Centromere-proximal suppression of meiotic crossovers in Drosophila is robust to changes in centromere number, repetitive DNA content, and centromere-clustering

Abstract Accurate segregation of homologous chromosomes during meiosis depends on both the presence and the regulated placement of crossovers (COs). The centromere effect, or CO exclusion in pericentromeric regions of the chromosome, is a meiotic CO patterning phenomenon that helps prevent nondisjunction, thereby protecting against chromosomal disorders and other meiotic defects. Despite being identified nearly a century ago, the mechanisms behind this fundamental cellular process remain unknown, with most studies of the Drosophila centromere effect focusing on local influences of the centromere and pericentric heterochromatin. In this study, we sought to investigate whether dosage changes in centromere number and repetitive DNA content affect the strength of the centromere effect, using phenotypic recombination mapping. Additionally, we studied the effects of repetitive DNA function on centromere effect strength using satellite DNA–binding protein mutants displaying defective centromere-clustering in meiotic nuclei. Despite what previous studies suggest, our results show that the Drosophila centromere effect is robust to changes in centromere number, repetitive DNA content, as well as repetitive DNA function. Our study suggests that the centromere effect is unlikely to be spatially controlled, providing novel insight into the mechanisms behind the Drosophila centromere effect.

Two experts in the field have reviewed your manuscript, and I have read it as well.I am pleased to inform you that, with minor revisions, it is potentially suitable for publication in GENETICS.
Both reviewers felt that the experiments were well thought out and the data were readily interpretable.Although the data are essentially negative throughout, there was prior evidence indicating that these experiments would uncover a mechanism.The fact no phenotype was uncovered supports that these aspects of chromosome biology are not involved in the centromere effect, which is an important result on its own.Both reviewers have comments and concerns that need to be addressed in a revised manuscript.You can read them at the end of this email.If possible, please strongly consider the experiment suggested by Reviewer #1 regarding centromere clustering in ProdK/+ females (and presumably D1 females) -this would be useful information for the field and would help to interpret the data from your experiment.Other suggested experiments would be welcome, but would not be necessary for the paper to be published.
We look forward to receiving your revised manuscript.Please let the editorial office know approximately how long you expect to need for revisions.
Upon resubmission, please include: 1.A clean version of your manuscript; 2. A marked version of your manuscript in which you highlight significant revisions carried out in response to the major points raised by the editor/reviewers (track changes is acceptable if preferred); 3. A detailed response to the editor's/reviewers' comments and to the concerns listed above.Please reference line numbers in this response to aid the editors.
Additionally, please ensure that your resubmission is formatted for GENETICS.https://academic.oup.com/genetics/pages/general-instructionsFollow this link to submit the revised manuscript: Link Not Available Sincerely, Jack Bateman Associate Editor GENETICS Approved by: Howard Lipshitz Editor in Chief GENETICS - -------------------------------------------------------------------Reviewer #1 (Comments for the Authors (Required)): Pazhayam et al. attempted to address a fundamental question regarding the influence of centromeres and repetitive DNA on the centromere effect (the absence of crossovers in the pericentromeric heterochromatin).The centromere effect has been investigated in Drosophila in previous work through different experimental approaches, but the mechanisms behind this process are still unknown.There was previous evidence suggesting centromere dosage and repetitive DNA play a role in the centromere effect.The authors extensively tested the importance of centromere and repetitive DNA dosage and unfortunately found crossovers were unchanged in any of the conditions they tested.This has been an open question in the field for a very long time and the work presented here (while negative data) is still an important step forward in increasing our understanding of the mechanisms behind the centromere effect.Additionally, the authors experiments were very thorough and I don't have any experimental improvements other than point 1.Overall, the paper was well written, clear, and easy to follow.I have put my comments below.

Comments:
1. Line 391-393: I was unclear if the authors know what centromere clustering looks like in oocytes in ProdK/+ females.I looked at the original paper from Jagannathan et al. and did not see oocyte data.If the authors don't know what centromere clustering looks like in females this is an experiment they could do.It is easy to examine centromere clustering via antibody staining.They did address the possibility that the phenotype in females might not be the same but it would be a more complete experiment if they are confident there is a clustering phenotype in oocytes in those mutants.
2. Line 133-135: "it is unclear whether this effect is due to a change in total centromere number from 16 to (one per sister chromatid for eight chromosomes) to 24 in triploids (one per sister chromatid for twelve chromosomes)" This description was confusing-the authors could rewrite this to be clearer.Also, the "to" is repeated.
3. Line 154-158: The sentences about recombination intervals should be rewritten to be more accessible to a non-Drosophila audience.It could be clearer.
4. Line 160: The genotype C(1)/0 ; C(4)/0 should be referenced earlier (probably at line 149) to make it clear what the genotype is for the compound X and 4 for readers not familiar with Drosophila genotypes.5.The descriptions of the increase in repetitive DNA was a bit confusing.I would recommend outlining the XXY and triplo-4 flies prior to the double.

Reviewer #2 (Comments for the Authors (Required)):
This paper investigates a widespread phenomenon in eukaryote genomes -specifically the suppression of meiotic crossovers in proximity to centromeres -taking a genetic approach in Drosophila.Specifically, they alter the dosage of centromeres, and repeat DNA content, and analyse how this influences crossovers.
First the authors use Drosophila stocks with compound X and Chr4, creating dicentric (at the sequence level) chromosomes.The authors use CENP-C staining to show that the fusion chromosomes only have a single centromere focus -this is interesting and shows that one of the centromeres has epigenetically inactivated.Is it always the same centromere which inactivates?Is the remaining centromere in its normal position?It might be interesting to perform CENP-A or CENP-C ChIP-seq in these lines to provide a higher resolution map of centromere location in these strains?Using these lines the authors then map crossovers on chromosome 2 across the centromere.Using segregation of phenotypic markers, no difference is observed on chromosome 2, in the presence of the X-Chr4 fusion.
Map distances are compared to physical distances based on genome assembly 6.53 -is this assembly known to be complete through the centromeres?
Next the authors analysed XXY triplo-4 flies and mapped crossovers on chromosomes 2 and 3, but only weak effects observed.I was wondering where the piRNA clusters are located in Drosophila?Are they located in crossover suppressed pericentromere intervals?I am aware that piRNA clusters are highly polymorphic between strains -perhaps that could be a useful way to address the question of varying heterochromatin on the crossover landscape.I believe recent studies have also used Cas9 to delete the piRNA clusters, which could also be useful?In terms of trans effects, maybe using known heterochromatic mutants might also be productive -eg suv3-9?Along these lines, the authors test the D1 and prod mutants, which encode proteins known to bind satellite repeats, but again no effects were seen.
In summary, the authors attempt a number of experiments to see whether the strength of the CE can be modified, but without any significant changes.What is known of the structural differences between the centromeres of the strains used?Could extensive SVs within the centromeres be causing crossover suppression, irrespective of whatever else is changing?The work overall is well performed and analysed appropriately -however, I though the authors could make more use of the available complete centromere assemblies available in Drosophila.
Minor points: Line 37 references Beadle 1932 for the CE -however, this effect is widespread and seen in diverse plant, animals and fungi.I think a more extensive set of references showing this effect is common across species, and across different types of centromeres, is worthaking (ie point, regional, satellite, RT-based).
Suppression of Meiotic Crossovers in Drosophila is Robust to Changes in Centromere Number and Repetitive DNA Content Dear Dr. Sekelsky: