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Heterozygous inversion breakpoints suppress meiotic crossovers by altering recombination repair outcomes [1]

['Haosheng Li', 'Department Of Biology', 'Case Western Reserve University', 'Cleveland', 'Ohio', 'United States Of America', 'Erica Berent', 'Savannah Hadjipanteli', 'Miranda Galey', 'Division Of Genetic Medicine']

Date: 2023-05

Heterozygous chromosome inversions suppress meiotic crossover (CO) formation within an inversion, potentially because they lead to gross chromosome rearrangements that produce inviable gametes. Interestingly, COs are also severely reduced in regions nearby but outside of inversion breakpoints even though COs in these regions do not result in rearrangements. Our mechanistic understanding of why COs are suppressed outside of inversion breakpoints is limited by a lack of data on the frequency of noncrossover gene conversions (NCOGCs) in these regions. To address this critical gap, we mapped the location and frequency of rare CO and NCOGC events that occurred outside of the dl-49 chrX inversion in D. melanogaster. We created full-sibling wildtype and inversion stocks and recovered COs and NCOGCs in the syntenic regions of both stocks, allowing us to directly compare rates and distributions of recombination events. We show that COs outside of the proximal inversion breakpoint are distributed in a distance-dependent manner, with strongest suppression near the inversion breakpoint. We find that NCOGCs occur evenly throughout the chromosome and, importantly, are not suppressed near inversion breakpoints. We propose a model in which COs are suppressed by inversion breakpoints in a distance-dependent manner through mechanisms that influence DNA double-strand break repair outcome but not double-strand break formation. We suggest that subtle changes in the synaptonemal complex and chromosome pairing might lead to unstable interhomolog interactions during recombination that permits NCOGC formation but not CO formation.

Meiosis is the specialized cell cycle used to generate genetic diversity and reduce the genome copy number in gametes. Successful meiosis requires homologous chromosomes to pair and recombine to form crossovers, which are necessary for proper chromosome segregation. However, chromosome rearrangements called inversions that reverse the order of genes on one of the homologous chromosomes suppress crossovers. While this phenomenon has been studied for 100 years, much is still unknown about the mechanisms that prevent crossovers from occurring. In our current work, we show that these heterozygous inversions suppress crossovers nearby but outside of the rearrangement boundaries by altering the regulation of recombination.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: DEM is engaged in a research agreement with Oxford Nanopore Technologies and they have paid for him to travel to speak on their behalf. DEM is also on the scientific advisory board of Oxford Nanopore Technologies.

Introduction

Chromosome inversions have far-ranging impacts on reproduction and speciation when paired with a non-inverted homolog. At the molecular level, heterozygous inversions disrupt fundamental aspects of meiosis by suppressing both the formation and recovery of meiotic crossovers (COs) within the inversion and in the regions nearby but outside the inversion breakpoints [1] (Fig 1B). At the population level, suppressing COs prevents genetic exchange between an inversion and its non-inverted homolog, dramatically reducing gene flow for that portion of the genome [2,3]. Within the inversion, recombination is unable to separate combinations of alleles, which can be beneficial for advantageous or adaptive alleles [4–6]. Alternatively, suppression of exchange can have negative outcomes such as harboring selfish genetic elements and meiotic drive systems (reviewed in [7]). Lastly, the ability of inversions to prevent gene flow locally in the genome is the basis of the chromosomal theory of speciation [8,9]. Given the far-ranging impacts of heterozygous inversions on reproduction and speciation, it is critical to understand how they disrupt meiosis at the molecular level. Despite a rich history of studying inversions, the mechanisms of how they suppress COs remain unknown. Here, we exploit the unique properties of inversion breakpoints to provide crucial insight into the mechanisms of CO suppression in Drosophila melanogaster.

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TIFF original image Download: Fig 1. A) Canonical recombination pathways used in meiosis. 1) Meiosis is initiated by an enzymatically mediated DSB. 2) The DSB is resected into single-stranded ends, one of which invades the homologous chromosome and primes DNA synthesis. This forms a displacement loop (D-loop) and during synthesis dependent strand annealing (SDSA), this structure can be unwound by a helicase into a NCO. 3) If the second end of the DSB is captured by the D-loop, it also primes synthesis. 4) The second-end capture intermediate is ligated into a double Holliday Junction, which is cleaved by a meiosis-specific endonuclease to form mostly COs, although some NCOs can occasionally form. B) Predicted pairing arrangement between an inversion and a standard arrangement homologous chromosome. Single COs within the inversion breakpoints lead to acentric and dicentric chromosomes with deletions and duplications. If these COs occur, they are not recovered in the offspring (the so-called transmission distortion effect). COs are also suppressed in the collinear regions outside of the inversion breakpoints, even though COs in these regions would not lead to chromosome rearrangements. This suggests that COs are prevented from forming, as opposed to being suppressed due to transmission distortion. https://doi.org/10.1371/journal.pgen.1010702.g001

