Unconventional kinetochore kinases KKT2 and KKT3 have unique centromere localization domains
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Abstract
Chromosome segregation in eukaryotes is driven by the kinetochore, the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Cells must tightly control the number and position of kinetochores so that all chromosomes assemble a single kinetochore. A central player in this process is the centromere-specific histone H3 variant CENP-A, which localizes constitutively at centromeres and promotes kinetochore assembly. However, CENP-A is absent from several eukaryotic lineages including kinetoplastids, a group of evolutionarily divergent eukaryotes that have an unconventional set of kinetochore proteins. There are six proteins that localize constitutively at centromeres in the kinetoplastid parasite Trypanosoma brucei, among which two homologous protein kinases (KKT2 and KKT3) have limited similarity to polo-like kinases. In addition to the N-terminal kinase domain and the C-terminal divergent polo boxes, KKT2 and KKT3 have a central domain of unknown function as well as putative DNA-binding motifs. Here we show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization in T. brucei . Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei . Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.
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Reply to the reviewers
We are very grateful to the three reviewers for their useful and constructive comments on our manuscript. All reviewers appreciated that our manuscript provides a good characterization of the KKT2/3 functional domains, especially in solving crystal structures of the KKT2 central domain and revealing the importance of KKT2/3 central domains for their centromere targeting. They also commented that additional experiments (e.g. testing DNA-binding activities using recombinant proteins and examining whether ectopically expressed KKT2 fragments localize at kinetochores transiently) would significantly strengthen the manuscript. In the revised manuscript, we are …
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Reply to the reviewers
We are very grateful to the three reviewers for their useful and constructive comments on our manuscript. All reviewers appreciated that our manuscript provides a good characterization of the KKT2/3 functional domains, especially in solving crystal structures of the KKT2 central domain and revealing the importance of KKT2/3 central domains for their centromere targeting. They also commented that additional experiments (e.g. testing DNA-binding activities using recombinant proteins and examining whether ectopically expressed KKT2 fragments localize at kinetochores transiently) would significantly strengthen the manuscript. In the revised manuscript, we are going to address their comments as follows.
Reviewer #1:
From the information presented, it seems like there are only two possibilities to explain the role of the zinc finger domains in directing centromere targeting. First, this could mediate a protein-protein interaction. The authors attempt to assess this using their mass spec experiments, but this does not absolutely rule this out as this interaction may not persist through their purification procedure (low affinity or requires the presence of DNA, such as for a nucleosome).
Response: We agree with the reviewer’s comment. We will add a sentence to discuss this possibility in the revised manuscript.
*Second, this could reflect direct DNA binding by the zinc finger. Although the existing paper is solid and highlights a role for the zinc finger domains in the localization of these proteins, it would be even better if the authors were to at least assess DNA binding in vitro with their recombinant protein. Comparing its behavior to a well characterized DNA-binding zinc finger protein would be powerful for assessing whether direct DNA binding could be responsible for its centromere localization. *
Response: We have tested DNA-binding activities for the KKT2 central domain from T. brucei, Bodo saltans, and *Perkinsela *using a fluorescent polarization assay. We tested three different DNA probes (50 bp each) that were fluorescently-labelled: a 50 bp DNA probe from the CIR147 sequence, which is the unit sequence of centromere repeats of several chromosomes in T. brucei (36% GC content), as well as two random DNA sequences of 25% and 74% GC content. We found that the *Perkinsela *KKT2a central domain binds these three different DNA probes with similar affinities (Kd ~100 nM), suggesting that the *Perkinsela *KKT2a central domain binds DNA in a sequence-independent manner. Although we have not been able to obtain reliable results for T. brucei and Bodo saltans proteins thus far (due to quenching of fluorescent signals by these proteins), it is likely that the T. brucei KKT2 central domain also binds DNA in a sequence-independent manner given the similarity of the Znf1 structure/sequence among kinetoplastids. This is consistent with the observation that there is no DNA sequence that is commonly found in the centromere of all chromosomes in T. brucei and other kinetoplastids. We are going to add the DNA-binding assay results for the Perkinsela KKT2a central domain in the revised manuscript. We do not feel it is informative to compare the KKT2 Znf1’s behavior to a well characterized DNA-binding zinc finger protein (that binds specific DNA sequence), because Perkinsela KKT2a binds DNA in a sequence-independent manner.
