Interactions with multiple inner kinetochore proteins determine mitotic localization of FACT
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Review Commons)
Abstract
The FAcilitates Chromatin Transcription (FACT) complex is a dimeric histone chaperone that operates on chromatin during transcription and replication. FACT also interacts with a specialized centromeric nucleosome containing the histone H3 variant centromere protein A (CENP-A) and with CENP-TW, two subunits of the constitutive centromere–associated network (CCAN), a 16-protein complex associated with CENP-A. The significance of these interactions remains elusive. Here, we show that FACT has multiple additional binding sites on CCAN. The interaction with CCAN is strongly stimulated by casein kinase II phosphorylation of FACT. Mitotic localization of FACT to kinetochores is strictly dependent on specific CCAN subcomplexes. Conversely, CENP-TW requires FACT for stable localization. Unexpectedly, we also find that DNA readily displaces FACT from CCAN, supporting the speculation that FACT becomes recruited through a pool of CCAN that is not stably integrated into chromatin. Collectively, our results point to a potential role of FACT in chaperoning CCAN during transcription or in the stabilization of CCAN at the centromere during the cell cycle.
Article activity feed
-
-
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Response to Reviewers
We thank all three reviewers for their time and engagement, for their generally supportive comments, and for raising some important concerns. We are pleased to submit a significantly revised manuscript where we tried to accommodate all suggested changes and extensions. Importantly, we have included additional experiments that support the relevance of FACT for the overall stability of the inner kinetochore. Below is a detailed point-to-point response. Changes to the manuscript relative to the original submission have been highlighted at the end of this response.
__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __
Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Response to Reviewers
We thank all three reviewers for their time and engagement, for their generally supportive comments, and for raising some important concerns. We are pleased to submit a significantly revised manuscript where we tried to accommodate all suggested changes and extensions. Importantly, we have included additional experiments that support the relevance of FACT for the overall stability of the inner kinetochore. Below is a detailed point-to-point response. Changes to the manuscript relative to the original submission have been highlighted at the end of this response.
__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __
Summary: The authors investigated molecular interactions between CCAN and FACT complexes. They revealed contact domains in FACT and the cognate subcomplexes of CCAN by in vitro reconstitution from recombinant proteins followed by SEC and pull-down assay.
They also revealed a couple of potential means to control interactions between FACT and the CCAN. They conclude that phosphorylation of FACT by CK2 is essential for binding to the CCAN; and CENP-A nucleosomes or DNA prevent CCAN from interacting with FACT.
Major comments:
The authors show that phosphorylation of FACT is essential for interaction with CCAN.
They argue that this phosphorylation is partly catalysed by CK2.
My concerns are:
-1- The authors assume that the sites phosphorylated in insect cell are also phosphorylated in human cells. However, it is not demonstrated which residues are phosphorylated in human cells and whether they match those from insect cells. Whether phosphorylation of recombinant proteins in insect cells is physiologically relevant to mammalian is uncertain. Kinetochore components are not very well conserved evolutionarily, thus their regulation may be different.
We thank the reviewer for these remarks, which we answer together with point 2 below.
-2- They identify several residues which are phosphorylated by CK2 in vitro. However, these are not necessarily the same sites as those phosphorylated in insect cells or more importantly in human cells. The in vitro phosphorylation by CK2 did not restore binding affinity in full, suggesting phosphorylation at other sites may be critical for interaction with CCAN. Further evidence is required to support the claim that those sites are phosphorylated in vivo and important for integrity of kinetochores in mitosis.
Our analysis of FACT phosphorylation represents a relatively small part of a very data-rich paper, and was not meant to be exhaustive. Nonetheless, the reviewer's comments are important and well received. We agree that we have no definitive evidence that the same sites are phosphorylated in insect cells, in vitro, and in human cells. However, it is quite remarkable, and supports specificity, that the interaction with FACT, lost after dephosphorylation in vitro, is restored with CK2 and not with three additional mitotic kinases (CDK1, Aurora B, and PLK1 - Figure S8D). We also note that S437, S444 and S667 of SSRP1, which were phosphorylated by CK2 in vitro, were also detected as phosphorylated sites on recombinant FACT purified from insect cells (Table S1). So collectively, while we agree with the reviewer that the analysis of FACT phosphorylation is not complete, it does significantly add to the manuscript and more generally to the FACT field.
