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Nature Genetics | Article

日本語要約

Mouse tetrad analysis provides insights into recombination mechanisms and hotspot evolutionary dynamics

Journal name:
Nature Genetics
Volume:
46,
Pages:
1072–1080
Year published:
DOI:
doi:10.1038/ng.3068
Received
Accepted
Published online

Abstract

The ability to examine all chromatids from a single meiosis in yeast tetrads has been indispensable for defining the mechanisms of homologous recombination initiated by DNA double-strand breaks (DSBs). Using a broadly applicable strategy for the analysis of chromatids from a single meiosis at two recombination hotspots in mouse oocytes and spermatocytes, we demonstrate here the unidirectional transfer of information—gene conversion—in both crossovers and noncrossovers. Whereas gene conversion in crossovers is associated with reciprocal exchange, the unbroken chromatid is not altered in noncrossover gene conversion events, providing strong evidence that noncrossovers arise from a distinct pathway. Gene conversion frequently spares the binding site of the hotspot-specifying protein PRDM9, with the result that erosion of the hotspot is slowed. Thus, mouse tetrad analysis demonstrates how unique aspects of mammalian recombination mechanisms shape hotspot evolutionary dynamics.

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  1. Mouse oocyte tetrad analysis provides direct evidence for meiotic gene conversion.
    Figure 1: Mouse oocyte tetrad analysis provides direct evidence for meiotic gene conversion.

    (a) Individual germinal vesicle stage oocytes from F1 hybrid mice were microdissected for PCR to amplify the Psmb9 hotspot (chromosome 17) from all four chromatids using primers that recognize both parental chromosomes (white arrows). These universal PCRs provided DNA for tetrad analysis of recombinants. (b) Identification of crossovers at the Psmb9 hotspot from B10 × R209 hybrids by RFLP analysis (representative of the four crossovers detected in this hybrid). DNA from the universal PCR of each oocyte was digested with TaqI and BstXI and electrophoresed to differentiate PCR products from parental and crossover chromatids. Polymorphisms are schematically represented by blue and red circles. CO, crossover. (c) Tetrad amplification strategy for crossovers. DNA from the universal PCR of each oocyte was used to seed four separate PCRs, each with a primer set to amplify the Psmb9 hotspot from either a parental or recombinant chromatid (red and blue arrows). For B10 × R209 hybrids, any oocyte suspected of containing a crossover (b) was tested by this assay; for the B10.A × SGR hybrids, all oocytes were tested this way. (d) Tetrad amplification strategy for noncrossovers. Universally amplified DNA from a was used to seed two separate PCRs, each with one primer directed to a polymorphism being queried for conversion (asterisk) and a second primer directed to a polymorphism at one end of the Psmb9 hotspot. (e) Reciprocal crossovers with associated gene conversion in the B10 × R209 hybrid. Meiotic DSBs can form at the Psmb9 hotspot on the B10 (red) or R209 (blue) chromosome, leading to strand invasion into the intact donor homolog, which serves as a template for repair synthesis (dashed line). Thus, gene conversion will occur in favor of the donor homolog, as delineated by dotted vertical lines. Only the two chromatids engaged in homologous recombination are shown for simplicity. Tetrad analysis (n = 119 oocytes, from 3 mice) identified reciprocal crossovers with associated gene conversion in which Psmb9 sequences on the B10 chromosome (1 crossover) or R209 chromosome (3 crossovers) were converted to those of the homolog. Thus, on a per-meiosis basis, the crossover frequency was 3.4% (95% CI = 1.1–8.9%), translating to a per-gamete frequency of 1.7% (8 crossover chromatids per 476 haploid genome equivalents; Table 1). The red arrowhead indicates the position of the PRDM9wm7 binding site32. The mean minimal and maximal gene conversion tracts are as indicated. Colored bar, minimal gene conversion tract; gray lines, maximal gene conversion tract; ticks, polymorphisms. (f) Reciprocal crossovers with associated gene conversion in the B10.A × SGR hybrid. DSBs form preferentially at the Psmb9 hotspot on the B10.A chromosome (red), such that repair is templated by the R209 (blue) chromosome. Tetrad analysis (n = 205 oocytes, from 3 mice) identified reciprocal crossovers in which the Psmb9 sequences on the B10.A chromosome were converted to those of the SGR chromosome. The 2.9% (95% CI = 1.2–6.6%) per-meiosis crossover frequency agrees with previous estimates from ovaries (Table 1)23. The red arrowhead indicates the two polymorphisms that confer preferential PRDM9wm7 binding to the hotspot center from the B10.A chromosome32. (g) Noncrossovers in the B10 × R209 hybrid. Tetrad analysis identified noncrossovers in which Psmb9 sequences on the B10 chromosome (four noncrossovers) or R209 chromosome (three noncrossovers) were converted to those of the homolog, whereas the homolog was unaltered in each case. The noncrossover frequency was 5.9% (95% CI = 2.6–12.2%) per meiosis. Asterisks indicate the four polymorphisms queried for noncrossovers, using the approach shown in d. Monitoring the same four polymorphisms and three others in either pooled sperm or oocytes, >95% of detected noncrossovers were restricted to the four polymorphisms (Supplementary Fig. 1 and Supplementary Table 1). (h) Noncrossovers in the B10.A × SGR hybrid. All five noncrossovers identified by tetrad analysis involved conversion of Psmb9 hotspot sequences on the B10.A chromosome to those of the SGR chromosome. The noncrossover frequency was 2.4% (95% CI = 0.9–5.9%) per meiosis. Asterisks indicate the polymorphisms queried for noncrossovers, as in g.

