protein”

Comments 25:--Lines 798-799: “…reduced negative super-coiling upon topoisomerase or RNAPII depletion may interfere cohesin extrusion activity“

Rather: “…interfere with cohesin extrusion activity“ or “disrupt cohesin extrusion activity.“ 

Response 25: Thank you for your suggestion. We refined this sentence to “...RNAPII depletion may interfere with cohesin extrusion activity”

Comments 26:--Line 805: Current limitations of the AID system

Two problems are discussed in this section, basal degradation and direct effects of auxin. Perhaps it should be mentioned, that the method requires introduction of three different components (ligase complex, tag, and auxin) in the cell, which greatly complicates the procedure.

Response 26: Thank you for your comment. We refined this part in “Current limitations” following your suggestions.

Comments 27:--Lines 836-838: “Moreover, one of the upregulated AHR pathway genes was found to change its position within the nucleus upon lamin B depletion [204], finding it difficult to distinguish  between lamin depletion effect and effect of auxin addition.“

I would suggest: “…which made it difficult to distinguish…“ instead of “finding it difficult to distinguish…“ 

Response 27: We changed this part of the sentence to “Moreover, one of the upregulated AHR pathway genes was reported to reposition within the nucleus upon lamin B depletion [204], complicating the distinction between effects caused by lamin depletion and those resulting from IAA treatment”

Comments 28:--The MS. has no Conclusion; instead there is only a section about the method limitations.

But adding general conclusive remarks would make the MS. more interesting. The section may include hypotheses about other proteins which may be treated by AID to investigate the processes of chromatin maintenance and transformation. For example, could this method be useful in the study of inactive X-chromosome folding? Or nucleolus assembly after mitosis?  

Response 28: Thanks for the observation. We have supplemented our review with a conclusion section (starting in lines 837) 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

This revised manuscript represents a significant improvement since the previous version. This reviewer supports its publication pending the following minor revisions:

1. On line 33, the genus names should be italicized according to typical convention (e.g Drosophila).

2. Starting on line 23, this reviewer thinks it would be beneficial to include 1-2 sentences related to the fact that AID technology is prominently used in the study of chromatin conformation factors (similar to one of the authors' responses). It was noted that the authors link the AID technology discussion with the chromatin conformation research starting in line 44. However, the gap between the Abstract and line 44 might be too large for audiences, especially at the very beginning. Perhaps some kind of rewording that states the importance of chromatin conformations and how the AID technology is currently the state-of-the-art approach for studying it in vivo. The authors can reiterate the idea again at the end of the section (similar to what they have now).

 

Author Response

Comments: This revised manuscript represents a significant improvement since the previous version. This reviewer supports its publication pending the following minor revisions:

Response: We thank the Reviewer for the evaluation of our work and the important suggestions. Below we include responses to the reviewer’s comments.

Comments 1: On line 33, the genus names should be italicized according to typical convention (e.g Drosophila).

Response 1: Thanks for the observation, we have corrected as advised.

Comments 2:  Starting on line 23, this reviewer thinks it would be beneficial to include 1-2 sentences related to the fact that AID technology is prominently used in the study of chromatin conformation factors (similar to one of the authors' responses). It was noted that the authors link the AID technology discussion with the chromatin conformation research starting in line 44. However, the gap between the Abstract and line 44 might be too large for audiences, especially at the very beginning. Perhaps some kind of rewording that states the importance of chromatin conformations and how the AID technology is currently the state-of-the-art approach for studying it in vivo. The authors can reiterate the idea again at the end of the section (similar to what they have now).

Response 2: Thanks for the suggestion, we have revised this paragraph per your advice.