Minor comments:
Figure 1H
I am confused with 4 stars shown at the top of the right plot. If the 4 stars are meant to show a significant difference, then the statement in the text (line 123) is not correct.
"SSRP1 localization was also largely unaffected ..."
Similar discrepancies are found in Figures 3H (line 212), Figures S2 (line 122), S5I (line 197), and S6I (line209). Figure S6H is not referred to anywhere.
There is no description for the numbers at the top. Are they mean values? Do red bars represent S.D.?
We thank the reviewer for these comments. In this revised version of the manuscript, we have substantially improved the quantification and statistical analysis. The main problem with the previous automated analysis is that the non-circular shape of the CREST-staining led to inconsistencies with the statistical analysis and the statement. In contrast, the same analysis works well when the CENP-C signal was used for KT identification (e.g. in Figure 3), as CENP-C staining yields well separated circular signals ideally suited for our automated identification of individual KTs and subsequent retrieval of fluorescence intensities. We have therefore modified our analysis macro for all experiments where CREST was used as a reference. We used Othsu-thresholding of the DAPI signal for generating a segmentation mask per each cell. Then, integrated cell intensities were calculated for each fluorescence channel based on the DAPI reference mask. With these adjustments, the statistical analyses (Figures 1, S2, S3) support the claim presented. We have updated the Methods and Results sections to reflect the revised analysis.
The numbers on top of the graphs are median values, bars represent interquartile ranges. We have now included the description in figure legends.
We appreciate your feedback, which prompted us to clarify and enhance the rigor of our approach.
We are now referring to Fig. S6H in the text.
Figure 1D
There is no description of R* to the right of gels.
We have added a description of R* to the relevant figure legend.
Figure S2
A 4 hour nocodazole treatment is too short to drive all cells into mitosis. Is the data taken from mitotic cells only?
Yes, the data are taken only from the mitotic population. We have now clarified this in the figure legend.
Reviewer #1 (Significance (Required)):
The interaction of FACT with kinetochore components has been known for several years. However how FACT contributes to architecture or function of kinetochore is not very well understood. How the FACT complex, which is known for its established role as a histone chaperone, is involved in kinetochore assembly/architecture will attract interest in several fields of basic research including epigenetics, mitosis, structural biology.
We are grateful to the reviewer for this supportive statement that recognizes the broad potential interest of the manuscript.
Identification of CCAN subunits that interact with FACT is important for future analysis to understand the kinetochore function of FACT. The authors identified OPQRU and CHIKM subcomplex in addition to TW as FACT-interacting domains. These subcomplexes are geographically scattered in a 3D model of CCAN holocomplex. Stoichiometry of CCAN and FACT might be informative whether a single or multiple FACT binds to the multiple sites of CCAN. The authors do not address whether these multiple sites are occupied simultaneously, separately or sequentially.
We thank the reviewer for raising this point. As mentioned in the discussion, we have not yet been able to perform a structural analysis of the FACT/CCAN complex to determine its stoichiometry. However, we have now added a newexperiment (Figure S1B,C) where we quantified in-gel tryptophan fluorescence after analytical size-exclusion chromatography. This strongly suggests that FACT and CCAN form a complex with a 1:1 stoichiometry. Nevertheless, we cannot comment on which sites are occupied.
The statement at the end of Abstract (lines 23-25) is a speculative hypothesis without evidence for "a pool of CCAN that is not stably integrated into chromatin", "chaperoning CCAN", and "stabilisation of CCAN".
We agree with the reviewer that this is speculative, and have therefore modified the Abstract to clearly indicate this point.
__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __
FACT is a histone chaperone and is involved in various events on chromatin such as transcription and replication. In addition, FACT interacts with various kinetochore components, suggesting potential functions at the kinetochore. However, it is largely unclear how FACT functions in the kinetochore. Authors of this MS took the biochemical approach to understand roles of FACT in the kinetochore.
Authors demonstrated that FACT forms a complex with the constitutive centromere associated network (CCAN), which contains 16 subunits on centromeric chromatin, using multiple binding sites. They also showed that casein kinase II (CK2) phosphorylated FACT and dephosphorylated FACT did not bind to CCAN. Furthermore, they displayed that DNA addition disrupt the stable FACT-CCAN complex.
