when heterochromatin undergoes unpacking it becomes?
Thank you for submitting your article "Topoisomerase II and Spo11 induce post-meiotic DNA double-strand breaks in Tetrahymena thermophila haploid pronuclei" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Reviewing Editor Kathleen Collins and Kevin Struhl as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Kazufumi Mochizuki (Reviewer #1); Elcin Unal (Reviewer #2).
The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.
Summary:
In this manuscript, Akematsu et al. provide compelling evidence that post-meiotic DNA double-strand breaks (DSBs) occur in Tetrahymena. They further demonstrate that the formation of the post-meiotic DSBs requires a germline-specific topoisomerase 2 (Top2G) as well as a meiotic endonuclease Spo11 and that the repair of these breaks facilitates chromatin remodeling and functional gamete formation. These are important observations because 1) post-meiotic DSB are suggested to occur in other eukaryotes including mammals and 2) no previous studies demonstrated the function of post-meiotic DSBs. A limited amount of additional work would improve the manuscript to be suitable for publication.
Essential revisions:
1) The imaging data can greatly benefit from quantitative analysis. It's unclear what percentage of the post-meiotic pronuclei display signs of DSBs and how this changes in spo11 and top2G mutants. Similar analysis for Asf1-GFP and H3K56Ac will be helpful.
2) The mechanism of post-meiotic DSB repair is unknown, but correlates with H3K56ac. If possible, it would be informative to check markers specific for HR and/or NHEJ-directed repair such as Rad51, Ku, Mre11. This analysis will at least indicate whether these pathways are on during the time of post-meiotic DSB induction/repair.
3) The authors suggest that Top2G might be the B subunit for Spo11 in forming post-meiotic DSBs. I think it will be helpful if they determine whether Spo11 and Top2G interact with one another in a stage specific manner (i.e. in post-meiotic cells but not during meiotic prophase). Since they generated epitope-tagged alleles for both of these genes and since there is good time resolution between the occurrence of meiotic and post-meiotic breaks, a co-immunoprecipitation experiment in two different stages can be performed to address this question. Otherwise the speculation that Top2g could be an as-yet-undiscovered B-subunit for Spo11 seems ungrounded: Spo11 is a member of the TopVI family (heterotetramers) whereas one would assume Top2g (like other eukaryotic TOP2) is, a homodimer; it seems very hard to imagine that Tetrahymena Spo11 functions biochemically as a heterodimer with Top2g.
4) Have the authors checked whether the decline in γH2AX and the accompanying increase in H3K56ac occur in the unselected pronuclei in ∆ATG8 mutants? This could help distinguish whether there is a specific signal that directs DSB repair and chromatin remodeling to the selected pronuclei or whether the lack of nuclear elimination per se is sufficient for DSB repair to occur in the unselected pronuclei.
Figure 1A/B – it would help to emphasize that the γH2Ax staining is specific to the pronuclei, and not observed in the MAC.
Observations of H2AX-GFP shown in Figure 2C says nothing about the specificity of the anti-gH2AX antibody. If I am wrong, the authors need to add some more explanation for why they think this experiment demonstrates the specificity. Otherwise, the authors may simply remove the figure.
5) Is it technically feasible to deplete Spo11 and/or Top2 activity only in the post-meiotic stage? This would directly test the requirement of Spo11/Top2 in PM-DSB formation independently of any role they may have in meiotic DSB formation (that indirectly influences PM-DSB formation).