*The code for KKT2 and KKT3 localization is complicated by the multiple regions that contribute to their targeting. This includes both the zinc finger domain that the authors identify here, as well as a second region that appears to act through associations with other constitutive centromere components. Due to this, it feels that there are several aspects of these proteins that are incompletely explored. First, the authors show that the Znf1 mutant in KKT2 localizes apparently normally to centromeres, but is unable to support KKT2 function in chromosome segregation. This suggests that this zinc finger domain could have a separable role in kinetochore function that is distinct from centromere targeting. *
Response: We agree with the reviewer that the mechanism of KKT2 kinetochore localization is complicated because there are at least three distinct domains that contribute to its targeting (Figure 2 in the original manuscript), but we showed that the centromere targeting of the ectopically-expressed KKT2 central domain fragment depends on Znf1 (Figure 6B in the original manuscript). Together with the finding that the Znf1-equivalent domain is essential for the localization of the full length KKT3 protein, we think that a function of the KKT2 Znf1 domain is to promote its centromere localization. In the future, it will be critical to understand the molecular mechanism of how the KKT2 central domain localizes specifically at centromeres.
*Second, although the authors identify these minimal zinc finger regions as sufficient for centromere localization, they do not test whether this behavior depends on the presence of other KKT proteins. This seems like a very important experiment to test whether recruitment of the zinc finger occurs through other factors, or whether it could act directly through binding to DNA or histones. *
Response: We do not have an experimental setup to test whether the centromere localization of KKT2/3 central domains depends on other KKT proteins (i.e. we cannot keep the expression of the central domain to a low level while inducing RNAi constructs at a high level). As an alternative approach, we have been testing the localization dependency of endogenously-tagged full-length KKT2/3 proteins using RNAi against various KKT proteins but our preliminary results have not found any kinetochore protein whose depletion affects the localization of KKT2 or KKT3 at centromeres. Although these results could be explained by inefficient protein depletion, they are consistent with the possibility that KKT2 and KKT3 central domains directly interact with centromere DNA. We could consider adding these data in the revised manuscript, although a significant amount of additional work will be necessary to confirm these results.
- Based on the description of kinetoplastid centromeres that the authors provide, it is actually unclear to whether these are indeed sequence independent. The authors state that "There is no specific DNA sequence that is common to all centromeres in each organism [Trypanosomes and Leishmania], suggesting that kinetoplastids also determine their kinetochore positions in a sequence-independent manner." However, it remains possible that there are features to this DNA that are responsible for defining the centromere. In principle, enriched clustering of a short motif that may elude sequence comparisons could be responsible for specifying these regions. It would be helpful to use caution with this statement, and I would also encourage the Aikyoshi lab to test this directly in future work, such as using strategies to remove a centromere or alter its position. *
Response: We agree with the reviewer that we cannot exclude the possibility that there might be an enrichment of a short motif that promotes the localization of kinetochore proteins. We will discuss this possibility in the revised manuscript.
- It would be helpful to provide a schematic of kinetoplastid kinetochore organization based on their studies to date (possibly in Figure 1) to provide a context for the relationships between the different KKT proteins tested in this paper.*
Response: While we agree with the referee that a model figure would be helpful, we feel that drawing a model for the overall organization of kinetoplastid kinetochores at this stage could be misleading because we still know very little about it. In fact, our published data (e.g. the microtubule-binding kinetochore protein KKT4 localizes at centromeres throughout the cell cycle and has DNA-binding activities) and our unpublished observations suggest that the design principle of kinetoplastid kinetochores may well be fundamentally different from that of canonical kinetochores in other eukaryotes. We therefore would like to obtain more data before drawing a model of kinetoplastid kinetochores. Instead of a model, we are going to include a summary of localization patterns for kinetoplastid kinetochore proteins in Figure 1 to help orient readers.