Overall, while authors have done solid and high-quality biochemical analyses (these are elegant), it is still unclear how FACT plays its roles in the kinetochore. Simple knockout or knockdown study on FACT might be complicated, because FACT has multi-functions. If authors can identify specific regions of FACT for interaction with CCAN, they would put specific mutations into FACT to analyze phenotype. Although they did not reach a high-resolution structure for the FACT-CCAN complex, they can utilize AlphaFold and test specific interaction regions, biochemically. Then, using such information, significance of FACT-CCAN interaction might be testable in cells. Such a kind of study would be expected. In summary, biochemical parts are beautiful, but the paper did not address significance of FACT-CCAN interaction.
We thank the reviewer for praising the biochemical work in our manuscript. The reviewer, however, also underscored the limits of our functional analysis. The reviewer proposes generating separation-of-function mutants in a minimal kinetochore-binding region. Indeed, we have identified the minimal domain for the interaction of FACT with kinetochores. However, this information is insufficient for a reliable functional analysis at this stage, as the region we identified encompasses the AIDs and the phosphorylation-rich region, both of which have been previously shown to be important for transcription and other functions. Furthermore, any suitable mutant should be tested in cells devoid of endogenous FACT, raising the concern that the resulting phenotype may be indirect.
Nonetheless, as we wanted to provide at least an initial answer to the reviewer's concern, we enriched the manuscript by adding experiments in a recently published cell line (K562-SSRP1-dTAG) where FACT levels can be controlled with a small molecule (Žumer et al. Mol Cell., 2024) and that the authors generously shared with us. In this line, which grows in suspension and that we had to adapt to grow on a substrate for imaging, we were able to deplete FACT while cells were arrested in mitosis. We are glad to report that we found a significant reduction in the kinetochore levels of CENP-TW after this treatment, which is consistent with other conclusions from our study. These experiments add an initial functional characterization of the interaction of FACT with kinetochores, and extend the significance of the manuscript. We refer to these results again below in response to specific point 5.
Specific point
Authors showed nice mitotic localization of FACT. Can they observe this localization by a usual IF? Using GFP fusion, do they observe kinetochore localization like IF experiments?
The localization of FACT was observed using pre-extraction and fixation followed by antibody staining. We have now added a panel demonstrating mitotic localization of GFP-SSRP1 at the kinetochore in transiently transfected RPE-1 cells (Fig. S2A).
On page 7, authors tested CENP-C binding to FACT and they conclude that C-teminal region of CENP-C preferentially binds to FACT. However, they used N-terminal region of CENP-C (2-545) for CCAN-FACT complex formation in entire MS. therefore, this is complicated, and story on CENP-C N-terminal region can be removed from this MS.
We were only able to purify full-length CENP-C with tags at the N- and C-terminus, including an MBP tag with a stabilizing effect. At the time of our first successful purification of full-length CENP-C, we had already established the solid phase assay using MBPFACT as a bait on amylose beads and CENP-C2-545HIKM as one of the preys. As we cannot obtain stable full-length CENP-C without MBP, this form of CENP-C is incompatible with our pull-down assay. Nevertheless, CENP-C2-545 still has low affinity for FACT, influencing the FACT/CCAN interaction independent of the PEST-rich region. We, therefore, opted for keeping this information in the manuscript.
On page 9, authors suddenly focus on N-terminal tails of CENP-Q and CENP-U. Why did they focus on this region. They should explain this. If they perform a structural prediction, they should describe this point.
Thanks for raising this point. We focused on the N-terminal tails of CENP-QU because they are known interaction hubs. We have now added a sentence to introduce this concept and citing the appropriate literature.
I agree the fact that FACT phosphorylation is required for FACT-CCAN interaction. They may explain how the phosphorylation contributes to stable FACT-CCAN interaction.
We have added a sentence explaining that FACT is known to mimic DNA, and negative charges due to phosphorylation could drive this effect. A more detailed mechanistic understanding will require identifying specific phosphorylation sites required for the interaction.
Readers really want to know phenotype, if FACT-CCAN interaction was compromised without disruption pf CCAN assembly in cells. Although I agree that FACT has some functions in the kinetochore, it is still unclear what FACT does in the kinetochore.
