Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains
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Evaluation Summary:
The cohesin complex is involved in both sister chromatid cohesion (SCC) and intra-chromatid loop formation. Combining molecular genetic and cytological tools with genome-wide calibrated ChIP and HiC analyses in budding yeast, the authors elegantly show that Eco1 and Eco1-mediated Smc3 acetylation promote the boundary formation of chromatin loops by cohesin, which is critical for both meiotic recombination in prophase I and sister chromatid segregation in meiosis II. Cohesin's role in the boundary formation is independent of meiotic DNA replication and of antagonizing a cohesion releasing protein, Wapl. Future studies will reveal the molecular mechanisms of how Eco1-mediated Smc3 acetylation stabilizes cohesin at convergent transcription sites for boundary formation.
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
Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here, we show that meiotic chromosomes are organized into functional domains by Eco1 acetyltransferase-dependent positioning of both chromatin loops and sister chromatid cohesion in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore mono-orientation, and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes.
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Author Response:
Reviewer #2 (Public Review):
This manuscript by Barton and colleagues explores the roles of the conserved Eco1 transacetylase in modulating cohesin function in meiosis in budding yeast. Numerous studies in mitotically dividing cells have shown that the Eco1 family of transacetylases acetylate the Smc3 subunit of cohesin and that this acetylation renders cohesin on chromosomes resistant to removal by the Wapl (Wpl1 in budding yeast) family of proteins. Cohesins play critical roles in both sister chromatid cohesion and chromatin organization (through the formation of intrachromosomal loops). How cohesins are regulated by Eco1 in meiosis to accommodate meiotic chromosome structures such as the synaptonemal complex, chromatin domains around centromeres, repair of programmed meiotic double strand DNA breaks in prophase, …
Author Response:
Reviewer #2 (Public Review):
This manuscript by Barton and colleagues explores the roles of the conserved Eco1 transacetylase in modulating cohesin function in meiosis in budding yeast. Numerous studies in mitotically dividing cells have shown that the Eco1 family of transacetylases acetylate the Smc3 subunit of cohesin and that this acetylation renders cohesin on chromosomes resistant to removal by the Wapl (Wpl1 in budding yeast) family of proteins. Cohesins play critical roles in both sister chromatid cohesion and chromatin organization (through the formation of intrachromosomal loops). How cohesins are regulated by Eco1 in meiosis to accommodate meiotic chromosome structures such as the synaptonemal complex, chromatin domains around centromeres, repair of programmed meiotic double strand DNA breaks in prophase, and sequential removal of cohesins - first at arms in meiosis I and centromeres at meiosis II - is largely unexplored. Thus, this manuscript is exploring important new areas.
The authors show that Eco1 persists thru prophase I (longer than it does in vegetative cell cycles), that it is not necessary for cohesin loading at centromeres but is needed to counteract Wpl1 to protect centromeric cohesion, that it is critical for the establishment of chromatin loops on meiotic chromosome arms and that it is critical for protection of the arm cohesin from removal by Wpl1. The authors also provide evidence that, in meiosis, Wpl1 exhibits underappreciated functions in cohesin loading or cohesion establishment in addition to its recognized role in cohesin removal.
The experiments demonstrate that Eco1 is necessary for sharp cohesin boundaries that flank the centromeres and suggest this might be a replication-independent function of Eco1 (the boundaries form in clb5, clb6 cells with no DNA replication phase) but it is unclear if the detectable, but diminished, boundaries in clb5,clb6 cells were formed in the replication-free meiosis or presist from the S-phase associated loading and cohesion establishment from the preceding mitotic cycle.
Entry into meiosis occurs from G1 when there is no cohesin on the chromosomes and boundaries are not present, therefore this would only be a concern if there were persistent mitotic cells in G2 (i.e. after DNA replication). Our flow cytometry shows that the cells used in the experiment were unreplicated, so even if mitotic cells were present, they would not have been through S phase.
Nevertheless, we addressed this point by analysis of pre-S phase meiotic cells (ime1/ime4 block) and by anchoring away Eco1 in unreplicated cells.
Immunofluorescence imaging assays are used to observe the behavior of sister chromatids in meiosis I and meiosis II as a function of Eco1 activity. In wild-type cells sister chromatids co-orient in meiosis I and move to the same pole of the spindle. In mammalian cells and fission yeast this co-orientation requires cohesin while studies in budding yeast have suggested the co-orientation is cohesin-independent. Here, the authors show that when Eco1 is depleted, the sisters often move to opposite poles at meiosis I, and suggest that cohesin (and Eco1) is indeed required for sister co-orientation. An alternate possibility is that the sisters have lost their association in meiotic prophase (due to cohesin failures) before attaching to microtubles and segregating randomly - often to opposite poles.
