Condensin I DC has a Functional ATPase That is Required for X-Chromosome Dosage Compensation in C. elegans

  1. Bahaar Chawla
  2. Suchi Jatia
  3. Dillon E Sloan
  4. Gyorgyi Csankovszki

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Abstract

Dosage compensation (DC) in C. elegans utilizes a condensin complex that resembles mitotic condensins, but differs by one subunit, DPY-27. DPY-27 replaces SMC-4, one of the Structural Maintenance of Chromosome (SMC) proteins that is responsible for hydrolyzing ATP, required for condensation of DNA and other mitotic condensin functions. To understand if the ATPase function is required in DC, we first demonstrated that DPY-27 is capable of hydrolyzing ATP in vitro. Then, we used CRISPR/Cas9-mediated genome editing to generate an ATPase mutation in dpy-27 and demonstrated that this mutation results in a loss of DC. Specifically, we found that without ATPase function, DPY-27 containing condensin I DC has reduced capacity to bind DNA, condense the X chromosomes, and facilitate H4K20me1 enrichment on the X-chromosomes. Our results suggest that condensin I DC , like mitotic condensins, uses ATP hydrolysis to perform its functions, making C. elegans DC a model for how activities attributed to mitotic condensins can be used to regulate gene expression.

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  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    We thank the reviewers for their thoughtful comments and suggestions. Our plans for revisions are first summarized. Below you can find the original reviews and our responses and detailed plans (indicated by "Response").

    Revision plan summary:

    1. Many of the concerns can be addressed by changes in the text and better explanations of how the experiments were done. These changes are detailed in the point-by-point responses.
    2. The reviewers suggested experiments such as ChIP-seq and immunoprecipitation which require collection of a large number of mutants. Since our mutants are sterile, the line needs to be maintained as heterozygotes, from which we can pick out individual mutant worms. Therefore, with the current reagents it is impossible to collect mutants in sufficient quantities for ChIP-seq or IP. We understand that it limits the conclusions that can be drawn.
    3. For some figures, additional quantification of fluorescence signal will be done to show differences between mutant and wild type.
    4. A few experiments will be repeated:
    5. We will repeat the ATPase assays shown on Fig 1 with additional independently prepared and purified protein samples.
    6. Additional replicates will be performed for the few immunofluorescence experiments that were only performed once. Point-by-point responses:

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Dosage compensation (DC) in C. elegans involves halving the gene expression from the two hermaphrodite X chromosomes to match the output of the single X in male worms. The key regulator of this repression is a specialized condensin complex, which is defined by a dedicated SMC-4 paralog, termed DPY-27. SMC-4 in other animals is an ATPase that functions as a motor of loop extrusion in cohesion complexes. In their current manuscript, Chawla et al. assessed whether DPY-27 has ATPase function and whether this activity is required for dosage compensation. It had previously been shown that an ATPase-deficient 'EQ' mutant DPY-27 protein interacts with other DC complex members, yet fails to localize to the X. This observation was made with an extra copy of DPY-GFP expressed in addition to the endogenous wildtype protein [Ref 77]. No dominant negative effect was observed. The authors have now engineered the 'EQ' mutation into the endogenous gene locus and genetically generated hetero- and homozygous ATPase mutant worms. Their data suggest that the ATPase activity is required or X-chromosome localization, complex assembly, chromosome compaction as well as enrichment of H4K20me1 on the dampened X chromosome.

    Major comments:

    1. ATPase assays, Figure 1.Preparations of individual recombinant proteins may vary significantly and may occasionally show much reduced enzymatic activity. A conclusion about the failure of an ATPase activity should not be concluded from a single preparation, but several protein preps need to be tested, which then serve as 'biological replicates' for the in vitro reaction. Apparently, the ATPase assays shown only involved technical replicates, which is not sufficient.

    Response: We will express and purify additional protein samples and will repeat the assay.

    CRISPR-mediated engineering may lead to unwanted reactions, exemplified by the 'indel' mutation that was recovered in one clone. As a good practice and important control, the sequences of the mutated alleles in the worms should be determined by sequencing of PCR products. Restrictions enzyme cleavage or gel electrophoresis of the PCR products is not sufficient to document the nature of the mutation.

    Response: The sequence of the edit was confirmed by Sanger sequencing. We will make it clear in the text.

    All IF data need to be collected from at least 2 biological replicates, i.e. the experiment must have been carried out independently on two different days. The replicates should deliver consistent results. The number of independent replicates should be mentioned in each figure legend.

    Response: Most of our experiments were performed multiple times. We will indicate the number of replicates in the figure legends. The one or two experiments that were only performed once, will be repeated an additional time.