We wholeheartedly agree with the reviewer. As depletion of FACT by RNAi required 48 h, an unreasonably long time for this multifunctional protein. We therefore turned to engineering RPE-1 cells for rapid degradation of SSRP1. While these attempts were unsucessful, earlier this year, Žumer et al. Mol Cell., 2024 reported generating a K562-SSRP1-dTAG cell line growing in suspension. As already reported, this cell line now allowed to demonstrate a significant effect on the kinetochore stability of CENP-TW upon mitotic depletion of FACT.
Reviewer #2 (Significance (Required)):
As mentioned above, biochemical parts are beautiful, but the paper did not address significance of FACT-CCAN interaction.
We thank the reviewer for this positive assessment. In this revision, we have obtained initial evidence that FACT contributes to kinetochore stability.
__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __
Main findings:
The major findings of this paper are:
Detailed dissection of CCAN subunit interactions and requirements to bind the FACT complex using in vitro reconstituted components Binding of FACT and nucleosomes to CENP-C are mutually exclusive FACT phosphorylation by CK2 enhances interaction with CCAN FACT localization in mitosis depends on the CCAN CCAN binding to FACT is outcompeted by DNA and CENP-A nucleosomes The claims and conclusions of the paper are supported by the data and do not require additional experiments. All experiments include biological replicates and appropriate controls.
We are thankful to the reviewer for this very positive assessment of our work.
Minor comments
Intro: • Line 81: In humans [...], here it is worth mentioning that in Drosophila, FACT subunits have been shown to interact directly with the CENP-A assembly factor CAL1 (Ref 61). This paper is perfunctorily cited once in the context of its implication of FACT in CENP-A deposition, but it merits more consideration when setting up the foundational context for the present work.
We have extended the Introduction and discuss the specified paper more thoroughly.
Figure 1:
1F: Add insets.
Done.
1G and all other figures containing IFs: Avoid red/green color scheme (red-green colorblindness is fairly common, affecting about 8% of men).
Done.
1E: Please add a table summarizing interactions.
We have included this table as Fig. S1E.
Results: • It's fine to direct readers to previous work in which you reconstituted the CCAN, but the text should mention how proteins are exogenously expressed and purified (as done for FACT in line 247).
Done.
Line 113: FACT has been shown to localize to the mitotic kinetochore also in Drosophila (Ref 61).
We have included this information now.
Line 132: The authors should cite work from the Drosophila system as well when they mention centromere transcriptional activity in mitosis (e.g. https://doi.org/10.1083/jcb.201404097; https://doi.org/10.1083/jcb.201611087; and Ref 61).
We have added these citations.
Figure 2F: The authors could use a line to mark the region interacting with FACT and that interacting with CENP-A to help summarize the data in this diagram.
Done.
Figure 4: Highlight constructs n.2 (FACT^TRUNC) since these are sufficient for interaction (e.g., use a box around them).
Done.
Line 276: "CCAN decodes CENP-A^NCP..." What do the authors mean by "decodes"? This whole sentence would benefit from clearer language.
We thank the reviewer for this suggestion and have aimed for clearer language.
Figure 6: There's a lot of information in these experiments that would benefit from two schematics, one showing the mechanism of FACT + CCAN binding with DNA and one with CENP-A nucleosomes.
Done.
Discussion: The authors discuss FACT localization at kinetochores in mitosis. In Drosophila Schneider cells, FACT is observed enriched at the centromeres in both mitosis and interphase (Ref 61). The authors mention their inability to detect FACT in interphase in the discussion, but I did not find this mentioned in the results. The authors state that FACT "redistributes to the entire chromosome" upon entry into interphase. They cite Figure 1F in reference to this statement, but the staining in the early G1 panel is difficult to interpret with the low signal/noise scaling of CENP-C and the lack of zoom insets. Their protocol uses a pre-extraction step with Triton prior to fixation. Apparently, this was not enough to reveal FACT in interphase, but better images and a brief description are warranted.
We have now added a staining of SSRP1 in interphase in the panel.
It is unlikely that FACT would change its localization pattern in mitosis. A more likely possibility is that in mitosis FACT is not redistributed, but rather more tightly bound (and thus less easily extracted by Triton treatment) at kinetochores, while along the arms FACT is more readily removed by extraction because at this time transcription is repressed and FACT is likely less engaged in transcription-mediated histone destabilization.
