The kleisin subunit controls the function of C. elegans meiotic cohesins by determining the mode of DNA binding and differential regulation by SCC-2 and WAPL-1
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eLife assessment
This important paper reveals distinct dynamics of two meiosis-specific cohesin complexes containing either REC-8 or CHO-3/4 in C. elegans: REC-8-cohesin is essential for sister chromatid cohesion in meiosis I and DNA double-strand break repair, while COH-3/4-cohesin, whose binding to meiotic chromosomes is stabilized by the cohesin accessory protein SCC-2, is necessary for loop-axis formation. The experimental evidence in the paper is solid based on cytological analysis using a conditional depletion of the gene. The work will be of interest to researchers working on meiosis and chromosome dynamics.
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Abstract
The cohesin complex plays essential roles in chromosome segregation, 3D genome organisation, and DNA damage repair through its ability to modify DNA topology. In higher eukaryotes, meiotic chromosome function, and therefore fertility, requires cohesin complexes containing meiosis-specific kleisin subunits: REC8 and RAD21L in mammals and REC-8 and COH-3/4 in Caenorhabditis elegans . How these complexes perform the multiple functions of cohesin during meiosis and whether this involves different modes of DNA binding or dynamic association with chromosomes is poorly understood. Combining time-resolved methods of protein removal with live imaging and exploiting the temporospatial organisation of the C. elegans germline, we show that REC-8 complexes provide sister chromatid cohesion (SCC) and DNA repair, while COH-3/4 complexes control higher-order chromosome structure. High-abundance COH-3/4 complexes associate dynamically with individual chromatids in a manner dependent on cohesin loading (SCC-2) and removal (WAPL-1) factors. In contrast, low-abundance REC-8 complexes associate stably with chromosomes, tethering sister chromatids from S-phase until the meiotic divisions. Our results reveal that kleisin identity determines the function of meiotic cohesin by controlling the mode and regulation of cohesin–DNA association, and are consistent with a model in which SCC and DNA looping are performed by variant cohesin complexes that coexist on chromosomes.
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Author Response
Reviewer #2 (Public Review):
During meiosis, mitotic cohesin complexes are replaced by meiosis-specific cohesins to enable a stepwise loss of sister chromatid cohesion. The identity of the cohesin complex is defined by its kleisin subunit. In the early meiotic prophase, the mitotic kleisin Scc1 is replaced by a meiotic counterpart Rec8. C. elegans expresses two additional meiotic kleisins, COH-3 and COH-4; however, how meiotic cohesin complexes differ in their loading and function has been unclear. In this paper, Castellano-Pozo and colleagues unveil their differential dynamics and functions using elegant approaches that include auxin-mediated depletion and TEV-mediated removal of meiotic kleisins. The association of COH-3/4 with chromosomes is dynamic and is under the control of two cohesin regulators, WAPL-1 and …
Author Response
Reviewer #2 (Public Review):
During meiosis, mitotic cohesin complexes are replaced by meiosis-specific cohesins to enable a stepwise loss of sister chromatid cohesion. The identity of the cohesin complex is defined by its kleisin subunit. In the early meiotic prophase, the mitotic kleisin Scc1 is replaced by a meiotic counterpart Rec8. C. elegans expresses two additional meiotic kleisins, COH-3 and COH-4; however, how meiotic cohesin complexes differ in their loading and function has been unclear. In this paper, Castellano-Pozo and colleagues unveil their differential dynamics and functions using elegant approaches that include auxin-mediated depletion and TEV-mediated removal of meiotic kleisins. The association of COH-3/4 with chromosomes is dynamic and is under the control of two cohesin regulators, WAPL-1 and SCC-2, while REC-8 remains more stably associated. The authors established that COH-3/4 is involved in maintaining the structural integrity of chromosome axes, whereas the REC-8 cohesin is solely responsible for sister chromatid cohesion throughout meiosis. They further demonstrated the role of REC-8 in the repair of meiotic DSBs.
Overall, this solid work unequivocally establishes the distinct regulation and requirements for REC-8 and COH-3/4 cohesin complexes during C. elegans meiosis.
We thank the reviewer for their overall support.
