Depletion or cleavage of cohesin during anaphase differentially affects chromatin structure and segregation
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Evaluation Summary:
Cohesin is an evolutionarily conserved protein complex that plays essential roles in mitotic chromosome structure and function. Previous studies suggest that multiple activities of cohesin are required only prior to the onset of chromosome segregation. Using a Mcd1-AID and a Mcd1-TEV to either degrade or cleave cohesin's kleisin subunit Mcd1 of the yeast Saccharomyces cerevisiae, this study shows that cohesion plays also a role in anaphase organizing the centromeric regions, providing new evidence that cohesin function is critical for chromosome structure and segregation during and after the onset of chromosome segregation. The work is of relevance for students of chromosome biology and cell division.
(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. The reviewers remained anonymous to the authors.)
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Abstract
Chromosome segregation requires both the separation of sister chromatids and the sustained condensation of chromatids during anaphase. In yeast cells, cohesin is not only required for sister chromatid cohesion but also plays a major role determining the structure of individual chromatids in metaphase. Separase cleavage is thought to remove all cohesin complexes from chromosomes to initiate anaphase. It is thus not clear how the length and organisation of segregating chromatids is maintained during anaphase in the absence of cohesin. Here, we show that degradation of cohesin at the anaphase onset causes aberrant chromatid segregation. Hi-C analysis on segregating chromatids demonstrates that cohesin depletion causes loss of intrachromatid organisation. Surprisingly, tobacco etch virus (TEV)-mediated cleavage of cohesin does not dramatically disrupt chromatid organisation in anaphase, explaining why bulk segregation is achieved. In addition, we identified a small pool of cohesin complexes bound to telophase chromosomes in wild-type cells and show that they play a role in the organisation of centromeric regions. Our data demonstrates that in yeast cells cohesin function is not over in metaphase, but extends to the anaphase period when chromatids are segregating.
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Evaluation Summary:
Cohesin is an evolutionarily conserved protein complex that plays essential roles in mitotic chromosome structure and function. Previous studies suggest that multiple activities of cohesin are required only prior to the onset of chromosome segregation. Using a Mcd1-AID and a Mcd1-TEV to either degrade or cleave cohesin's kleisin subunit Mcd1 of the yeast Saccharomyces cerevisiae, this study shows that cohesion plays also a role in anaphase organizing the centromeric regions, providing new evidence that cohesin function is critical for chromosome structure and segregation during and after the onset of chromosome segregation. The work is of relevance for students of chromosome biology and cell division.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also …
Evaluation Summary:
Cohesin is an evolutionarily conserved protein complex that plays essential roles in mitotic chromosome structure and function. Previous studies suggest that multiple activities of cohesin are required only prior to the onset of chromosome segregation. Using a Mcd1-AID and a Mcd1-TEV to either degrade or cleave cohesin's kleisin subunit Mcd1 of the yeast Saccharomyces cerevisiae, this study shows that cohesion plays also a role in anaphase organizing the centromeric regions, providing new evidence that cohesin function is critical for chromosome structure and segregation during and after the onset of chromosome segregation. The work is of relevance for students of chromosome biology and cell division.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study is mainly based on the comparative analysis of the impact that an Mcd1-AID degron and an Mcd1-TEV have on S. cerevisiae chromosome organization and segregation. After solving the unexpected situation generated by the Mcd1-TEV construct due to the N-rule of protein degradation, the authors confirm that a rapid depletion of cohesin achieved with the Mcd1-AID allele causes disruption of chromosome organization and segregation as well as mitotic catastrophe, whereas the TEV-cleaved Mcd1 leaves an active fragment at the CEN regions of telophase chromosomes and shows a defect in the fidelity of chromosome segregation, but does not prevent bulk nuclear separation. TEV-cleaved cohesins remain bound to chromatin for longer than degraded chromatin. The number of cohesin-binding sites per chromosome is …
Reviewer #1 (Public Review):
This study is mainly based on the comparative analysis of the impact that an Mcd1-AID degron and an Mcd1-TEV have on S. cerevisiae chromosome organization and segregation. After solving the unexpected situation generated by the Mcd1-TEV construct due to the N-rule of protein degradation, the authors confirm that a rapid depletion of cohesin achieved with the Mcd1-AID allele causes disruption of chromosome organization and segregation as well as mitotic catastrophe, whereas the TEV-cleaved Mcd1 leaves an active fragment at the CEN regions of telophase chromosomes and shows a defect in the fidelity of chromosome segregation, but does not prevent bulk nuclear separation. TEV-cleaved cohesins remain bound to chromatin for longer than degraded chromatin. The number of cohesin-binding sites per chromosome is strongly diminished in telophase-arrested cells, with only a few regions exhibiting binding that indeed belongs to telomeric regions. The authors conclude that cohesin complexes affect cohesin contacts at telophase centromeres. In addition, using a conditional mcd1-73 allele the study shows that inactivation of cohesin at telophase also causes decondensation of chromosomes in the rDNA region. The study is clear, well explained, and data convincing. However, a few points should be considered to make conclusions stronger.
