Meiotic chromosome structures constrain and respond to designation of crossover sites

Abstract

Crossover recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologues that ensure their proper segregation at meiosis I1. Despite this requirement for crossovers and an excess of the double-strand DNA breaks that are the initiating events for meiotic recombination, most organisms make very few crossovers per chromosome pair2. Moreover, crossovers tend to inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly understood phenomenon known as crossover interference3,4. Here we show that the synaptonemal complex, a meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by crossover recombination events. Using a cytological marker of crossover sites in Caenorhabditis elegans5, we show that partial depletion of the synaptonemal complex central region proteins attenuates crossover interference, increasing crossovers and reducing the effective distance over which interference operates, indicating that synaptonemal complex proteins limit crossovers. Moreover, we show that crossovers are associated with a local 0.4–0.5-micrometre increase in chromosome axis length. We propose that meiotic crossover regulation operates as a self-limiting system in which meiotic chromosome structures establish an environment that promotes crossover formation, which in turn alters chromosome structure to inhibit other crossovers at additional sites.

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Figure 1: SYP-1 partial depletion increases numbers of COSA-1 foci and chiasmata.
Figure 2: SYP-1 partial depletion attenuates crossover interference.
Figure 3: SYP-1 partial depletion decreases crossover interference strength.
Figure 4: Crossover designation causes a local expansion of chromosome axis length.

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Acknowledgements

We thank A. Dernburg and M. Zetka for antibodies and the CGC (funded by National Institutes of Health (NIH) P40 OD010440) for strains. We thank K. Hillers and K. Zawadzki for comments on the manuscript. This work was supported by a Helen Hay Whitney Foundation Postdoctoral Fellowship, a Katharine McCormick Advanced Postdoctoral Fellowship, and NIH K99 HD076165 to D.E.L. and by NIH R01 GM067268 to A.M.V. B.J.M. is an investigator of the Howard Hughes Medical Institute.

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Contributions

D.E.L. and A.M.V. conceived and designed the experiments, analysed the data and wrote the paper. D.E.L. performed the experiments. S.U. deconvolved confocal images. S.U. and B.J.M. provided scientific discussions and technical expertise for computational straightening of chromosomes.

Corresponding author

Correspondence to Anne M. Villeneuve.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Quantification of SYP-1 partial depletion by attenuated RNAi.

a, Representative western blot analysis of protein lysates from control, syp-1 partial RNAi and syp-1 null worms. A dilution series of control samples was used to estimate that the level of SYP-1 protein was reduced to approximately 25–30% of the control SYP-1 level following syp-1 partial RNAi under our experimental conditions. All panels shown are from the same membrane probed with indicated antibodies. Similar results were obtained for three independent experiments. b, Representative immunofluorescence images of late pachytene nuclei co-stained for SYP-1 (green), chromosome axis marker HTP-3 (red) and GFP::COSA-1 (blue), showing reduction of SYP-1 fluorescence relative to HTP-3 fluorescence and increase in GFP::COSA-1 foci in syp-1 partial RNAi nucleus compared to control nucleus. Except for right-most panels, images shown are sum projections through three-dimensional data stacks encompassing whole nuclei. For the first four pairs of control and syp-1 partial RNAi panels, identical exposure times and dynamic range settings for image display were used to highlight the reduction in the SYP-1:HTP-3 ratio. In the last two panels, SYP-1 signal levels were adjusted for syp-1 partial RNAi images to facilitate visualization of the SYP-1 tracts. Because some SCs from the top and bottom halves of the nuclei are superimposed in the full projections encompassing whole nuclei, partial projections showing half nuclei are also provided (right-most images). Scale bar, 2 μm. c, Graphs showing quantification of the reduction in SYP-1 fluorescence relative to HTP-3 fluorescence following syp-1 partial RNAi. Two different methods for analysing the data (see Methods) yield similar results, indicating that under the syp-1 partial RNAi conditions used for our experimental analysis, SYP-1 levels are reduced to approximately 30–40% of control levels. Error bars indicate s.d. Numbers of gonads assessed: experiment no. 1: control = 7 gonads, syp-1 RNAi = 7 gonads; experiment no. 2: control = 15 gonads, syp-1 RNAi = 6 gonads; experiment no. 3: control = 10 gonads, syp-1 RNAi = 8 gonads.

Extended Data Figure 2 Partial depletion of SYPs increases numbers of COSA-1 foci.

Graph depicting the mean numbers of GFP::COSA-1 foci per late pachytene nucleus detected following exposure to syp-1/F26D2.2, syp-2/C24G6.1 or syp-3/F39H2.4 RNAi or empty vector control28. RNAi and control conditions were identical to those described in Methods, except that worms were dissected for immunofluorescence at 24 h post-L4 stage on RNAi or control plates at 25 °C. Error bars indicate s.d. Control nuclei had an average of six COSA-1 foci per nucleus and a very low standard deviation, indicating operation of the highly robust crossover control system. Partial RNAi treatment for any of the syp genes resulted both in a significant increase in the average number of GFP::COSA-1 foci >6 per nucleus (Mann–Whitney, two-tailed P > 0.0001 for syp-1, syp-2 and syp-3 RNAi) and in a much higher s.d., indicating impairment of crossover control. Numbers of nuclei counted were: control, n = 78; syp-1, n = 64; syp-2, n = 129; syp-3, n = 87.