We agree with this point, but would argue that the “alternative possibility” (which our data support) still leads to the conclusion that cohesin and Eco1 are required for sister co-orientation. A prior study (Monje-Casas et al., 2007) had suggested that monopolin could link sister kinetochores even without cohesin. We now show that this is not the case, which we believe to be an important conclusion.
Our results indicate that establishment of monoorientation requires the cohesin that is localized at centromeres. WPL1 deletion in eco1-aa rescues centromeric cohesion (Figure 2F, Figure 8E), but not chromosome arm cohesion (Figure 2H) or sister chromatid segregation in meiosis II (Figure 8F), indicating that pericentromeric cohesion must still be defective.
For clarity, please note that the relevant data is not immunofluorescence, but live cell imaging (now shown in Figure 8) so these conclusions are based on observation of single chromosomes in individual live cells from prophase I until anaphase II.
In summary the authors show that Eco1 has distinct roles on chromosome arms and centromeres and probably in both replication-linked and replication-independent events, acts to modulate cohesin location and function in meiosis.
Reviewer #3 (Public Review):
This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.
The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that Eco1 is important for establishing the meiotic segregation pattern. The paper is well written and the data largely support the conclusions. As such, this paper is expected to be of substantial interest to the field.
One notable weakness is the poor definition of the eco1 anchor-away allele (eco1-aa), on which much of the eco1 phenotypic analysis is based. The presented data indicate that addition of the FRB-GFP tag alone causes most of the phenotypes, regardless of nuclear depletion. It is well possible that the tag creates a meiosis-specific loss-of-function allele, although it is surprising that the tag does not have mitotic defects even though Eco1 presumably has the same substrate (the cohesin subunit Smc3) in both situations. Encouragingly, some of the phenotypes could be confirmed using a non-acetylatable smc3 mutant. However, the tag may also create neomorphic effects that may contribute to the Wpl1-independent effects and the apparent stronger defects of the eco1-aa allele compared to the non-acetylatable smc3 mutant.
Available evidence suggests that eco1-aa is a loss of function allele.
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Evaluation Summary:
The cohesin complex is involved in both sister chromatid cohesion (SCC) and intra-chromatid loop formation. Combining molecular genetic and cytological tools with genome-wide calibrated ChIP and HiC analyses in budding yeast, the authors elegantly show that Eco1 and Eco1-mediated Smc3 acetylation promote the boundary formation of chromatin loops by cohesin, which is critical for both meiotic recombination in prophase I and sister chromatid segregation in meiosis II. Cohesin's role in the boundary formation is independent of meiotic DNA replication and of antagonizing a cohesion releasing protein, Wapl. Future studies will reveal the molecular mechanisms of how Eco1-mediated Smc3 acetylation stabilizes cohesin at convergent transcription sites for boundary formation.
This preprint has been reviewed by eLife. We …
Evaluation Summary:
The cohesin complex is involved in both sister chromatid cohesion (SCC) and intra-chromatid loop formation. Combining molecular genetic and cytological tools with genome-wide calibrated ChIP and HiC analyses in budding yeast, the authors elegantly show that Eco1 and Eco1-mediated Smc3 acetylation promote the boundary formation of chromatin loops by cohesin, which is critical for both meiotic recombination in prophase I and sister chromatid segregation in meiosis II. Cohesin's role in the boundary formation is independent of meiotic DNA replication and of antagonizing a cohesion releasing protein, Wapl. Future studies will reveal the molecular mechanisms of how Eco1-mediated Smc3 acetylation stabilizes cohesin at convergent transcription sites for boundary formation.
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):
Cohesin complex is involved in both sister chromatid cohesion (SCC) and intra-chromatid loop formation. Combined of molecular genetic and cytological tools with genome-wide calibrated ChIP and HiC analyses, the authors elegantly showed that Eco1, thus, Smc3 acetylation, promotes the boundary formation of the chromatin loop by the cohesin, which is critical for both meiotic recombination in prophase I and sister chromatid segregation in meiosis II. This role in the boundary formation is independent of its role in SCC. However, it still remains to be solved how Eco1-mediated Smc3 acetylation stabilizes the cohesin to convergent transcription sites for the boundary formation at a molecular level, due to the lack of biochemical analysis of the acetylated cohesion complex in loop-extrusion activity.
This paper …
Reviewer #1 (Public Review):
Cohesin complex is involved in both sister chromatid cohesion (SCC) and intra-chromatid loop formation. Combined of molecular genetic and cytological tools with genome-wide calibrated ChIP and HiC analyses, the authors elegantly showed that Eco1, thus, Smc3 acetylation, promotes the boundary formation of the chromatin loop by the cohesin, which is critical for both meiotic recombination in prophase I and sister chromatid segregation in meiosis II. This role in the boundary formation is independent of its role in SCC. However, it still remains to be solved how Eco1-mediated Smc3 acetylation stabilizes the cohesin to convergent transcription sites for the boundary formation at a molecular level, due to the lack of biochemical analysis of the acetylated cohesion complex in loop-extrusion activity.