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Reviewer #2 (Public Review):
Using yeast genetics and Hi-C the authors aim to assess the potential role of the cohesin complex in ordering post-anaphase mitotic chromosomes. This is a stage of the cell cycle where chromosomal cohesin was considered to be absent due to the cleavage of the Scc1/Mcd1 subunit of cohesin by the Separase enzyme post anaphase. They start by comparing the chromosome segregation kinetics of cells where anaphase is induced by artificial cleavage of the Scc1 to anaphase induced by AID-mediated degradation of Scc1 in metaphase. They observe a strong mis-segregation phenotype in the metaphase induced by degradation of Scc1 compared to that induced by artificial cleavage of Scc1. They also observe that artificial cleavage of Scc1 leaves the cleaved interactions with other subunits of cohesin intact, potentially …
Reviewer #2 (Public Review):
Using yeast genetics and Hi-C the authors aim to assess the potential role of the cohesin complex in ordering post-anaphase mitotic chromosomes. This is a stage of the cell cycle where chromosomal cohesin was considered to be absent due to the cleavage of the Scc1/Mcd1 subunit of cohesin by the Separase enzyme post anaphase. They start by comparing the chromosome segregation kinetics of cells where anaphase is induced by artificial cleavage of the Scc1 to anaphase induced by AID-mediated degradation of Scc1 in metaphase. They observe a strong mis-segregation phenotype in the metaphase induced by degradation of Scc1 compared to that induced by artificial cleavage of Scc1. They also observe that artificial cleavage of Scc1 leaves the cleaved interactions with other subunits of cohesin intact, potentially leaving a population of cohesin on chromosomes where it can promote looping and compaction. In contrast, the complete degradation of cohesin does not leave residual cohesin on chromosomes. From this, they hypothesize that residual cohesin left on chromosomes following Scc1 cleavage promotes faithful segregation of chromosomes during anaphase. By using Hi-C they demonstrate that the structure of chromosomes in the anaphase induced by TEV cleavage retains more contacts in a range consistent with cohesin-dependent looping than observed following Scc1 degradation. This is consistent with the cohesin-dependent organisation being partially retained in anaphase. To confirm that normally processed cohesin supports anaphase chromosome structure they assess the association of cohesin and the extent of cohesin regulation of chromosome structure in anaphase cells arrested before mitotic exit (cdc15-2) by ChIP, Hi-C, and microscopy. They convincingly show that some cohesin is detected at centromeres at anaphase cells and that cohesin regulates chromosome structure in anaphase at both the centromeres and at and around the rDNA repeats in budding yeast.
The strengths of this manuscript are that it convincingly shows that a cohesin subpopulation is still available in anaphase to associate and organise mitotic chromosomes. Such activity was previously not suspected and I think is a major conceptual advance. These findings raise a number of interesting questions about how this mitotic subpopulation escapes the normal degradation of cohesin that occurs in anaphase. The weakness of the manuscript is the assertion that some looping is retained all along anaphase chromosomes. The analysis of cdc15-2 cells provides strong evidence that the organisation of centromeres and rDNA chromosomes is regulated by cohesin in anaphase. The evidence that cohesin also partially maintains looping along chromosome arms is weaker and dependent on the interpretation of the activity of the TEV cleavage system.
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Reviewer #3 (Public Review):
The dogma in the literature is that cohesin's role in chromosome function is completed by the onset of anaphase because the destruction of cohesin activity by artificial cleavage of its kleisin subunit at metaphase induces normal anaphase with proper chromosome segregation. The authors show that artificial cleavage does not destroy all kleisin functions. When the kleisin subunit is completely destroyed, the induction of anaphase in the metaphase arrested cells exhibit chromosome missegregation and chromosome bridges. These results suggest that cohesin has novel functions post metaphase cells. Consistent with this conclusion the authors show that a small fraction of cohesin is present on chromosomes after metaphase. Inactivation of this cohesin fraction in telophase leads to changes in chromosome structure. …
Reviewer #3 (Public Review):
The dogma in the literature is that cohesin's role in chromosome function is completed by the onset of anaphase because the destruction of cohesin activity by artificial cleavage of its kleisin subunit at metaphase induces normal anaphase with proper chromosome segregation. The authors show that artificial cleavage does not destroy all kleisin functions. When the kleisin subunit is completely destroyed, the induction of anaphase in the metaphase arrested cells exhibit chromosome missegregation and chromosome bridges. These results suggest that cohesin has novel functions post metaphase cells. Consistent with this conclusion the authors show that a small fraction of cohesin is present on chromosomes after metaphase. Inactivation of this cohesin fraction in telophase leads to changes in chromosome structure. For the most part, the authors' conclusions are based upon well-designed experiments with clear results. Demonstrating a role for cohesin post metaphase would be an important contribution to the field of mitosis. However currently several important aspects of the work need clarifying, particularly the analysis of the Hi C results.
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