  2. Mouse spermatocyte tetrads demonstrate that noncrossovers result from the unidirectional transfer of information.
    Figure 2: Mouse spermatocyte tetrads demonstrate that noncrossovers result from the unidirectional transfer of information.

    (a) Single-cell suspensions from the testes of adult F1 hybrid mice were stained with Hoechst 33342 and propidium iodide. Cells with the highest blue and red fluorescence intensities were sorted using the indicated gate (oval). In three independent experiments, all sorted cells were primary spermatocytes, with ~98% in diplonema or MLH1-positive pachynema, on the basis of staining for the axial element marker SYCP3 and the crossover marker MLH1 (refs. 37,58,59), as shown in the representative images chosen from 188 analyzed spermatocytes. Scale bar, 10 μm. (b) A3 hotspot amplification strategy for spermatocytes. Left, to amplify both noncrossovers and crossovers, cells were plated in pools of ~20 cells per well, and DNA was universally amplified across the A3 hotspot on chromosome 1. The amplified DNA was used to seed two separate PCRs using an allele-specific forward primer and a universal reverse primer. Right, to amplify only crossovers, cells were plated in larger pools of ~100 cells per well for universal amplification. Universally amplified DNA was used to seed two separate PCRs using primer sets to detect both recombinant chromatids. (c) Representative crossover and noncrossover recombinants from spermatocyte analysis. Replicate blots were generated from PCRs in the A/J-to-universal (U) orientation (top) or the DBA/2J-to-universal orientation (bottom) and probed with allele-specific oligonucleotides to genotype polymorphisms across the A3 hotspot. The genotype of a representative crossover with the length of the gene conversion tract is shown between the blots and that of a representative noncrossover is shown below the blots. Dot blot legend: solid colored circles or squares, genotype determined by blotting; dashed colored circles or squares, inferred genotype; black squares in the upper right corner, loading control of amplified DBA/2J or A/J DNA; black rectangles in the lower left corner, dilutions of the loading control; #, well containing noncrossovers on both the A/J and DBA/2J chromosomes. (d) Spermatocyte noncrossovers at the A3 hotspot. Because recombination initiates preferentially on the DBA/2J chromosome (red), the majority of noncrossovers involve conversion of DBA/2J polymorphisms to the A/J genotype (blue). The noncrossover frequency was 1.25% (95% CI = 1.0–1.5%) per meiosis. A schematic of the central polymorphisms is shown at the top. Red arrowheads indicate polymorphisms implicated in differential PRDM9 binding between A/J and DBA/2J; yellow shading indicates the predicted PRDM9 binding site (Fig. 3b). Indel 3 is located within a direct repeat such that only the A/J polymorphism can be genotyped; <4% of noncrossovers on the DBA/2J chromosome were converted only at this polymorphism. An asterisk indicates the noncrossover event highlighted in c. (e) Total Poisson-adjusted noncrossover frequencies on the DBA/2J (top) and A/J (bottom) chromosomes. Noncrossover frequencies at each tested polymorphism are normalized for co-conversion events. Ticks in the center represent the 22 polymorphisms tested.