Extended Data Figure 3 GFP::COSA-1 foci in syp-1 RNAi nuclei correspond to inter-homologue crossovers.

a, Quantification of chiasmata on the mnT12 bivalent in diakinesis-stage oocytes, showing that the incidence of chiasmata corresponds well with the incidence of GFP::COSA-1 foci observed at late pachytene (Fig. 2a). For bivalents with only one or two chiasmata, each individual chiasma was readily scored; bivalents with ≥ 3 chiasmata were pooled into a single category owing to their highly contorted structures, which in some cases made it difficult to discriminate whether 3, 4 or 5 chiasmata were present. In control oocytes, all mnT12 bivalents had one or two chiasmata. By contrast, 47% of syp-1 RNAi oocytes had mnT12 bivalents with ≥ 3 chiasmata, corresponding well with 49% of late pachytene syp-1 RNAi mnT12 having ≥ 3 GFP::COSA-1 foci. As formation of each chiasma requires an inter-homologue crossover event, the close correspondence between the numbers of GFP::COSA-1 foci at late pachytene and chiasmata at diakinesis on syp-1 RNAi mnT12 bivalents indicates that most, and probably all, GFP::COSA-1 foci in syp-1 RNAi late pachytene nuclei are marking inter-homologue crossovers. Numbers of diakinesis nuclei scored: control, 86 nuclei from 35 gonads; syp-1 RNAi, 156 nuclei from 53 gonads. b, GFP::COSA-1 foci are not detected on asynapsed chromosome segments. In the regions of the mnT12 control and mnT12 syp-1 RNAi germ lines that were imaged for analyses of GFP::COSA-1 foci, we identified a subset of nuclei in which portions of the mnT12 fusion chromosome pair were asynapsed. These asynapsed segments were found at comparable frequencies among analysed nuclei from control (8%) and syp-1 RNAi (12%) germ lines and may represent early stages of desynapsis as cells transition from late pachynema to early diplonema. (Within these nuclei, the asynapsed mnT12 segments comprised approximately 18% of the total mnT12 axis length for control and 26% for syp-1 RNAi.) The mnT12 bivalents for all control (top left) and syp-1 RNAi (top right) nuclei in this category are represented in schematic form, with the chromosome axes (HTP-3, red) cartooned to depict both the approximate location and size of the asynapsed segment(s) relative to the total axis length, and the positions of GFP::COSA-1 (green) and HIM-8 (blue) foci. Notably, all GFP::COSA-1 foci on these partially asynapsed chromosome pairs were associated with synapsed segments, located either within a synapsed segment or at the boundary between a synapsed segment and an asynapsed segment; GFP::COSA-1 foci were never found on the asynapsed axis segments. Given the fraction of total axis length that was asynapsed in these syp-1 RNAi mnT12 nuclei, the observed restriction of GFP::COSA-1 foci to synapsed segments (where homologues are closely juxtaposed) represents a highly significant (χ2 test; P = 0.0002) departure from the distribution expected if GFP::COSA-1 foci were equally likely to occur on synapsed segments and asynapsed segments (where homologues are separated), consistent with the interpretation that these GFP::COSA-1 foci correspond to inter-homologue recombination events.

Extended Data Figure 4 Distribution of GFP::COSA-1 foci among evenly spaced intervals along mnT12.

a, Bar graph for eight-interval analysis of mnT12 (X;IV) fusion chromosomes, indicating the frequencies of GFP::COSA-1 foci in each interval for control (blue) and syp-1 partial RNAi (purple) worms. b, Table for eight-interval analysis indicating both the focus frequencies and the numbers of GFP::COSA-1 foci in each interval. c, Table for four-interval analysis indicating for each interval both the numbers and the percentages of mnT12 chromosome pairs with ≥ 1 GFP::COSA-1 focus in that interval (used for interference strength calculations in Fig. 3a and Extended Data Table 1).

Extended Data Figure 5 Gamma probability distribution modelling of inter-COSA-1 focus distances.

a, b, Histograms of the distribution of inter-focus distances (reported as percentage of total axis length) for binned control data (n = 47) (a) and syp-1 RNAi data (n = 183) (b). The best-fit gamma probability distribution curves generated from modelling the binned data sets (Fig. 3a) are overlaid on the histograms.

Extended Data Figure 6 Association between local axis length and GFP::COSA-1 foci at 20 °C.

Scatter plot of length measurements (μm) for the segment of mnT12 chromosome axis from the left end of mnT12 to the centre of the HIM-8 focus (as seen in Fig. 4d), for spo-11/+ nuclei without (blue diamonds) or with (green diamonds) a GFP::COSA-1 focus in this chromosome segment and for the spo-11 mutant (red triangles), which lacks meiotic DSBs and crossovers. Middle lines indicate mean and error bars indicate s.d. Mean length measurements for spo-11 nuclei (0.31 μm, n = 92) and spo-11/+ nuclei (0.35 μm, n = 88) lacking a focus in this chromosome segment were not significantly different from each other (Mann–Whitney, two-tailed P = 0.062), whereas both were significantly lower (Mann–Whitney, two-tailed P = 0.0010; P = 0.0011) than for spo-11/+ nuclei that had a GFP::COSA-1 focus in this segment (0.83 μm, n = 4).

Extended Data Table 1 Four-interval analysis of interference.

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Libuda, D., Uzawa, S., Meyer, B. et al. Meiotic chromosome structures constrain and respond to designation of crossover sites. Nature 502, 703–706 (2013). https://doi.org/10.1038/nature12577

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