    The expression levels of wildtype and mutant proteins are concluded from IFM. This is very qualitative; quantitative measurements would strengthen the paper.

    Response: We will quantify fluorescence intensity on our existing images to show differences between mutant and wild type.

    Figure 4B: What are the criteria for classification of the three classes of mutant nuclei? To the uninitiated eye they look very similar. I am a bit worried about the human bias, if such diffuse staining are to be categorized. The two categories of localization need be documented better.

    Response: We will provide more images to show the range of phenotypes and provide a better explanation of how they were classified. We will also try a few ways to quantify “diffuseness” to provide a numerical readout.

    Figure 5: volume of the X chromosome. Related to (5): Apparently, the mask that contains the X chromosome was drawn by hand on each individual nucleus? I find it very difficult to see how the X chromosomal territory would be assessed in the examples shown. I would be good to see a panel of nuclei, in which the masks are visible. I think the analysis should be blinded, in which a researcher not involved in the analysis draws masks on coded nuclei and their classes are only revealed later. The same concern holds for the FISH/IP overlaps or DPY-27/SDC-2 overlaps.

    Response: The masks used were not drawn by hand but were based on fluorescence intensity thresholds. We will make a supplementary figure that shows the masks used for quantification to help clarify how the experiment and quantification were performed.

    For figure 5, age-matched hermaphrodites were analyzed. How was the age determined and what would be the consequence of age-variations? What is the effect of the mutations on development?

    Response: For our staining experiments, we routinely use young adult which we define as 24 hr past larval L4 stage. At this stage, young adults have started laying eggs. We have unpublished data that shows that dosage compensation and chromosome compaction deteriorates with age. To avoid using old worms in our assays, we pick L4 larvae, and then use them for experiments the following day.

    Minor comments:

    1. The labeling of p-values as a-f in the figures with the values listed in a supplemental table is not comfortable. The p-values corresponding to the letters should be listed in the corresponding legends.

    Response: p values can be added to the figure or the figure legend (they are currently in supplementary tables).

    How were the concentrations of the ATPase preparations determined? It would help to see a proteins gel in the supplement to assess their purity.

    Response: Concentrations were determined using a spectrometer. We can show protein gels of the preparations as a supplementary figure.

    In figure 1, heterodimers are assumed, but not shown. Do they dimerize under these conditions?

    Response: We can cite papers from others that show heterodimerization in these conditions (for example, Hassler et al, 2019).

    Reviewer #1 (Significance (Required)):

    Significance: The involvement of the ATPase function of DPY-27 was somewhat expected, in light of the earlier findings published in reference 77 using a transgene. The current study confirms and extends these earlier findings. In principle, the genetic experiment presented here is stronger, if documented better.

    Strengths: The study investigates endogenous proteins and measures different phenomena known to be correlated from previous work. The data are internally consistent.

    Limitations: The lack of biological replicates, and unclear procedures of how to draw the IF masks that underlie the conclusions about X chromosome (co)localization and nuclear volume determination render the argument less convincing. For this reviewer, who is not in the C. elegans field, the analysis of mutant phenotypes is difficult to follow. The conclusions are based on only one type of experiment. In reference 77, the X chromosome binding was done by ChIP-seq, clearly a superior, complementary method.

    Response: As explained above, since the strain has to be maintained as a heterozygote, we are unable to collect enough mutants for a ChIP-seq experiment. We can perform and better document the experimental replicates and we can better explain the quantification methods used.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary: The authors analyzed the ATPase function of an SMC-4 variant required for dosage compensation in C. elegans. They made a single amino acid mutation that significantly reduced ATPase activity of the protein as shown by in vitro ATP hydrolysis. They showed that the mutation results in the phenotypic consequences of those shown for other DC mutants, including viability assay, immunofluorescence and DNA FISH. These results demonstrate the important role of ATPase activity in transcription repression.

    Major comments:

    • Are the key conclusions convincing? The key conclusion that DPY-27 has ATPase activity and using a classic mutation that reduces it largely eliminates its function is convincing. The interpretation of the IF experiments to build the model in the final figure requires stronger evidence, as commented below in additional experiment section.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Yes, as explained below.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    The main issue with the current model is that the authors assume that the EQ proteins that they are analyzing is in complex with the rest of the condensin IDC subunits. However, there is no evidence in the paper suggesting that this occurs. The results are consistent with the possibility that a large portion of the DPY27-EQ is not in a complex.