This paper discloses new findings on regulation/functions of meiotic cohesion complex at two distinct chromosome regions in meiosis: the centromere and chromosome arm (peri-centromeric borders). The experiments are of good quality and the results are very much convincing.
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Reviewer #2 (Public Review):
This manuscript by Barton and colleagues explores the roles of the conserved Eco1 transacetylase in modulating cohesin function in meiosis in budding yeast. Numerous studies in mitotically dividing cells have shown that the Eco1 family of transacetylases acetylate the Smc3 subunit of cohesin and that this acetylation renders cohesin on chromosomes resistant to removal by the Wapl (Wpl1 in budding yeast) family of proteins. Cohesins play critical roles in both sister chromatid cohesion and chromatin organization (through the formation of intrachromosomal loops). How cohesins are regulated by Eco1 in meiosis to accommodate meiotic chromosome structures such as the synaptonemal complex, chromatin domains around centromeres, repair of programmed meiotic double strand DNA breaks in prophase, and sequential …
Reviewer #2 (Public Review):
This manuscript by Barton and colleagues explores the roles of the conserved Eco1 transacetylase in modulating cohesin function in meiosis in budding yeast. Numerous studies in mitotically dividing cells have shown that the Eco1 family of transacetylases acetylate the Smc3 subunit of cohesin and that this acetylation renders cohesin on chromosomes resistant to removal by the Wapl (Wpl1 in budding yeast) family of proteins. Cohesins play critical roles in both sister chromatid cohesion and chromatin organization (through the formation of intrachromosomal loops). How cohesins are regulated by Eco1 in meiosis to accommodate meiotic chromosome structures such as the synaptonemal complex, chromatin domains around centromeres, repair of programmed meiotic double strand DNA breaks in prophase, and sequential removal of cohesins - first at arms in meiosis I and centromeres at meiosis II - is largely unexplored. Thus, this manuscript is exploring important new areas.
The authors show that Eco1 persists thru prophase I (longer than it does in vegetative cell cycles), that it is not necessary for cohesin loading at centromeres but is needed to counteract Wpl1 to protect centromeric cohesion, that it is critical for the establishment of chromatin loops on meiotic chromosome arms and that it is critical for protection of the arm cohesin from removal by Wpl1. The authors also provide evidence that, in meiosis, Wpl1 exhibits underappreciated functions in cohesin loading or cohesion establishment in addition to its recognized role in cohesin removal.
The experiments demonstrate that Eco1 is necessary for sharp cohesin boundaries that flank the centromeres and suggest this might be a replication-independent function of Eco1 (the boundaries form in clb5, clb6 cells with no DNA replication phase) but it is unclear if the detectable, but diminished, boundaries in clb5,clb6 cells were formed in the replication-free meiosis or presist from the S-phase associated loading and cohesion establishment from the preceding mitotic cycle.
Immunofluorescence imaging assays are used to observe the behavior of sister chromatids in meiosis I and meiosis II as a function of Eco1 activity. In wild-type cells sister chromatids co-orient in meiosis I and move to the same pole of the spindle. In mammalian cells and fission yeast this co-orientation requires cohesin while studies in budding yeast have suggested the co-orientation is cohesin-independent. Here, the authors show that when Eco1 is depleted, the sisters often move to opposite poles at meiosis I, and suggest that cohesin (and Eco1) is indeed required for sister co-orientation. An alternate possibility is that the sisters have lost their association in meiotic prophase (due to cohesin failures) before attaching to microtubles and segregating randomly - often to opposite poles.
In summary the authors show that Eco1 has distinct roles on chromosome arms and centromeres and probably in both replication-linked and replication-independent events, acts to modulate cohesin location and function in meiosis.
-
Reviewer #3 (Public Review):
This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.
The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that …
Reviewer #3 (Public Review):
This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.
The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that Eco1 is important for establishing the meiotic segregation pattern. The paper is well written and the data largely support the conclusions. As such, this paper is expected to be of substantial interest to the field.
One notable weakness is the poor definition of the eco1 anchor-away allele (eco1-aa), on which much of the eco1 phenotypic analysis is based. The presented data indicate that addition of the FRB-GFP tag alone causes most of the phenotypes, regardless of nuclear depletion. It is well possible that the tag creates a meiosis-specific loss-of-function allele, although it is surprising that the tag does not have mitotic defects even though Eco1 presumably has the same substrate (the cohesin subunit Smc3) in both situations. Encouragingly, some of the phenotypes could be confirmed using a non-acetylatable smc3 mutant. However, the tag may also create neomorphic effects that may contribute to the Wpl1-independent effects and the apparent stronger defects of the eco1-aa allele compared to the non-acetylatable smc3 mutant.
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