Reviewer #2: *The experiments are in general well presented but some could be better controlled: * - localization of KKT2 and KKT3 mutants is never verified to be centromeres, we have to believe the dots in the DAPI region are centromeres.
Response: We have assumed that the KKT2 and KKT3 mutants that had dots very likely localized at centromeres because they behaved similarly to wild-type proteins (i.e. align at metaphase plate in some 2K1N cells and localize at the leading edge of separating chromosomes). We will confirm this assumption by imaging the KKT2/3 mutants with a kinetochore protein marker (e.g. tdTomato-KKT1).
in some cases mutants are made in full-length (FL) background (viability, sometimes localization), but in other cases only in isolated domains. The former should be done for all assays. This is also important to show that central domain of KKT2 and KKT3 is necessary for localization.
Response: It is very laborious to create point mutants in full-length background at an endogenous locus. This is why we first tested a number of mutants in our ectopic expression of truncated (for KKT2) or full-length (for KKT3) proteins to identify the most critical mutations, which were subsequently tested in the endogenous context. Although not included in the original manuscript, we have performed an ectopic expression of additional KKT2 mutants (C597A/C600A, C616A/C619A, C624A/C627A, C640A/C643A, and H656A/C660A) in the full-length protein and found that all of them had apparently normal localization pattern, which is consistent with the results we obtained in the endogenous expression experiments (C576A, D622A, and C640A/C643A: Figure 6c in the original manuscript).
*The data of F2 are interpreted to mean that PDB-like domain and middle region get to kinetochores by binding transient KT components, even though KKT2 itself is constitutive. That interpretation would really be strenghthened by showing the KKT2 fragments are now transient also. **
Response: Our observations suggest that these KKT2 fragments indeed localize at centromeres transiently (from S phase to anaphase). We will confirm this result by imaging with a transiently-localized kinetochore protein, KKT1 tagged with tdTomato, and include in the revised manuscript.
*The paper could do with some attempts to get to this, based on the presented data. For example, does Znf1 bind centromeric DNA, does it bind nucleosomes, is it essential for recruiting the other KKTs, etc. *
Response: As we responded to Reviewer 1, we have found that Perkinsela KKT2a central domain Znf1 has DNA-binding activities. We agree that it will be important to test whether KKT2 binds nucleosomes but it will be necessary for us to reconstitute nucleosomes using recombinant T. brucei histones. It will also be important to test whether KKT2/3 are essential for recruiting other kinetochore proteins but we think that they are beyond the scope of this manuscript.
Reviewer #3: ***Major Comments:** * *- No page numbers - this makes it difficult to refer to different parts of the text... *
Response: We sincerely apologize for the lack of page numbers in the original manuscript. We will add page numbers and line numbers in the revised manuscript.
*Introduction (page 2), fourth-from bottom line: the authors refer here to "regional centromere" but have not defined this term (I assume, as opposed to point-centromeres of budding yeast?). I suggest rephrasing. *
Response: We thank the reviewer for pointing it out. We will rephrase it in the revised manuscript.
*Page 4, bottom: The discussion of KKT2 kinetochore localization brings up a lot or questions. * *First, can the authors use an assay like yeast two-hybrid to test for pairwise interactions between KKT2 domains and other kinetochore proteins? This could provide direct functional data on the role of these various domains in kinetochore localization. *
Response: Based on the mass spectrometry of immunoprecipitated KKT2 fragments that localized at kinetochores, we are currently trying to identify direct protein-protein interactions between the KKT2 domains and other kinetochore proteins (e.g. does KKT2-DPB directly interact with KKT1, KKT6, or KKT7 proteins?). While we agree that it is important to address these questions, we think that it is beyond the scope of this manuscript because its focus is the characterization of KKT2/3 central domains. As we mentioned in the manuscript, these central domains failed to co-purify with other kinetochore proteins, and the experiment therefore did not give us any clue about how they might localize specifically at centromeres.