    IP-western experiments comparing the proportion of other subunits pulled down by the wild type versus the EQ mutant (perhaps extract from ~50% EQ containing population could be reached) is needed to understand the incorporation of the EQ mutant in the complex. This is particularly important for the interpretation of the data in Figure 4A, where 70% of the nuclei show diffuse CAPG-1 and DPY-27 EQ. Is this signal due to disassembled subunits diffusing freely, or as depicted in the model figure, bound less stably everywhere? The immunofluorescence results are consistent with both EQ mutation 1) forming a full complex and unstably binding or 2) destabilizing the complex but incompletely assembled complexes sustaining a pool of free EQ detected by the immunofluorescence experiments.

    Response: We agree that to conclusively show interactions, an IP would be necessary. However, as explained above for ChIP, it is not possible to collect enough mutants to make enough protein extract for an IP. An IP in heterozygous worms is also not ideal, as it would be nearly impossible to distinguish wild protein from the mutant. The antibody we used recognizes the N terminus, which is identical in the two proteins. The only way to distinguish them would be mass spec. However, during the fragmentation process for mass spec, Q can deaminate to E, which would complicate interpretation of our data. To do this experiment properly, we would need to introduce a different tag into the mutant protein. With the current reagents, an IP is not possible.

    Instead, we have to rely on indirect evidence. The fact that DPY-27 and CAPG-1 colocalize (figure 4) does provide some support for the hypothesis. From previous studies,including our recent publication Trombley et al PLoS Genetics 2025, we know that the condensin IDC complex is not stable unless all subunits are present. It is therefore highly unlikely, although not impossible, that what we detect is diffuse individual subunits.

    We can make changes in the text to soften this claim and better discuss the caveats of the experiment and the conclusions.

    Along the same point, authors show that EQ protein that binds to the X is incapable of bringing H4K20me1, which is consistent with the possibility that a large portion of the EQ protein is not in a complex. : "To our surprise, we observed that there was no discernable enrichment of H4K20me1, even though there is discernable enrichment of DPY-27 EQ on the X chromosomes in the dpy-27 EQ mutants (Figure 8A).

    Response: There is an important difference. CAPG-1 and DPY-27 are both members of condensin IDC. The five subunits of this complex depend on each other for stability. DPY-21, the protein that introduces the H4K20me1 mark, also localizes to the X chromosomes, but is not part of condensin IDC. Condensin IDC is able to localize to the X chromosomes in the absence of DPY-21, and is not dependent on DPY-21 for stability. However, DPY-21 is dependent on condensin IDC for X localization (Yonker et al 2003). It is then possible that the mutant condensin IDC is X-bound, but it is unable to recruit DPY-21. We can clarify this in the text.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. It is unclear how long it would take to collect enough het/mutant worms can be collected for IP-western. Without additional evidence, interpretation of the data would be affected.

    Response: As explained above, collecting enough mutant worms is essentially impossible. Collecting enough heterozygotes is possible, but distinguishing the mutant protein from the wild type in hets is not.

    • Are the data and the methods presented in such a way that they can be reproduced? Yes
    • Are the experiments adequately replicated and statistical analysis adequate? Yes, except the presentation of the test (see minor comment below)

    Minor comments:

    • Specific experimental issues that are easily addressable. The use of letters for statistical test result is confusing and the figure legend is not clear about what actual p values were produced "Letters represent multiple comparison p values, with different letters indicating statistically significant differences, and any repeated letter demonstrating no significance. " Providing the values at a reasonably concise manner in the legend will help the reader a lot.

    Response: P values can be added to the figures, or the legend

    • Are prior studies referenced appropriately? The authors state that "Surprisingly, this mutant did not phenocopy the transgenic EQ mutant in [77], .." however in the previous paragraph, the authors state that the transgenic was expressed in the presence of wild type copy. Therefore, the endogenous mutant showing phenotypes rather than the transgenic is rather expected.

    Response: What we referred to were ways in which the protein behaved (for example in ability to bind to the X at all), and not mutant phenotypes of worms. We can clarify this in the text.

    The authors state that "One possible explanation could be that mitotic condensation has multiple drivers of equal consequence including changes in histone modifications [129], whereas condensation of dosage compensated X chromosomes is predominantly dependent on the DCC. " In a dpy-21 mutant, X chromosome decondenses but DPY-27 stays on the chromosome. Therefore, the effect of the EQ mutation may be due to lack of H4K20me1 enrichment in addition to the lack of loop extrusion.

    Response: We can add the role of H4K20me1 to the discussion.

    • Are the text and figures clear and accurate? Yes
    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions? The Pearson correlation coefficient for assessing colocalization between SDC-2 and DPY-27 was helpful for quantification, because there is a lot of background signal that makes the support for or lack of colocalization with the X in the other IF/FISH figures difficult to assess. Additionally, please provide information on how chromatic aberration was assessed when analyzing colocalization experiments.