  3. PRDM9 binds to the center of the A3 hotspot.
    Figure 3: PRDM9 binds to the center of the A3 hotspot.

    (a) Spermatocyte crossovers with associated gene conversion at the A3 hotspot. Tetrad analysis identified four reciprocal crossovers in which A3 sequences on the A/J chromosome were converted to those of the DBA/2J chromosome and eight reciprocal crossovers in which A3 sequences on the DBA/2J chromosome were converted to those of the A/J chromosome. Crossovers were isolated from experiments with 20 cells (2 crossovers) or 100 cells (10 crossovers) per well. The crossover frequency per meiosis was 0.08% (95% CI = 0.05–0.13%). An asterisk indicates the crossover highlighted in Figure 2c; the yellow bar represents predicted PRDM9 binding. (b) Deviation from the mendelian 50% transmission frequency due to crossover and noncrossover gene conversion. The A/J transmission frequency per gamete at each indicated polymorphism is shown. Anything over 50% represents the non-mendelian transmission of A/J sequences, that is, transmission distortion. The consensus PRDM9b binding motif derived by Brick et al.28 is indicated below the graph. The sequence of the 57-bp probes used in d is shown at the bottom. A match with the indicated P value to the consensus binding motif for the PRDM9b allele is shown. The yellow bar and yellow shading represent the predicted PRDM9 binding site. Red arrowheads indicate polymorphisms implicated in differential PRDM9 binding. Noncrossover gene conversion tracts incorporated the PRDM9 binding site polymorphisms in 26 of 131 events (20%), 22 of which were conversions to the A/J genotype (Fig. 2d). Among these events, the C>T transition (~4-fold) and the 2-bp insertion-deletion (≥7-fold) were much more likely to be converted in favor of A/J sequences (Fig. 2d). (c) Mapping the PRDM9 binding site at the A3 hotspot. DBA/2J and A/J mice carry the M. musculus domesticus Prdm9b allele that is identical to that in C57BL/6J mice. The horizontal bars represent the positions of the eight overlapping ~250-bp DNA probes generated for the A/J and DBA/2J genotypes. Representative southwesterns for each probe against His-tagged PRDM9b are shown along with a loading control (probed with antibody to the His6 affinity tag) on the far right. The lower-molecular-weight bands correspond to PRDM9 degradation products. Quantification of the relative binding of the PRDM9b protein (on the basis of 2–5 independent experiments) is shown at the bottom (± s.e.m.). (d) Southwestern analysis localizing PRDM9b but not PRDM9wm7 binding to the center of the A3 hotspot. The 57-bp central probe (sequence shown in b) was used against His-tagged PRDM9b (b) and PRDM9wm7 (w) with quantification of relative binding on the right (± s.e.m.; based on three independent measurements). Note that PRDM9wm7 but not PRDM9b binds to the center of Psmb9 in strains where this hotspot is active (Fig. 1, red arrowhead)32.

  4. Models of recombination and hotspot loss.
    Figure 4: Models of recombination and hotspot loss.

    (a) A model for how intersister recombination can result in the displacement of gene conversion away from DSB sites. (b) Modeling the speed of hotspot loss at A3. Transmission frequencies were used to estimate the number of generations until loss of the indicated PRDM9 binding site polymorphisms using Wright-Fisher modeling. A violin plot is used to show the median number of generations (white dot), the first to third quartiles (black bar) and s.d. (whiskers) and is framed by the probability density at each point.

  5. Noncrossovers at the Psmb9 hotspot in B10.A [times] SGR hybrids determined by analysis of DNA isolated from ovaries and sperm, respectively.
    Supplementary Fig. 1: Noncrossovers at the Psmb9 hotspot in B10.A × SGR hybrids determined by analysis of DNA isolated from ovaries and sperm, respectively.