*Second, if individual domains are being recruited to kinetochores by their non-constitutive binding partners, wouldn't this be evident if the authors looked at localization at different points in the cell cycle, and/or with dual localization tracking the putative binding partners? Could transient localization of some of the domains explain the intermediate localization phenotype observed for some domains in KKT2? *
Response: As we responded to Reviewer 2, our observations suggest that these KKT2 fragments indeed localize at centromeres transiently (from S phase to anaphase). We will confirm this result by imaging with a transiently-localized kinetochore protein, KKT1 tagged with tdTomato.
*Page 6: The authors note that KKT2 Znf2 bears strong similarity to DNA-binding canonical Zinc fingers, and even note the high conservation of some putative DNA-binding residues. Have the authors tested for DNA binding by this protein? *
Response: As we responded to Reviewer 1 and 2, we used a fluorescence polarization assay and found that the Perkinsela KKT2a central domain binds DNA in a sequence-independent manner.
*Can the authors at least model DNA binding and see if that would result in a clash, given the packing of Znf2 against the larger Znf1? *
Response: As suggested, we superimposed the structure of Bodo saltans KKT2 Znf2 with that of a zinc finger 268 bound to DNA (PDB:1AAY), which shows a possible mechanism by which Znf2 might bind DNA. It also revealed a clash between DNA and Znf1 (in the crystal packing of the solved structure), implying that the position of Znf2 would need to change in order to bind DNA. We will add a supplementary figure showing a hypothetical DNA-binding mechanism by Znf2 and discuss the possibility of a necessary structural change in the Znf1 position to accommodate the DNA binding by Znf2.
***Minor Comments:** * *- Page 5: I'm skeptical as to whether these zinc-binding domains, especially Znf1, should really be referred to as "fingers". *
Response: To our knowledge, the word “zinc finger” could be used for any protein that binds one or more zinc ions. Given that we still do not understand the molecular mechanism by which this domain functions, we wanted to use a very general term, Znf1. However, we do appreciate the reviewer’s point that calling this domain as a zinc finger could be misleading, so we will refer Znf1 and Znf2 in the original manuscript as the CL domain (for centromere localizing domain) and a classic C2H2 zinc finger in the revised manuscript.
*Page 8: At the beginning of the section describing KKT3 cellular experiments, I think the authors need to make it much more explicit that T. brucei KKT3 shares both Znf1 and Znf2 with KKT2. *
Response: We will add the suggested sentence before describing the functional assay for KKT3.
*Figure S1A: The gap between lanes in the middle of the major peak is really confusing (it's not even clear that this is two different SDS-PAGE gels next to one another). I initially thought that KKT2 was in both peaks, given the labeling of this figure. I suggest labeling the lanes specifically, or cropping the picture, to avoid confusion. *
Response: As suggested, we will prepare an image that shows only those lanes (from two separate gels) that were used for loading protein samples. We also like to retain the whole gel images in the same figure because those gels have rather low background signal (even without any contrast manipulation).
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Referee #3
Evidence, reproducibility and clarity
Summary:
The manuscript "Unconventional kinetochore kinases KKT2 and KKT3 have a unique zinc finger that promotes their kinetochore localization" by Marciano et al. describes functional and structural work on two unique kinetochore-localized proteins in kinetoplastids, KKT2 and KKT3. While the kinetochores of most eukaryotes are built on top of a histone H3 variant known as CENP-A (or CenH3), kinetoplastids lack CENP-A. Kinetoplastids also lack homologs of most conserved kinetochore proteins and instead possess an unique complement of kinetochore proteins, as described in earlier work by the lead author, B. Akiyoshi.
The current …
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Referee #3
Evidence, reproducibility and clarity
Summary:
The manuscript "Unconventional kinetochore kinases KKT2 and KKT3 have a unique zinc finger that promotes their kinetochore localization" by Marciano et al. describes functional and structural work on two unique kinetochore-localized proteins in kinetoplastids, KKT2 and KKT3. While the kinetochores of most eukaryotes are built on top of a histone H3 variant known as CENP-A (or CenH3), kinetoplastids lack CENP-A. Kinetoplastids also lack homologs of most conserved kinetochore proteins and instead possess an unique complement of kinetochore proteins, as described in earlier work by the lead author, B. Akiyoshi.