However, as the authors acknowledged, the role of REC-8 cohesins in sister chromatid cohesion has been shown previously using genetic mutants (Crawley et al., 2016 eLife). While the authors highlighted the advantages of removing cohesin subunits in establishing their distinct requirements, many of the results were recapitulated from their previous work (e.g. rec-8; spo-11 and coh-3/4; spo-11). It might be helpful for the readers to compare the results between the two studies and point out uniquely illuminating results.
Although we and others have previously suggested that REC-8 cohesin provides SCC in worms based on observations made in different meiotic mutants, a convincing demonstration of this possibility was lacking and an alternative model proposing that COH-3/4 cohesin do provide SCC had been proposed (Severson et al 2014). Using TEV-tagged versions of REC-8 and COH-3/4 we unequivocally establish that SCC is uniquely provided by REC-8 complexes in metaphase I oocytes. We have introduced modifications in the text and figures (including a model shown in Figure 5) to highlight the main results of our study.
The role of REC-8 in DNA repair has also been shown in different contexts. Chromosomes fragmentation and DNA bridges are observed in rec-8; syp-1 or rec-8; syp-2 (RNAi) animals (Colaiacovo et al., 2003 Dev Cell; Crawley et al., 2016 eLife), suggesting a role of REC-8 in inter-sister repair. Persistent RAD-51 foci are also observed on asynapsed chromosomes in rec-8 mutants, suggesting a role for REC-8 in DNA repair (Cahoon et al., 2019 Genetics). The authors must cite these papers and discuss the results in the context of prior work.
We agree with the reviewer that the studies mentioned above are consistent with the possibility that REC-8 complexes contribute to inter-sister repair. We now include citations of the manuscripts mentioned by the reviewer. The experiments presented in Figures 4A-B are different from those in the studies mentioned by the reviewer in that by introducing exogenous DSBs by IR (including in a spo-11 mutant background (Figure 4B)) we can more directly address the contribution of REC-8 and COH-3/4 complexes in pachytene nuclei under a situation in which similar numbers of DSBs are introduced. These experiments show that low abundance REC-8 complexes play a much more prominent role in DSB repair than highly-abundant COH-3/4 complexes and suggest that this activity is coupled to REC-8’s role in SCC.
Reviewer #3 (Public Review):
The study, performed in the animal model C. elegans, aims at characterizing functional differences in the meiosis-specific kleisins, REC-8 and COH-3/4.
The authors conclude that in worms the identity of the kleisin subunit of the cohesin complex determines whether cohesin promotes cohesion, or controls higher-order chromosome structure. COH-3/4 is highly abundant and dynamic and responds to SCC-2 and WAPL-1. In contrast, REC-8 complexes associate stably and in low abundance and are resistant to SCC-2 and WAPL-1 perturbations.
Main points:
This study is a continuation and partially a repeat of a study Castellano-Pozo & Martinez-Perez published in Nat. Comm. 2020, in which they depleted COH-3/4 and REC-8 by injecting TEV and cleaved artificially engineered TEV sites in these kleisins.The results were slightly different though, as the authors concluded: "Disassembly of axial elements requires simultaneous removal of REC-8 and COH-3/4."
The current study uses a degron instead of TEV and SIM to revisit the same result. This time, degradation of COH-3/4 alone, but not of Rec8 alone completely eliminates axial elements. It seems that, if the conclusion is now correct, the previous headline must be incorrect, showing that more care has to be taken in the conclusions.
The reviewer is referring to data shown in Figure 1C saying that we used a degron system to degrade COH-3/4 and REC-8 from pachytene nuclei. This is incorrect, images in this figure correspond to rec-8 and coh-3 coh-4 double mutants (as indicated in main text and figure legend) and therefore to germlines lacking REC-8 or COH-3/4 from the onset of meiosis. In contrast, in the Castellano-Pozo et al 2020 study REC-8 or COH-3/4 were removed from pachytene chromosomes using the TEV approach following normal chromosome morphogenesis at meiosis onset to specifically address how kleisin removal in nuclei at the pachytene stage impacted on meiotic progression. In addition to this, Figure 1C does not show that lack of COH-3/4 “completely eliminates axial elements”, as stated by the reviewer, but rather that “SMC-1::GFP signals appeared as discontinuous weak signals in pachytene nuclei” (see description of this result in lines 103-104 of first version). This finding is consistent with the Castellano-Pozo et al 2020 study where we reported that staining of HORMADs (used to visualise axial elements) became weaker and more discontinuous following removal of COH-3/4 than REC-8 from pachytene axial elements (this observation is also mentioned in lines 96-97 of the first version of our manuscript).