    Noncrossovers were identified on the B10.A chromosome at the seven queried polymorphisms (asterisks) in pooled ovaries (a) and sperm (b) as described23. See Supplementary Table 1 for detailed results. Once a noncrossover was identified at the queried polymorphism, the extent of gene conversion was assessed by sequencing the PCR product. For pooled ovaries, DNA was extracted from the ovaries of 2 newborn litters (6 and 5 females, respectively), and an estimated ~4,739 genomes from oocytes were tested. For sperm, ~8,981 genomes from a single 4-month-old mouse were tested for the –87 polymorphism and ~15,739 genomes were tested for the other 6 polymorphisms. The polymorphisms known to affect PRDM9 binding are 70 and, to a lesser extent, 87, which are located 17 bp apart (red arrowhead32). The mean minimal and maximal gene conversion tracts are as indicated. In these particular experiments, the overall frequencies of detected noncrossovers (on the B10.A chromosome; representing >90% of noncrossover events in this hybrid) and crossovers (SGR-B10.A orientation only), respectively, were 0.89% and 0.40% in oocytes and 0.45% and 0.16% in sperm.

  6. Noncrossovers from sperm DNA at the A3 hotspot in A/J [times] DBA/2J hybrids.
    Supplementary Fig. 2: Noncrossovers from sperm DNA at the A3 hotspot in A/J × DBA/2J hybrids.

    (a) Noncrossovers were identified using established methods36 on the DBA/2J chromosome in sperm DNA isolated from the same mice used for tetrad analysis. The mean minimal and maximal gene conversion tracts are as indicated. A nearly identical frequency (Table 1) and similar distribution of noncrossovers were obtained. Co-conversions were observed at a lower frequency in spermatocytes (6 of 111 noncrossovers) than in sperm (13 of 78 noncrossovers; P = 0.0142), although whether this difference reflects a biological distinction between the cell types is unclear. (b) For each polymorphism, the frequency and distribution of noncrossovers on the DBA/2J chromosome was compared between tetrad analysis (top; the same data as shown in Fig. 2e) to sperm (bottom). Polymorphisms analyzed with oligonucleotide probes new to this study are indicated by dots.

  7. Single-chromatid analysis cannot distinguish recombination mechanisms because gene conversion tracts are not identified.
    Supplementary Fig. 3: Single-chromatid analysis cannot distinguish recombination mechanisms because gene conversion tracts are not identified.

    (a) Examples of two possible crossovers initiated by a DSB on the frequently cleaved chromosome. In the example on the left, the crossover breakpoints flank the site of the DSB such that the gene conversion tract encompasses the center of the hotspot. In the example on the right, both crossover breakpoints occur to one side of the DSB, such that the conversion tract does not overlap the center of the hotspot. (b) Example of a crossover initiated by a DSB on the infrequently cleaved chromosome in which the crossover breakpoints flank the site of the DSB. Note that the breakpoint on the lower chromosome (breakpoint 3b) is similar to that from the off-center conversion in a (breakpoint 2b). (c) Distribution of crossover breakpoints at the A3 hotspot from A/J × DBA/2J mice previously determined from sperm analysis18. The A/J to DBA/2J (top) and DBA/2J to A/J (bottom) breakpoints are shifted relative to each other (asymmetric), consistent with substantially more frequent initiation on the DBA/2J chromosome. Vertical lines represent the midpoint for each orientation. The intervals in which the crossover breakpoints from a and b occur are indicated. (d) Southwestern analysis of the PRDM9wm7 protein for the A3 hotspot. The horizontal bars represent the positions of the eight overlapping ~250-bp DNA probes generated for the A/J and DBA/2J genotype. Representative southwesterns for each probe against His-tagged PRDM9wm7 are shown along with a loading control (probed with antibody to His) on the far right.

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Author information

  1. These authors contributed equally to this work.

    • Francesca Cole &
    • Frédéric Baudat

Affiliations

  1. Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Francesca Cole &
    • Maria Jasin

  2. Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, Texas, USA.

    • Francesca Cole

  3. Institut de Génétique Humaine, Centre National de la Recherche Scientifique, Unité Propre de Recherche 1142, Montpellier, France.

