Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm

Abstract

Recombination is critical to meiosis and evolution, yet many aspects of the physical exchange of DNA via crossovers remain poorly understood. We report an approach for single-cell whole-genome DNA sequencing by which we sequenced 217 individual hybrid mouse sperm, providing a kilobase-resolution genome-wide map of crossovers. Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-initiating template homolog and telomere proximity. These factors also influence the time for sites of recombination-initiating DNA double-strand breaks to find and engage their homologs, with rapidly engaging sites more likely to form crossovers. We show that chromatin environment on the template homolog affects positioning of crossover breakpoints. Our results also offer insights into recombination in the pseudoautosomal region.

Figure 1. Experimental design for inferring crossovers from single sperm cells.

A. An illustration of the method for whole-genome amplification (WGA) of isolated single sperm cells (25). Random RNA oligonucleotides act as primers for WGA mediated by Klenow fragment, which displaces adjacent synthesized fragments to form overlapping single-stranded DNA copies. These, in turn, serve as templates for primer annealing and chain extension. The resulting amplicons are converted into double-stranded DNA for sequencing. B: Sequencing depth and genome coverage achieved for each of 217 sperm. C: An Integrative Genomics Viewer (IGV) illustration of how a crossover was called by our method. The horizontal light gray lines show the reads that mapped in a region of chromosome 13 for a particular sperm. Vertical dark gray bars highlight variants found only in B6, while orange bars highlight variants found only in CAST. The crossover breakpoint lies within a region of uncertainty (green). This crossover overlapped a PRDM9^HUM hotspot, identified by DMC1 ChIP-seq (25), whose center was inferred to be at 113,864,493. A good match to the PRDM9^HUM binding motif occurs in the purple region.

Figure 2. Properties of crossovers and recombination hotspots.

A: Average number of crossovers called per chromosome per sperm (bars show 1 standard error), showing at least, and in many cases almost exactly, one crossover per chromosome per meiosis (equivalently 0.5 crossovers per haploid sperm). B: Distribution of H3K4me3 intensity in all autosomal recombination hotspots identified by DMC1 ChIP-seq (blue), after removing hotspots showing evidence of PRDM9-independent H3K4me3 (e.g. transcription-start sites, (25)). If crossovers occurred in proportion to the hotspot heat, the distribution of H3K4me3 in hotspots with crossovers should be the corresponding size-biased distribution (green). The observed distribution of H3K4me3 in hotspots with crossovers (red) is skewed further towards hotspots with greater H3K4me3 (p = 10⁻⁹⁰). C: The most active autosomal hotspot for crossover is on the centromere-distal end of chromosome 19. DMC1 binds the 3’ ssDNA overhangs on either side of the DSB, which leads to a shift between DMC1 coverage on the forward (blue) and reverse (red) strands (200 bp smoothing). Regions containing the crossover breakpoint in each sperm are in black. Crossovers at the same locus in distinct sperm can have different resolution, depending on the actual sequencing coverage achieved in each case. D: PRDM9 binding at a hotspot is a stochastic event in a cell. In a population of cells, some proportion of cells will have one, both, or neither homologue bound by PRDM9 (sky blue). Here we show the proportion of times each of these possibilities occurs at two illustrative symmetric hotspots. In the very active hotspot (top row), PRDM9 binds the B6 (red) and CAST (blue) homologues with probability 80% each. As a result, PRDM9 is bound to both homologues in the majority of cells (64%). In the less active hotspot, the probability of PRDM9 binding each homologue is 40%. The proportion of cells in which PRDM9 is bound to both homologues is lower (16%). E. As in D, a comparison of the proportion of cells in which PRDM9 (sky blue) binds one or both homologues, B6 (red) and CAST (dark blue) but at an illustrative asymmetric hotspot. The probability of PRDM9 binding the B6 homologue is ˜80% but only ˜4% for the CAST homologue. This is due to a SNP (yellow) in the PRDM9 motif on the CAST homologue, which partially disrupts binding. Only a small minority of cells have PRDM9 bound to both homologues.

Figure 3. PRDM9 binding on the non-initiating, template homologue affects crossover resolution.

A: Hotspots were binned into five groups depending on the level of asymmetry in PRDM9 binding of the homologues (25). The crossover resolution probability, which accounts for differences in H3K4me3, in each bin (normalized relative to the bin with the most symmetric hotspots, y-axis, 1 standard error bars), is plotted against the mean asymmetry of hotspots in that bin (x-axis). Predicted effects on crossover resolution if PRDM9 binding on the template homologue was irrelevant (black) and if it was essential (red) are shown for comparison. B: Hotspots were grouped into four bins depending on the number of SNP differences between B6 and CAST chromosomes in the central 200 bases of the hotspot. The crossover resolution probability in each bin (blue) was inferred relative to the bin containing hotspots with zero SNPs. Red points show the same quantity after correcting for asymmetry in PRDM9 binding. Bars show 1 standard error. C: Crossover resolution probability is significantly higher for DSBs initiated on the “less-bound” homologue than the “more-bound” homologue in asymmetric hotspots. Crossover resolution probabilities for initiation on the more-bound (red, n=47) and less-bound homologues (blue, n=13), after accounting for differences in H3K4me3 on them. Probabilities were normalized against the average for symmetric hotspots (dashed black line), bars show 95% confidence intervals (25). D: Fraction of crossovers (green), H3K4me3 (blue) and DMC1 (red) originating on the less-bound chromosome in asymmetric hotspots, with dashed line marking the proportion expected from H3K4me3. While the fraction of crossovers initiating on the less-bound chromosome is significantly greater than expected from H3K4me3 (p=5x10^-6), the fraction of DMC1 is significantly lower than expected from H3K4me3 (p<10^-16). Bars show 1 standard error. E: Illustration that the probability of PRDM9 having bound the template depends on which homologue is initially cut for the same asymmetric hotspot as in Fig. 2E. A DSB is more likely to occur on the more-bound homologue B6 (red). When it does, fewer than 4% of cells (3/80) will have the template CAST homologue (blue) bound. In the less likely event that the CAST homologue is cut, the B6 homologue will have been bound in 75% of cells (3/4). (Note that cells with PRDM9 bound on both homologues are twice as likely to be cut at this hotspot than cells with only one homologue bound.) F: Crossover resolution is influenced by PRDM9 binding on the template homologue. All potential sites for recombination initiation, i.e., the B6 and CAST homologous sites in each hotspot, were sorted according to the H3K4me3 on their respective template homologues. The initiating sites were then binned into 7 bins, such that the total H3K4me3 intensity on the initiating sites in each bin is the same. The proportion of crossovers that initiated in each bin (out of 685 crossovers where the initiating homologue could be inferred) is shown against the average H3K4me3 on the corresponding template homologues (x-axis). Dotted red line shows the expected relationship if H3K4me3 on the template were unrelated to crossover outcome.