Early work on heterozygous inversions focused on two possible explanations for how they might suppress COs. The first possibility is that when inversions are heterozygous with a non-inverted chromosome, meiotic chromosome pairing and synapsis are defective, which prevents CO formation [10,11]. The second possibility is that COs can form, but because COs within the inversion will lead to chromosome rearrangements, the gametes containing such chromosomes will be inviable [1,11] (Fig 1B). This will make it appear as if COs are suppressed, when in reality, those chromosomes simply are not recovered in the offspring. This debate was mostly settled in 1936 [12,13] when Sturtevant demonstrated that COs do form within an inversion yet, paradoxically, there is no increased embryonic lethality as expected; he suggested a model where chromosomes resulting from COs within an inversion are aligned on the meiotic spindle such that they are preferentially placed in the polar bodies and eliminated (see [14] for a summary of this model). Despite the lack of direct cytological evidence for Sturtevant’s model, the field has generally accepted the notion that heterozygous inversions suppress COs because the aneuploid offspring are not recovered [15–17].

Concomitant with the early observation that COs are suppressed within heterozygous inversions was the observation that they are also severely reduced in regions immediately outside the inversion breakpoints [11,12,18]. For example, on a chromosome carrying the dl-49 inversion in D. melanogaster, COs are reduced to approximately 30% of wildtype in the proximal interval and to about 4% of wildtype in the distal interval [11]. This phenomenon is not exclusive to D. melanogaster as it was also seen in D. pseudoobscura by Dobzhansky and Epling [4]. Additionally, in interspecies crosses between D. pseudoobscura and D. persimilis, crossover frequency ranges from 0.0% to 0.5% in regions up to approximately 2.5 Mb outside of the inversion breakpoints, depending on the inversion [19,20]. Since COs that occur outside of the inversion breakpoints will not lead to chromosome rearrangements, there must be a mechanistic explanation for why they do not form in these regions.

COs are made by repairing a DNA double-strand break (DSB) made during meiosis (Fig 1A). However, only a few select DSBs will be repaired into COs, while the majority of DSBs will be repaired into noncrossovers (reviewed in [21]). If these noncrossovers are associated with gene conversions, then there are small tracts of unidirectional gene transfer from one homolog to the other. A major open question is if heterozygous inversions impact noncrossover gene conversions (NCOGCs) in a manner similar to crossovers.

Data are sparse on how heterozygous inversions affect NCOGCs because they are notoriously difficult to detect due to their small size, less than 1 kb in Drosophila species [22–24]. It is possible to select for NCOGCs using purine selection for intragenic recombination events at the rosy locus in D. melanogaster, but these events are so rare that it requires selecting against 500,000 to 1 million offspring in each experiment [23]. Previous work using this approach to study NCOGC frequencies in the interior of an inversion selected against 5,000,000 offspring to recover 66 NCOGCs; this work demonstrated that NCOGCs occur within a heterozygous inversion at the same frequency as in standard arrangement chromosomes [25]. Even attempts at comprehensively assessing the frequency and location of NCOGCs using genome-wide analyses are limited by small sample sizes. Sequencing-based analyses in D. pseudoobscura and D. persimilis showed that NCOGC frequencies inside inversions were at least as high as in collinear regions, but the number of unique NCOGC events was only 32 [26]. Similar sequencing analyses in D. melanogaster show that NCOGCs within inversions occur at rates higher than on standard arrangement chromosomes [27]. While this work was able to analyze the location of 79 unique NCOGCs, it was done using multiply inverted balancer chromosomes, which may not be representative of how single or naturally occurring inversions behave.

The same sequencing-based analyses described above provide some limited insight into how NCOGCs behave outside of inversion breakpoints. In D. melanogaster, 20 of the 79 unique NCOGCs occurred between 0.5 and 1 Mb away from the inversion breakpoints and one NCOGC was detected only 14kb away [27]. In the D. pseudoobscura and D. persimilis analysis, there was one NCOGC 37 kb away from an inversion breakpoint [26]. The low—but non-zero—frequency of NCOGCs within 500 kb of inversion breakpoints suggests that these regions are at least competent to form DSBs, although it remains to be seen if DSBs form at the same frequency in these regions as in collinear portions of the genome. Critically, the datasets on NCOGCs outside inversion breakpoints are too small to draw robust mechanistic conclusions.

The emerging picture of recombination outside of inversion breakpoints is that CO frequency is severely reduced and that NCOGCs can occur. However, it remains unclear whether NCOGCs are reduced by similar levels as COs, preventing crucial insight into whether CO suppression is mediated by a reduction in DSBs or whether recombination is biased away from CO repair (Fig 1A). To address this, we generated a high-resolution map of rare CO and NCOGC events outside of a single X chromosome inversion in D. melanogaster. Critically, we built full-sibling wildtype and inversion stocks and recovered recombination events in syntenic regions, enabling us to directly compare frequencies and distributions of recombination events. We find that COs are suppressed in a distance-dependent manner from the inversion breakpoint and that NCOGCs occur at wildtype frequencies outside of inversion breakpoints. Together, these data suggest that inversion breakpoints suppress COs by altering recombination outcomes as opposed to suppressing DSB formation.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010702

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