The current manuscript follows up this earlier work and seeks to understand how two putative kinases, KKT2 and KKT3, localize to the kinetochores of kinetoplastids. They begin by mapping the regions of both proteins (in Trypanosoma brucei) that are required for kinetochore localization. In both cases, a conserved "central domain" is sufficient for kinetochore localization. They then purify and determine the structure of a KKT2 central domain from a related species (Bodo saltans), and show that it possess two zinc-binding domains, termed Znf1 and Znf2. A more diverged KKT2 from Perkinsela has Znf1, but not Znf2. The authors go on to show that the Znf1 region in particular is important for localization of both KKT2 and KKT3 to kinetochores, and for long-term cell survival, in Trypanosoma brucei.
Major Comments:
The work is well done, well described, and described in such a way that it should be reproducible.
No page numbers - this makes it difficult to refer to different parts of the text...
Introduction (page 2), fourth-from bottom line: the authors refer here to "regional centromere" but have not defined this term (I assume, as opposed to point-centromeres of budding yeast?). I suggest rephrasing.
Page 4, bottom: The discussion of KKT2 kinetochore localization brings up a lot or questions. First, can the authors use an assay like yeast two-hybrid to test for pairwise interactions between KKT2 domains and other kinetochore proteins? This could provide direct functional data on the role of these various domains in kinetochore localization. Second, if individual domains are being recruited to kinetochores by their non-constitutive binding partners, wouldn't this be evident if the authors looked at localization at different points in the cell cycle, and/or with dual localization tracking the putative binding partners? Could transient localization of some of the domains explain the intermediate localization phenotype observed for some domains in KKT2?
Page 6: The authors note that KKT2 Znf2 bears strong similarity to DNA-binding canonical Zinc fingers, and even note the high conservation of some putative DNA-binding residues. Have the authors tested for DNA binding by this protein? Can the authors at least model DNA binding and see if that would result in a clash, given the packing of Znf2 against the larger Znf1?
Minor Comments:
Page 5: I'm skeptical as to whether these zinc-binding domains, especially Znf1, should really be referred to as "fingers"
Page 8: At the beginning of the section describing KKT3 cellular experiments, I think the authors need to make it much more explicit that T. brucei KKT3 shares both Znf1 and Znf2 with KKT2.
Figure S1A: The gap between lanes in the middle of the major peak is really confusing (it's not even clear that this is two different SDS-PAGE gels next to one another). I initially thought that KKT2 was in both peaks, given the labeling of this figure. I suggest labeling the lanes specifically, or cropping the picture, to avoid confusion.
Significance
This work is interesting, well done, and described nicely. It highlights how unique and different the kinetochores of kinetoplastid species are, and brings up a number of questions about how these kinetochores are specified and how they function. The structural work is also interesting and well-done. Unfortunately, the work as a whole does not make any strong mechanistic conclusions, leading to a somewhat dissatisfying conclusion.
The work could be significantly strengthened if the authors were able to make a direct functional conclusion about the roles of the Znf regions of KKT2 and/or KKT3, for example detecting DNA binding in vitro, or detecting a specific pairwise interaction between this region and another kinetochore protein.
This work will most likely appeal to researchers in the cell division and kinetochore architecture fields, although since kinetoplastids are so unique the link between this work and most other kinetochore work is unclear. This is in a way exciting: we don't yet know much about how these kinetochores relate to other eukaryotes' kinetochores.
My field of expertise is structural biology and biochemistry, with experience in kinetochore architecture and structure.