Figure 4. Crossover resolution is affected by local GC-content and Telomere proximity.

A: Each autosomal hotspot with a crossover was paired with another hotspot lacking a crossover for the same PRDM9 variant, on the same chromosome and with very similar H3K4me3 on both homologues (25). The distribution of local GC-content (500 bp around the hotspot center) is compared between the two matched sets (n=1355, p=1.2×10⁻¹⁴, paired t-test). B: Hotspots were divided into 7 bins depending on their distance from the distal telomere of their respective chromosome. Crossover resolution probability (relative to the leftmost bin) is shown for each bin (1 standard error bars). Chromosomes with more than one crossover in an individual sperm were removed to avoid confounding with crossover interference (see Figs. S20-S21 for additional views).

Figure 5. Factors affecting homologue-engagement time in the repair of DSBs.

A: Hotspots in the B6 mouse were ordered by their H3K4me3 intensity and divided into 10 bins. Average homologue-engagement time, the ratio of total DMC1 with total SPO11 per bin (y-axis), is shown relative to the average H3K4me3 per hotspot in each bin (x-axis). B: Hotspots in the B6 mouse were divided into 8 bins depending on their distance from the distal telomere of their respective chromosome. Average homologue-engagement time (ratio of total DMC1 with total SPO11 in each bin) is shown (1 standard error bars). C: Hotspots in the B6 mouse were divided into 6 bins depending on their local GC-content (± 500 bp around the hotspot center). Average homologue-engagement time per bin is shown (bars show 95% confidence intervals). D: Comparison of estimated homologue-engagement time for the more-bound homologue (red) and less-bound homologue (blue) in asymmetric hotspots (corresponding to Fig. 3C, 95% confidence intervals, (25)). Estimated homologue-engagement time (ratio of DMC1 with H3K4me3) is normalized against the average for symmetric hotspots (dashed black line).

Figure 6. Positioning of crossover breakpoints is influenced by nucleosome positioning on the template chromosome.

A: Distribution of crossover breakpoints from the motif center (green) for crossovers that overlap symmetric PRDM9^CAST hotspots with a well-identified motif site, and have breakpoint resolution ≤250bp (n=132). To deal with the uncertainty in crossover breakpoint location in each sperm we assign equal weight to all possible breakpoint positions in that sperm (25). H3K4me3 ChIP-seq with MNase averaged over PRDM9^CAST hotspots in red (20bp smoothing). Red bars at top show average inferred positions of nucleosomes, black bar shows the PRDM9^CAST binding site. B: As (A) but for crossovers that overlap asymmetric PRDM9^CAST hotspots (n=33). Average MNase-seq for the less-bound chromosome of asymmetric hotspots (blue, 50bp smoothing), with blue bars at top showing average inferred nucleosome positions. This is an estimate of the nucleosome positioning at hotspot sites when PRDM9 is not bound (25). The peak in MNase-seq at the hotspot center is consistent with the presence of a nucleosome in PRDM9^CAST hotspots in the absence of PRDM9 binding (Fig. S37). C: Illustration of nucleosome positions when the template homologue is bound by PRDM9^CAST. DNA (dark brown) around histones (light brown), with red dots indicating H3K4me3 mark. Nucleosome positions on the DSB-initiating and template homologues are the same. This is more likely in symmetric hotspots (A). D: Illustration of nucleosome positions when the template homologue is not bound by PRDM9^CAST. Colours as in (C). Typical nucleosome positioning at sites bound by PRDM9 is shifted relative to unbound sites, resulting in a difference between the DSB-initiating and template chromosomes. This is more likely in asymmetric hotspots (B). The shift in crossover breakpoints between (A) and (B) is consistent with the shift in nucleosome positions on the template homologue, as illustrated in (C) and (D).

Figure 7. Differences in recombination in the pseudoautosomal region.

A: Histogram of the fraction of DMC1 reads on the CAST chromosome across hotspots (red, n=38). For the same regions, the corresponding histogram for reads from sequencing of bulk sperm is shown (blue) as a control to assess potential mapping artefacts. While DMC1 is significantly biased towards the CAST haplotype (p<10^-4, (25)), there is no significant bias in bulk sequencing (median=0.51, p=0.85, (25)). B: The most active hotspot for crossovers in the entire genome is in the het-PAR and is PRDM9-independent. DMC1 coverage (200bp smoothing) is shown for the forward (blue) and reverse (red) strands. Crossover breakpoints are in black. See Fig. S43 for a further het-PAR hotspot.