    • Frédéric Baudat,
    • Corinne Grey &
    • Bernard de Massy

  4. Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Scott Keeney

  5. Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

    • Scott Keeney

Contributions

F.C., F.B., S.K., B.d.M. and M.J. conceived the study, interpreted the data and wrote the manuscript. F.C. and F.B. performed the spermatocyte and oocyte (and associated) recombination experiments, respectively. F.B. and C.G. performed the southwestern blotting. F.C., M.J. and S.K. estimated the fixation rates.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Noncrossovers at the Psmb9 hotspot in B10.A × SGR hybrids determined by analysis of DNA isolated from ovaries and sperm, respectively. (104 KB)

    Noncrossovers were identified on the B10.A chromosome at the seven queried polymorphisms (asterisks) in pooled ovaries (a) and sperm (b) as described23. See Supplementary Table 1 for detailed results. Once a noncrossover was identified at the queried polymorphism, the extent of gene conversion was assessed by sequencing the PCR product. For pooled ovaries, DNA was extracted from the ovaries of 2 newborn litters (6 and 5 females, respectively), and an estimated ~4,739 genomes from oocytes were tested. For sperm, ~8,981 genomes from a single 4-month-old mouse were tested for the –87 polymorphism and ~15,739 genomes were tested for the other 6 polymorphisms. The polymorphisms known to affect PRDM9 binding are 70 and, to a lesser extent, 87, which are located 17 bp apart (red arrowhead32). The mean minimal and maximal gene conversion tracts are as indicated. In these particular experiments, the overall frequencies of detected noncrossovers (on the B10.A chromosome; representing >90% of noncrossover events in this hybrid) and crossovers (SGR-B10.A orientation only), respectively, were 0.89% and 0.40% in oocytes and 0.45% and 0.16% in sperm.

  2. Supplementary Figure 2: Noncrossovers from sperm DNA at the A3 hotspot in A/J × DBA/2J hybrids. (215 KB)

    (a) Noncrossovers were identified using established methods36 on the DBA/2J chromosome in sperm DNA isolated from the same mice used for tetrad analysis. The mean minimal and maximal gene conversion tracts are as indicated. A nearly identical frequency (Table 1) and similar distribution of noncrossovers were obtained. Co-conversions were observed at a lower frequency in spermatocytes (6 of 111 noncrossovers) than in sperm (13 of 78 noncrossovers; P = 0.0142), although whether this difference reflects a biological distinction between the cell types is unclear. (b) For each polymorphism, the frequency and distribution of noncrossovers on the DBA/2J chromosome was compared between tetrad analysis (top; the same data as shown in Fig. 2e) to sperm (bottom). Polymorphisms analyzed with oligonucleotide probes new to this study are indicated by dots.

  3. Supplementary Figure 3: Single-chromatid analysis cannot distinguish recombination mechanisms because gene conversion tracts are not identified. (333 KB)

    (a) Examples of two possible crossovers initiated by a DSB on the frequently cleaved chromosome. In the example on the left, the crossover breakpoints flank the site of the DSB such that the gene conversion tract encompasses the center of the hotspot. In the example on the right, both crossover breakpoints occur to one side of the DSB, such that the conversion tract does not overlap the center of the hotspot. (b) Example of a crossover initiated by a DSB on the infrequently cleaved chromosome in which the crossover breakpoints flank the site of the DSB. Note that the breakpoint on the lower chromosome (breakpoint 3b) is similar to that from the off-center conversion in a (breakpoint 2b). (c) Distribution of crossover breakpoints at the A3 hotspot from A/J × DBA/2J mice previously determined from sperm analysis18. The A/J to DBA/2J (top) and DBA/2J to A/J (bottom) breakpoints are shifted relative to each other (asymmetric), consistent with substantially more frequent initiation on the DBA/2J chromosome. Vertical lines represent the midpoint for each orientation. The intervals in which the crossover breakpoints from a and b occur are indicated. (d) Southwestern analysis of the PRDM9wm7 protein for the A3 hotspot. The horizontal bars represent the positions of the eight overlapping ~250-bp DNA probes generated for the A/J and DBA/2J genotype. Representative southwesterns for each probe against His-tagged PRDM9wm7 are shown along with a loading control (probed with antibody to His) on the far right.

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  1. Supplementary Text and Figures (1,845 KB)

    Supplementary Figures 1–3 and Supplementary Tables 1–3.

Additional data