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Referee #2
Evidence, reproducibility and clarity
Kinetoplastids have unconventional kinetochores that lack CENPA nucleosomes that normally dictates the position of the kinetochore in most other eukaryotes. Marciano and colleagues analyse KKT2 and KKT3, two consistutively localized kinetoplastid kinetochore proteins that may contribute to kinetochore positioning on centromeric DNA. They find that in both proteins the central, cysteine-rich domains are sufficient to support centromere localization but that in KKT2 also other domains can do so by themselves. They then obtain crystal structures of the KKT2 central domain from bodo saltans and show it consists of 2 Zinc-finger …
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Referee #2
Evidence, reproducibility and clarity
Kinetoplastids have unconventional kinetochores that lack CENPA nucleosomes that normally dictates the position of the kinetochore in most other eukaryotes. Marciano and colleagues analyse KKT2 and KKT3, two consistutively localized kinetoplastid kinetochore proteins that may contribute to kinetochore positioning on centromeric DNA. They find that in both proteins the central, cysteine-rich domains are sufficient to support centromere localization but that in KKT2 also other domains can do so by themselves. They then obtain crystal structures of the KKT2 central domain from bodo saltans and show it consists of 2 Zinc-finger structures (Znf1 and Znf2) of which the first is conserved in Perkinsella. Mutations of Znf1 and Znf2 in KKT2 and homologous mutations in KKT3 show that Znf1 is crucial for centromere localization and viability, while Znf2 is dispensible for both.
The paper presents a pretty straighforward characerization of functional domains in KKT2 and KKT3 with respect to centromere localization. The authors nicely show a unique Zn-finger structure (Znf1) of KKT2 and show it is crucial for localization. The study does not end up delivering an answer to the questions posed in the manuscript, namely how centromeres and therefore kinetochores are specified in kinetoplastids. The paper could do with some attempts to get to this, based on the presented data. For example, does Znf1 bind centromeric DNA, does it bind nucleosomes, is it essential for recruiting the other KKTs, etc.
The experiments are in general well presented but some could be better controlled:
- localization of KKT2 and KKT3 mutants is never verified to be centromeres, we have to believe the dots in the DAPI region are centromeres.
- in some cases mutants are made in full-length (FL) background (viability, sometimes localization), but in other cases only in isolated domains. The former should be done for all assays. This is also important to show that central domain of KKT2 and KKT3 is necessary for localization.
- The data of F2 are interpreted to mean that PDB-like domain and middle region get to kinetochores by binding transient KT components, even though KKT2 itself is constitutive. That interpretation would really be strenghtened by showing the KKT2 fragments are now transient also.
Significance
The paper presents a pretty straighforward characerization of functional domains in KKT2 and KKT3 with respect to centromere localization. The authors nicely show a unique Zn-finger structure (Znf1) of KKT2 and show it is crucial for localization. The study does not end up delivering an answer to the questions posed in the manuscript, namely how centromeres and therefore kinetochores are specified in kinetoplastids. The paper could do with some attempts to get to this, based on the presented data. For example, does Znf1 bind centromeric DNA, does it bind nucleosomes, is it essential for recruiting the other KKTs, etc.
-
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Referee #1
Evidence, reproducibility and clarity
Although most studied eukaryotes display similarities in their overall kinetochore structures to mediate chromosome segregation, kinetoplastid species display highly divergent kinetochores with no clear relationships to canonical kinetochore components. Prior work from the Akiyoshi lab and others has identified kinetochore proteins in Trypanosomes and other kinetoplastids. The identification of these proteins has provided a toolkit to begin to reveal the features that guide the function and assembly of these structures during chromosome segregation. Despite differences in protein composition, all kinetochores must display key …
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Referee #1
Evidence, reproducibility and clarity
Although most studied eukaryotes display similarities in their overall kinetochore structures to mediate chromosome segregation, kinetoplastid species display highly divergent kinetochores with no clear relationships to canonical kinetochore components. Prior work from the Akiyoshi lab and others has identified kinetochore proteins in Trypanosomes and other kinetoplastids. The identification of these proteins has provided a toolkit to begin to reveal the features that guide the function and assembly of these structures during chromosome segregation. Despite differences in protein composition, all kinetochores must display key properties including their ability to bind to both microtubules and chromosomal DNA. This paper focuses on the mechanisms by which kinetoplastid kinetochore components are targeted to centromere regions, an exciting question due to the apparent DNA sequence-independent nature of these associations. In other eukaryotes, this sequence independent association is specified through the action of histone variants. In contrast, it is unclear how DNA interactions occur in kinetoplastids.
