Molecular dissection of condensin II-mediated chromosome assembly using in vitro assays
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Evaluation Summary:
This paper is of broad interest to researchers studying chromosome structure. Using a powerful reconstitution system, the authors dissect the function of the chromosome organising complex, condensin II. Several findings, if supported by some additional analyses, are surprising and thus have the potential to fuel further mechanistic studies of condensin function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Abstract
In vertebrates, condensin I and condensin II cooperate to assemble rod-shaped chromosomes during mitosis. Although the mechanism of action and regulation of condensin I have been studied extensively, our corresponding knowledge of condensin II remains very limited. By introducing recombinant condensin II complexes into Xenopus egg extracts, we dissect the roles of its individual subunits in chromosome assembly. We find that one of two HEAT subunits, CAP-D3, plays a crucial role in condensin II-mediated assembly of chromosome axes, whereas the other HEAT subunit, CAP-G2, has a very strong negative impact on this process. The structural maintenance of chromosomes ATPase and the basic amino acid clusters of the kleisin subunit CAP-H2 are essential for this process. Deletion of the C-terminal tail of CAP-D3 increases the ability of condensin II to assemble chromosomes and further exposes a hidden function of CAP-G2 in the lateral compaction of chromosomes. Taken together, our results uncover a multilayered regulatory mechanism unique to condensin II, and provide profound implications for the evolution of condensin II.
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Evaluation Summary:
This paper is of broad interest to researchers studying chromosome structure. Using a powerful reconstitution system, the authors dissect the function of the chromosome organising complex, condensin II. Several findings, if supported by some additional analyses, are surprising and thus have the potential to fuel further mechanistic studies of condensin function.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The authors aim to understand how condensin I contributes to chromosome structure. The approach they take is to add back recombinant condensin II complexes to Xenopus extract depleted for both condensin I and condensin II and visualise the ability of the extracts to condense exogenously added chromatin. To test the requirement for individual condensin subunits CAP-D3 and/or CAP-G2 , the authors added back recombinant complex lacking these subunits, or with truncated versions. This led to the surprising conclusion that although the CAP-G2 subunit is required for condensin association with chromosomes, CAP-D3 is not. Indeed, the absence of CAP-D3 enhances condensin association with chromosomes. Similarly, deletion of the C-terminal region of CAP-G2 also increases condensin association with chromosomes. The …
Reviewer #1 (Public Review):
The authors aim to understand how condensin I contributes to chromosome structure. The approach they take is to add back recombinant condensin II complexes to Xenopus extract depleted for both condensin I and condensin II and visualise the ability of the extracts to condense exogenously added chromatin. To test the requirement for individual condensin subunits CAP-D3 and/or CAP-G2 , the authors added back recombinant complex lacking these subunits, or with truncated versions. This led to the surprising conclusion that although the CAP-G2 subunit is required for condensin association with chromosomes, CAP-D3 is not. Indeed, the absence of CAP-D3 enhances condensin association with chromosomes. Similarly, deletion of the C-terminal region of CAP-G2 also increases condensin association with chromosomes. The authors also recapitulate these findings in an extract-free assay, adding strength to their conclusions, although future work will be required to determine the importance of their findings in vivo. Overall, the experiments in this manuscript support the conclusions for the most part and provide interesting insight into condensin II function which will be of great interest to those in the field.
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Reviewer #2 (Public Review):
Condensins promote the condensation of chromosomes during mitosis and meiosis. In many eukaryotes, including humans, there are two condensin complexes, condensin I and II, that are thought to alter chromosome morphology in different yet poorly understood ways. Key studies from the Hirano lab have previously parsed the roles of the condensin I subunits in condensation, but much less was known about condensin II at the mechanistic level. Thus, the authors examined the different contributions of each of the subunits of the five-member condensin II complex to mitotic chromosome condensation in Xenopus egg extracts, a powerful system that recapitulates the hallmarks of condensation and allows manipulations of condensin makeup that would be difficult or impossible in other systems. This important study provides an …
Reviewer #2 (Public Review):
Condensins promote the condensation of chromosomes during mitosis and meiosis. In many eukaryotes, including humans, there are two condensin complexes, condensin I and II, that are thought to alter chromosome morphology in different yet poorly understood ways. Key studies from the Hirano lab have previously parsed the roles of the condensin I subunits in condensation, but much less was known about condensin II at the mechanistic level. Thus, the authors examined the different contributions of each of the subunits of the five-member condensin II complex to mitotic chromosome condensation in Xenopus egg extracts, a powerful system that recapitulates the hallmarks of condensation and allows manipulations of condensin makeup that would be difficult or impossible in other systems. This important study provides an excellent foundation for our understanding of vertebrate condensin II function.
Key findings of this work are the surprising roles of the condensin II G2 and D3 subunits in the regulation of condensin II ATPase activity, condensin interaction with chromatin, and chromosome morphology, many of which are different than the previously elucidated roles of condensin I subunits using a similar experimental setup. Specifically, the authors establish a positive, ATP-hydrolysis dependent role for CAP-D3 in condensin II chromosomal accumulation and chromosome axis formation while demonstrating a negative role for CAP-G2 in those processes, as well as in condensin II ATP-hydrolysis. These results demonstrate that condensin II is acting in a distinct manner from condensin I, which is a big advance over our previously limited understanding of the differences between these complexes. The results here, especially the demonstration that removal of the C-terminal tail of the D3 subunit can promote a condensin I-like shaping activity, suggests regulatory events that are unique to condensin II. As condensin II is also involved in other processes, including the shaping of genome architecture, and centromere identity, these findings will undoubtedly be of broad interest. The experiments are generally well-conducted and clearly and logically presented. However, there are some potential weaknesses of the current work, which include the use of mouse sperm nuclei, instead of frog sperm nuclei, as the chromatin source, and the incomplete depletion of some condensin II subunits.
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Reviewer #3 (Public Review):
Condensin I plays a dominant role in chromosome condensation (at least globally). To uncover and investigate the specific functions of condensin II, the authors compare the phenotypes caused by condensin I-depletion with those of combined depletion of condensin I and condensin II. They show that condensin II (alone) promotes the formation of 'chenille' chromatids (lacking lateral compaction).
The work underscores the different functions of condensin I and II in shaping chromatids and reveals distinct roles of their HEAT subunits in regulating chromosome condensation in mitosis. They uncover a putative self-inhibitory mechanism via one of the HEAT subunits (CAP-G2) which requires the C-terminal tail of the other HEAT subunit (CAP-D3) and potentially involves phosphorylation by mitotic CDK.
The work also …
Reviewer #3 (Public Review):
Condensin I plays a dominant role in chromosome condensation (at least globally). To uncover and investigate the specific functions of condensin II, the authors compare the phenotypes caused by condensin I-depletion with those of combined depletion of condensin I and condensin II. They show that condensin II (alone) promotes the formation of 'chenille' chromatids (lacking lateral compaction).
The work underscores the different functions of condensin I and II in shaping chromatids and reveals distinct roles of their HEAT subunits in regulating chromosome condensation in mitosis. They uncover a putative self-inhibitory mechanism via one of the HEAT subunits (CAP-G2) which requires the C-terminal tail of the other HEAT subunit (CAP-D3) and potentially involves phosphorylation by mitotic CDK.
The work also highlights differences in the role of conserved motifs (III and IV) in CAP-H and CAP-H2 (condensin I and II) with mutations hindering chromosome association in the latter but not the former.
Of note, many conclusions rely on the efficient depletion of endogenous condensin I and II subunits from the extracts.
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