We thank the reviewer for these remarks and have updated the Discussion.
Given the well-known function of FACT in transcription and the many studies linking transcription to centromere maintenance, including with the involvement of FACT, the model that "the localization of FACT at the kinetochore coincides with active centromeric transcription in mitosis and interphase" is very tempting. A speculative model would go a long way to help the reader visualize all these complex aspects of FACT's interactions and possible functions.
We agree with the reviewer that such a model is tempting. However, we also feel that it would be rather speculative at this stage and we feel that the manuscript does not provide sufficient data to support the model.
Reviewer #3 (Significance (Required)):
The strongest aspect of the study is the detailed characterization of protein-protein interactions, as well as competition with DNA and CENP-A nucleosomes. The siRNA experiments in cells complement this largely in vitro study. However, a limitation of the study is that it does not shed light on what FACT might be doing at the centromere. Additionally, it does not sufficiently provide context for these findings in relation to previous studies that have demonstrated the roles of FACT at the centromere in budding yeast, fission yeast, and Drosophila. Nonetheless, this study provides valuable insights into the details of FACT interactions at the kinetochore and will be of interest to readers interested in centromeres and kinetochore. I am a centromere biologist with molecular and cell biology expertise.
We are very grateful to the reviewer for his/her support.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Main findings:
The major findings of this paper are:
- Detailed dissection of CCAN subunit interactions and requirements to bind the FACT complex using in vitro reconstituted components
- Binding of FACT and nucleosomes to CENP-C are mutually exclusive
- FACT phosphorylation by CK2 enhances interaction with CCAN
- FACT localization in mitosis depends on the CCAN
- CCAN binding to FACT is outcompeted by DNA and CENP-A nucleosomes The claims and conclusions of the paper are supported by the data and do not require additional experiments. All experiments include biological replicates and appropriate controls.
Minor comments
Intro:
- Line 81: In humans …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Main findings:
The major findings of this paper are:
- Detailed dissection of CCAN subunit interactions and requirements to bind the FACT complex using in vitro reconstituted components
- Binding of FACT and nucleosomes to CENP-C are mutually exclusive
- FACT phosphorylation by CK2 enhances interaction with CCAN
- FACT localization in mitosis depends on the CCAN
- CCAN binding to FACT is outcompeted by DNA and CENP-A nucleosomes The claims and conclusions of the paper are supported by the data and do not require additional experiments. All experiments include biological replicates and appropriate controls.
Minor comments
Intro:
- Line 81: In humans [...], here it is worth mentioning that in Drosophila, FACT subunits have been shown to interact directly with the CENP-A assembly factor CAL1 (Ref 61). This paper is perfunctorily cited once in the context of its implication of FACT in CENP-A deposition, but it merits more consideration when setting up the foundational context for the present work.
Figure 1:
- 1F: Add insets.
- 1G and all other figures containing IFs: Avoid red/green color scheme (red-green colorblindness is fairly common, affecting about 8% of men).
- 1E: Please add a table summarizing interactions.
Results:
- It's fine to direct readers to previous work in which you reconstituted the CCAN, but the text should mention how proteins are exogenously expressed and purified (as done for FACT in line 247).
- Line 113: FACT has been shown to localize to the mitotic kinetochore also in Drosophila (Ref 61).
- Line 132: The authors should cite work from the Drosophila system as well when they mention centromere transcriptional activity in mitosis (e.g., https://doi.org/10.1083/jcb.201404097; https://doi.org/10.1083/jcb.201611087; and Ref 61).
- Figure 2F: The authors could use a line to mark the region interacting with FACT and that interacting with CENP-A to help summarize the data in this diagram.
- Figure 4: Highlight constructs n.2 (FACT^TRUNC) since these are sufficient for interaction (e.g., use a box around them).
- Line 276: "CCAN decodes CENP-A^NCP..." What do the authors mean by "decodes"? This whole sentence would benefit from clearer language.
- Figure 6: There's a lot of information in these experiments that would benefit from two schematics, one showing the mechanism of FACT + CCAN binding with DNA and one with CENP-A nucleosomes.