One new experiment in this study is the degradation of scc-2::AID::GFP. The authors treat the germline with auxin for 14 hours. How long scc-2::AID actually needs for degradation and thus, how long cells actually remain without SCC-2, is unknown. What is definitely needed is a serious analysis of the speed of degradation of Scc2 in the various stages.
It is currently not possible to estimate, as the authors do, how long cells have been without SCC-2. This estimation assumes an immediate depletion of SCC-2.
If this were indeed the case, then depletion intervals should be much shorter, because the important primary phenotypes occur immediately after depletion, not 14 hours later.
We now analyse REC-8::HA and COH-3/4 staining after auxin treatment for 8 and 14 hours, showing that 8 hours results in weaker effect on COH-3/4 depletion in pachytene nuclei and a smaller section of the germline lacking REC-8::HA staining in early prophase. We also include cartoons in Figure 2B to explain how nuclei progress through pachytene (35 hours in total).
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eLife assessment
This important paper reveals distinct dynamics of two meiosis-specific cohesin complexes containing either REC-8 or CHO-3/4 in C. elegans: REC-8-cohesin is essential for sister chromatid cohesion in meiosis I and DNA double-strand break repair, while COH-3/4-cohesin, whose binding to meiotic chromosomes is stabilized by the cohesin accessory protein SCC-2, is necessary for loop-axis formation. The experimental evidence in the paper is solid based on cytological analysis using a conditional depletion of the gene. The work will be of interest to researchers working on meiosis and chromosome dynamics.
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Reviewer #1 (Public Review):
Meiosis uses distinct cohesin complexes for chromosome morphogenesis and segregation such as cohesins with meiosis-specific REC-8 and COH-3/4 in the nematode. In this important paper, by using stage-specific depletion of the cohesin component, the authors nicely showed that REC-8-cohesin stably binds to meiotic chromosomes and plays an essential role in sister chromatid cohesion in diakinesis and meiosis I. Moreover, COH-3/4-cohesin, whose chromosome binding is stabilized by the SCC-2 cohesin regulator, is more dynamic than Rec8-cohesin in prophase I and plays a role in loop-axis formation.
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Reviewer #2 (Public Review):
During meiosis, mitotic cohesin complexes are replaced by meiosis-specific cohesins to enable a stepwise loss of sister chromatid cohesion. The identity of the cohesin complex is defined by its kleisin subunit. In the early meiotic prophase, the mitotic kleisin Scc1 is replaced by a meiotic counterpart Rec8. C. elegans expresses two additional meiotic kleisins, COH-3 and COH-4; however, how meiotic cohesin complexes differ in their loading and function has been unclear. In this paper, Castellano-Pozo and colleagues unveil their differential dynamics and functions using elegant approaches that include auxin-mediated depletion and TEV-mediated removal of meiotic kleisins. The association of COH-3/4 with chromosomes is dynamic and is under the control of two cohesin regulators, WAPL-1 and SCC-2, while REC-8 …
Reviewer #2 (Public Review):
During meiosis, mitotic cohesin complexes are replaced by meiosis-specific cohesins to enable a stepwise loss of sister chromatid cohesion. The identity of the cohesin complex is defined by its kleisin subunit. In the early meiotic prophase, the mitotic kleisin Scc1 is replaced by a meiotic counterpart Rec8. C. elegans expresses two additional meiotic kleisins, COH-3 and COH-4; however, how meiotic cohesin complexes differ in their loading and function has been unclear. In this paper, Castellano-Pozo and colleagues unveil their differential dynamics and functions using elegant approaches that include auxin-mediated depletion and TEV-mediated removal of meiotic kleisins. The association of COH-3/4 with chromosomes is dynamic and is under the control of two cohesin regulators, WAPL-1 and SCC-2, while REC-8 remains more stably associated. The authors established that COH-3/4 is involved in maintaining the structural integrity of chromosome axes, whereas the REC-8 cohesin is solely responsible for sister chromatid cohesion throughout meiosis. They further demonstrated the role of REC-8 in the repair of meiotic DSBs.