    Response: Chromatic aberration was not considered for these experiments.

    Reviewer #2 (Significance (Required)):

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. Although long assumed to be a functional SMC, the demonstration of DPY-27 function depending on ATPase activity is important. This demonstrates that an X-specific condensin retained its SMC activity.

    • Place the work in the context of the existing literature (provide references, where appropriate). The authors do an adequate job in doing this in their discussion.

    • State what audience might be interested in and influenced by the reported findings. The field of 3D genome organization and function would be influenced by the reported findings.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Genomic analyses of 3D genome organization and gene expression.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors analyzed the ATPase function of an SMC-4 variant required for dosage compensation in C. elegans. They made a single amino acid mutation that significantly reduced ATPase activity of the protein as shown by in vitro ATP hydrolysis. They showed that the mutation results in the phenotypic consequences of those shown for other DC mutants, including viability assay, immunofluorescence and DNA FISH. These results demonstrate the important role of ATPase activity in transcription repression.

    Major comments:

    • Are the key conclusions convincing? The key conclusion that DPY-27 has ATPase activity and using a classic mutation that reduces it largely eliminates its function is convincing. The interpretation of the IF experiments to build the model in the final figure requires stronger evidence, as commented below in additional experiment section.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Yes, as explained below.

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    The main issue with the current model is that the authors assume that the EQ proteins that they are analyzing is in complex with the rest of the condensin IDC subunits. However, there is no evidence in the paper suggesting that this occurs. The results are consistent with the possibility that a large portion of the DPY27-EQ is not in a complex.

    IP-western experiments comparing the proportion of other subunits pulled down by the wild type versus the EQ mutant (perhaps extract from ~50% EQ containing population could be reached) is needed to understand the incorporation of the EQ mutant in the complex. This is particularly important for the interpretation of the data in Figure 4A, where 70% of the nuclei show diffuse CAPG-1 and DPY-27 EQ. Is this signal due to disassembled subunits diffusing freely, or as depicted in the model figure, bound less stably everywhere? The immunofluorescence results are consistent with both EQ mutation 1) forming a full complex and unstably binding or 2) destabilizing the complex but incompletely assembled complexes sustaining a pool of free EQ detected by the immunofluorescence experiments.

    Along the same point, authors show that EQ protein that binds to the X is incapable of bringing H4K20me1, which is consistent with the possibility that a large portion of the EQ protein is not in a complex. : "To our surprise, we observed that there was no discernable enrichment of H4K20me1, even though there is discernable enrichment of DPY-27 EQ on the X chromosomes in the dpy-27 EQ mutants (Figure 8A).

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. It is unclear how long it would take to collect enough het/mutant worms can be collected for IP-western. Without additional evidence, interpretation of the data would be affected.

    • Are the data and the methods presented in such a way that they can be reproduced? Yes

    • Are the experiments adequately replicated and statistical analysis adequate? Yes, except the presentation of the test (see minor comment below)

    Minor comments:

    • Specific experimental issues that are easily addressable. The use of letters for statistical test result is confusing and the figure legend is not clear about what actual p values were produced "Letters represent multiple comparison p values, with different letters indicating statistically significant differences, and any repeated letter demonstrating no significance. " Providing the values at a reasonably concise manner in the legend will help the reader a lot.

    • Are prior studies referenced appropriately? The authors state that "Surprisingly, this mutant did not phenocopy the transgenic EQ mutant in [77], .." however in the previous paragraph, the authors state that the transgenic was expressed in the presence of wild type copy. Therefore, the endogenous mutant showing phenotypes rather than the transgenic is rather expected.

    The authors state that "One possible explanation could be that mitotic condensation has multiple drivers of equal consequence including changes in histone modifications [129], whereas condensation of dosage compensated X chromosomes is predominantly dependent on the DCC. " In a dpy-21 mutant, X chromosome decondenses but DPY-27 stays on the chromosome. Therefore, the effect of the EQ mutation may be due to lack of H4K20me1 enrichment in addition to the lack of loop extrusion.

    • Are the text and figures clear and accurate? Yes
    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions? The Pearson correlation coefficient for assessing colocalization between SDC-2 and DPY-27 was helpful for quantification, because there is a lot of background signal that makes the support for or lack of colocalization with the X in the other IF/FISH figures difficult to assess. Additionally, please provide information on how chromatic aberration was assessed when analyzing colocalization experiments.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field. Although long assumed to be a functional SMC, the demonstration of DPY-27 function depending on ATPase activity is important. This demonstrates that an X-specific condensin retained its SMC activity.