This paper begins by reasoning that the proteins responsible for DNA interactions and defining the location of the centromere would localize persistently to centromeres. Thus, they focus on two constitutively localized proteins with sequence similarity to each other, KKT2 and KKT3. The authors analyze these proteins using a combination of domain analysis to test the localization requirements for these proteins, mass spectrometry analysis of interacting proteins, mutational analysis to test specific residues for localization and function, and most importantly determination of the structure of a kinetochore targeting domain, which reveals a zinc finger structure. The structural work in particular is both interesting and reveals a feature of these proteins that was not obvious based on initial sequence analysis. Overall, this paper appears to be carefully executed, rigorous, and well controlled, but could benefit from additional experiments that would extend the impact of their findings.
- From the information presented, it seems like there are only two possibilities to explain the role of the zinc finger domains in directing centromere targeting. First, this could mediate a protein-protein interaction. The authors attempt to assess this using their mass spec experiments, but this does not absolutely rule this out as this interaction may not persist through their purification procedure (low affinity or requires the presence of DNA, such as for a nucleosome). Second, this could reflect direct DNA binding by the zinc finger. Although the existing paper is solid and highlights a role for the zinc finger domains in the localization of these proteins, it would be even better if the authors were to at least assess DNA binding in vitro with their recombinant protein. Comparing its behavior to a well characterized DNA-binding zinc finger protein would be powerful for assessing whether direct DNA binding could be responsible for its centromere localization.
- The code for KKT2 and KKT3 localization is complicated by the multiple regions that contribute to their targeting. This includes both the zinc finger domain that the authors identify here, as well as a second region that appears to act through associations with other constitutive centromere components. Due to this, it feels that there are several aspects of these proteins that are incompletely explored. First, the authors show that the Znf1 mutant in KKT2 localizes apparently normally to centromeres, but is unable to support KKT2 function in chromosome segregation. This suggests that this zinc finger domain could have a separable role in kinetochore function that is distinct from centromere targeting. Second, although the authors identify these minimal zinc finger regions as sufficient for centromere localization, they do not test whether this behavior depends on the presence of other KKT proteins. This seems like a very important experiment to test whether recruitment of the zinc finger occurs through other factors, or whether it could act directly through binding to DNA or histones.
- Based on the description of kinetoplastid centromeres that the authors provide, it is actually unclear to whether these are indeed sequence independent. The authors state that "There is no specific DNA sequence that is common to all centromeres in each organism [Trypanosomes and Leishmania], suggesting that kinetoplastids also determine their kinetochore positions in a sequence-independent manner." However, it remains possible that there are features to this DNA that are responsible for defining the centromere. In principle, enriched clustering of a short motif that may elude sequence comparisons could be responsible for specifying these regions. It would be helpful to use caution with this statement, and I would also encourage the Aikyoshi lab to test this directly in future work, such as using strategies to remove a centromere or alter its position.
- It would be helpful to provide a schematic of kinetoplastid kinetochore organization based on their studies to date (possibly in Figure 1) to provide a context for the relationships between the different KKT proteins tested in this paper.
Significance
This paper provides a nice advance in understanding the molecular architecture and functional organization of kinetoplastid kinetochores. As these remain understudied, this work is valuable for revealing the chromosome segregation behaviors in these medically-relevant parasites. In addition, due to the divergence in overall kinetochore function from other eukaryotes, this work will help provide insights into the logic by which kinetochores function and are organized. The existing paper represents a solid advance in understanding the structure and requirements for KKT2 and KKT3 kinetochore targeting through this novel zinc finger domain. However, conducting some of the additional experiments made above, such as testing DNA binding and the requirements for other KKT proteins for zinc finger localization, would allow the authors to make stronger statements and a more impactful advance.
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