Discussion:
The authors discuss FACT localization at kinetochores in mitosis. In Drosophila Schneider cells, FACT is observed enriched at the centromeres in both mitosis and interphase (Ref 61). The authors mention their inability to detect FACT in interphase in the discussion, but I did not find this mentioned in the results. The authors state that FACT "redistributes to the entire chromosome" upon entry into interphase. They cite Figure 1F in reference to this statement, but the staining in the early G1 panel is difficult to interpret with the low signal/noise scaling of CENP-C and the lack of zoom insets. Their protocol uses a pre-extraction step with Triton prior to fixation. Apparently, this was not enough to reveal FACT in interphase, but better images and a brief description are warranted. It is unlikely that FACT would change its localization pattern in mitosis. A more likely possibility is that in mitosis FACT is not redistributed, but rather more tightly bound (and thus less easily extracted by Triton treatment) at kinetochores, while along the arms FACT is more readily removed by extraction because at this time transcription is repressed and FACT is likely less engaged in transcription-mediated histone destabilization. Given the well-known function of FACT in transcription and the many studies linking transcription to centromere maintenance, including with the involvement of FACT, the model that "the localization of FACT at the kinetochore coincides with active centromeric transcription in mitosis and interphase" is very tempting. A speculative model would go a long way to help the reader visualize all these complex aspects of FACT's interactions and possible functions.
Significance
The strongest aspect of the study is the detailed characterization of protein-protein interactions, as well as competition with DNA and CENP-A nucleosomes. The siRNA experiments in cells complement this largely in vitro study. However, a limitation of the study is that it does not shed light on what FACT might be doing at the centromere. Additionally, it does not sufficiently provide context for these findings in relation to previous studies that have demonstrated the roles of FACT at the centromere in budding yeast, fission yeast, and Drosophila. Nonetheless, this study provides valuable insights into the details of FACT interactions at the kinetochore and will be of interest to readers interested in centromeres and kinetochore.
I am a centromere biologist with molecular and cell biology expertise.
-
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
FACT is a histone chaperone and is involved in various events on chromatin such as transcription and replication. In addition, FACT interacts with various kinetochore components, suggesting potential functions at the kinetochore. However, it is largely unclear how FACT functions in the kinetochore. Authors of this MS took the biochemical approach to understand roles of FACT in the kinetochore.
Authors demonstrated that FACT forms a complex with the constitutive centromere associated network (CCAN), which contains 16 subunits on centromeric chromatin, using multiple binding sites. They also showed that casein kinase II (CK2) …
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
FACT is a histone chaperone and is involved in various events on chromatin such as transcription and replication. In addition, FACT interacts with various kinetochore components, suggesting potential functions at the kinetochore. However, it is largely unclear how FACT functions in the kinetochore. Authors of this MS took the biochemical approach to understand roles of FACT in the kinetochore.
Authors demonstrated that FACT forms a complex with the constitutive centromere associated network (CCAN), which contains 16 subunits on centromeric chromatin, using multiple binding sites. They also showed that casein kinase II (CK2) phosphorylated FACT and dephosphorylated FACT did not bind to CCAN. Furthermore, they displayed that DNA addition disrupt the stable FACT-CCAN complex.
Overall, while authors have done solid and high-quality biochemical analyses (these are elegant), it is still unclear how FACT plays its roles in the kinetochore. Simple knockout or knockdown study on FACT might be complicated, because FACT has multi-functions. If authors can identify specific regions of FACT for interaction with CCAN, they would put specific mutations into FACT to analyze phenotype. Although they did not reach a high-resolution structure for the FACT-CCAN complex, they can utilize AlphaFold and test specific interaction regions, biochemically. Then, using such information, significance of FACT-CCAN interaction might be testable in cells. Such a kind of study would be expected. In summary, biochemical parts are beautiful, but the paper did not address significance of FACT-CCAN interaction.
Specific point
- Authors showed nice mitotic localization of FACT. Can they observe this localization by a usual IF? Using GFP fusion, do they observe kinetochore localization like IF experiments?
- On page 7, authors tested CENP-C binding to FACT and they conclude that C-teminal region of CENP-C preferentially binds to FACT. However, they used N-terminal region of CENP-C (2-545) for CCAN-FACT complex formation in entire MS. therefore, this is complicated, and story on CENP-C N-terminal region can be removed from this MS.