    • Place the work in the context of the existing literature (provide references, where appropriate). The authors do an adequate job in doing this in their discussion.

    • State what audience might be interested in and influenced by the reported findings. The field of 3D genome organization and function would be influenced by the reported findings.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    Genomic analyses of 3D genome organization and gene expression.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Dosage compensation (DC) in C. elegans involves halving the gene expression from the two hermaphrodite X chromosomes to match the output of the single X in male worms. The key regulator of this repression is a specialized condensin complex, which is defined by a dedicated SMC-4 paralog, termed DPY-27. SMC-4 in other animals is an ATPase that functions as a motor of loop extrusion in cohesion complexes. In their current manuscript, Chawla et al. assessed whether DPY-27 has ATPase function and whether this activity is required for dosage compensation. It had previously been shown that an ATPase-deficient 'EQ' mutant DPY-27 protein interacts with other DC complex members, yet fails to localize to the X. This observation was made with an extra copy of DPY-GFP expressed in addition to the endogenous wildtype protein [Ref 77]. No dominant negative effect was observed. The authors have now engineered the 'EQ' mutation into the endogenous gene locus and genetically generated hetero- and homozygous ATPase mutant worms. Their data suggest that the ATPase activity is required or X-chromosome localization, complex assembly, chromosome compaction as well as enrichment of H4K20me1 on the dampened X chromosome.

    Major comments:

    1. ATPase assays, Figure 1.Preparations of individual recombinant proteins may vary significantly and may occasionally show much reduced enzymatic activity. A conclusion about the failure of an ATPase activity should not be concluded from a single preparation, but several protein preps need to be tested, which then serve as 'biological replicates' for the in vitro reaction. Apparently, the ATPase assays shown only involved technical replicates, which is not sufficient.

    2. CRISPR-mediated engineering may lead to unwanted reactions, exemplified by the 'indel' mutation that was recovered in one clone. As a good practice and important control, the sequences of the mutated alleles in the worms should be determined by sequencing of PCR products. Restrictions enzyme cleavage or gel electrophoresis of the PCR products is not sufficient to document the nature of the mutation.

    3. All IF data need to be collected from at least 2 biological replicates, i.e. the experiment must have been carried out independently on two different days. The replicates should deliver consistent results. The number of independent replicates should be mentioned in each figure legend.

    4. The expression levels of wildtype and mutant proteins are concluded from IFM. This is very qualitative; quantitative measurements would strengthen the paper.

    5. Figure 4B: What are the criteria for classification of the three classes of mutant nuclei? To the uninitiated eye they look very similar. I am a bit worried about the human bias, if such diffuse staining are to be categorized. The two categories of localization need be documented better.

    6. Figure 5: volume of the X chromosome. Related to (5): Apparently, the mask that contains the X chromosome was drawn by hand on each individual nucleus? I find it very difficult to see how the X chromosomal territory would be assessed in the examples shown. I would be good to see a panel of nuclei, in which the masks are visible. I think the analysis should be blinded, in which a researcher not involved in the analysis draws masks on coded nuclei and their classes are only revealed later. The same concern holds for the FISH/IP overlaps or DPY-27/SDC-2 overlaps.

    7. For figure 5, age-matched hermaphrodites were analyzed. How was the age determined and what would be the consequence of age-variations? What is the effect of the mutations on development?

    Minor comments:

    1. The labeling of p-values as a-f in the figures with the values listed in a supplemental table is not comfortable. The p-values corresponding to the letters should be listed in the corresponding legends.

    2. How were the concentrations of the ATPase preparations determined? It would help to see a proteins gel in the supplement to assess their purity.

    3. In figure 1, heterodimers are assumed, but not shown. Do they dimerize under these conditions?

    Significance

    Significance: The involvement of the ATPase function of DPY-27 was somewhat expected, in light of the earlier findings published in reference 77 using a transgene. The current study confirms and extends these earlier findings. In principle, the genetic experiment presented here is stronger, if documented better.

    Strengths: The study investigates endogenous proteins and measures different phenomena known to be correlated from previous work. The data are internally consistent.

    Limitations: The lack of biological replicates, and unclear procedures of how to draw the IF masks that underlie the conclusions about X chromosome (co)localization and nuclear volume determination render the argument less convincing. For this reviewer, who is not in the C. elegans field, the analysis of mutant phenotypes is difficult to follow. The conclusions are based on only one type of experiment. In reference 77, the X chromosome binding was done by ChIP-seq, clearly a superior, complementary method.

  5. Version published to 10.1101/2025.02.05.636654 on bioRxiv