Overall, this solid work unequivocally establishes the distinct regulation and requirements for REC-8 and COH-3/4 cohesin complexes during C. elegans meiosis. However, as the authors acknowledged, the role of REC-8 cohesins in sister chromatid cohesion has been shown previously using genetic mutants (Crawley et al., 2016 eLife). While the authors highlighted the advantages of removing cohesin subunits in establishing their distinct requirements, many of the results were recapitulated from their previous work (e.g. rec-8; spo-11 and coh-3/4; spo-11). It might be helpful for the readers to compare the results between the two studies and point out uniquely illuminating results.
The role of REC-8 in DNA repair has also been shown in different contexts. Chromosomes fragmentation and DNA bridges are observed in rec-8; syp-1 or rec-8; syp-2 (RNAi) animals (Colaiacovo et al., 2003 Dev Cell; Crawley et al., 2016 eLife), suggesting a role of REC-8 in inter-sister repair. Persistent RAD-51 foci are also observed on asynapsed chromosomes in rec-8 mutants, suggesting a role for REC-8 in DNA repair (Cahoon et al., 2019 Genetics). The authors must cite these papers and discuss the results in the context of prior work.
-
Reviewer #3 (Public Review):
The study, performed in the animal model C. elegans, aims at characterizing functional differences in the meiosis-specific kleisins, REC-8 and COH-3/4.
The authors conclude that in worms the identity of the kleisin subunit of the cohesin complex determines whether cohesin promotes cohesion, or controls higher-order chromosome structure. COH-3/4 is highly abundant and dynamic and responds to SCC-2 and WAPL-1. In contrast, REC-8 complexes associate stably and in low abundance and are resistant to SCC-2 and WAPL-1 perturbations.Main points:
This study is a continuation and partially a repeat of a study Castellano-Pozo & Martinez-Perez published in Nat. Comm. 2020, in which they depleted COH-3/4 and REC-8 by injecting TEV and cleaved artificially engineered TEV sites in these kleisins.The results were slightly …
Reviewer #3 (Public Review):
The study, performed in the animal model C. elegans, aims at characterizing functional differences in the meiosis-specific kleisins, REC-8 and COH-3/4.
The authors conclude that in worms the identity of the kleisin subunit of the cohesin complex determines whether cohesin promotes cohesion, or controls higher-order chromosome structure. COH-3/4 is highly abundant and dynamic and responds to SCC-2 and WAPL-1. In contrast, REC-8 complexes associate stably and in low abundance and are resistant to SCC-2 and WAPL-1 perturbations.Main points:
This study is a continuation and partially a repeat of a study Castellano-Pozo & Martinez-Perez published in Nat. Comm. 2020, in which they depleted COH-3/4 and REC-8 by injecting TEV and cleaved artificially engineered TEV sites in these kleisins.The results were slightly different though, as the authors concluded: "Disassembly of axial elements requires simultaneous removal of REC-8 and COH-3/4."
The current study uses a degron instead of TEV and SIM to revisit the same result. This time, degradation of COH-3/4 alone, but not of Rec8 alone completely eliminates axial elements. It seems that, if the conclusion is now correct, the previous headline must be incorrect, showing that more care has to be taken in the conclusions.
One new experiment in this study is the degradation of scc-2::AID::GFP. The authors treat the germline with auxin for 14 hours. How long scc-2::AID actually needs for degradation and thus, how long cells actually remain without SCC-2, is unknown. What is definitely needed is a serious analysis of the speed of degradation of Scc2 in the various stages.
It is currently not possible to estimate, as the authors do, how long cells have been without SCC-2. This estimation assumes an immediate depletion of SCC-2.
If this were indeed the case, then depletion intervals should be much shorter, because the important primary phenotypes occur immediately after depletion, not 14 hours later. -