- On page 9, authors suddenly focus on N-terminal tails of CENP-Q and CENP-U. Why did they focus on this region. They should explain this. If they perform a structural prediction, they should describe this point.
- I agree the fact that FACT phosphorylation is required for FACT-CCAN interaction. They may explain how the phosphorylation contributes to stable FACT-CCAN interaction.
- Readers really want to know phenotype, if FACT-CCAN interaction was compromised without disruption pf CCAN assembly in cells. Although I agree that FACT has some functions in the kinetochore, it is still unclear what FACT does in the kinetochore.
Significance
As mentioned above, biochemical parts are beautiful, but the paper did not address significance of FACT-CCAN interaction.
-
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
Summary:
The authors investigated molecular interactions between CCAN and FACT complexes. They revealed contact domains in FACT and the cognate subcomplexes of CCAN by in vitro reconstitution from recombinant proteins followed by SEC and pull-down assay.
They also revealed a couple of potential means to control interactions between FACT and the CCAN. They conclude that phosphorylation of FACT by CK2 is essential for binding to the CCAN; and CENP-A nucleosomes or DNA prevent CCAN from interacting with FACT.
Major comments:
The authors show that phosphorylation of FACT is essential for interaction with CCAN. They argue that this …
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
Summary:
The authors investigated molecular interactions between CCAN and FACT complexes. They revealed contact domains in FACT and the cognate subcomplexes of CCAN by in vitro reconstitution from recombinant proteins followed by SEC and pull-down assay.
They also revealed a couple of potential means to control interactions between FACT and the CCAN. They conclude that phosphorylation of FACT by CK2 is essential for binding to the CCAN; and CENP-A nucleosomes or DNA prevent CCAN from interacting with FACT.
Major comments:
The authors show that phosphorylation of FACT is essential for interaction with CCAN. They argue that this phosphorylation is partly catalysed by CK2.
My concerns are:
- The authors assume that the sites phosphorylated in insect cell are also phosphorylated in human cells. However, it is not demonstrated which residues are phosphorylated in human cells and whether they match those from insect cells. Whether phosphorylation of recombinant proteins in insect cells is physiologically relevant to mammalian is uncertain. Kinetochore components are not very well conserved evolutionarily, thus their regulation may be different.
- They identify several residues which are phosphorylated by CK2 in vitro. However, these are not necessarily the same sites as those phosphorylated in insect cells or more importantly in human cells. The in vitro phosphorylation by CK2 did not restore binding affinity in full, suggesting phosphorylation at other sites may be critical for interaction with CCAN. Further evidence is required to support the claim that those sites are phosphorylated in vivo and important for integrity of kinetochores in mitosis.
Minor comments:
Figure 1H
I am confused with 4 stars shown at the top of the right plot. If the 4 stars are meant to show a significant difference, then the statement in the text (line 123) is not correct. "SSRP1 localization was also largely unaffected ..." Similar discrepancies are found in Figures 3H (line 212), Figures S2 (line 122), S5I (line 197), and S6I (line209). Figure S6H is not referred to anywhere. There is no description for the numbers at the top. Are they mean values? Do red bars represent S.D.?
Figure 1D
There is no description of R* to the right of gels.
Figure S2
A 4 hour nocodazole treatment is too short to drive all cells into mitosis. Is the data taken from mitotic cells only?
Significance
The interaction of FACT with kinetochore components has been known for several years. However how FACT contributes to architecture or function of kinetochore is not very well understood. How the FACT complex, which is known for its established role as a histone chaperone, is involved in kinetochore assembly/architecture will attract interest in several fields of basic research including epigenetics, mitosis, structural biology.
Identification of CCAN subunits that interact with FACT is important for future analysis to understand the kinetochore function of FACT. The authors identified OPQRU and CHIKM subcomplex in addition to TW as FACT-interacting domains. These subcomplexes are geographically scattered in a 3D model of CCAN holocomplex. Stoichiometry of CCAN and FACT might be informative whether a single or multiple FACT binds to the multiple sites of CCAN. The authors do not address whether these multiple sites are occupied simultaneously, separately or sequentially.
The statement at the end of Abstract (lines 23-25) is a speculative hypothesis without evidence for "a pool of CCAN that is not stably integrated into chromatin", "chaperoning CCAN", and "stabilisation of CCAN".
-
