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. 2020 Oct 13;9:e57117. doi: 10.7554/eLife.57117

Figure 4. 5hmC is enriched at PRDM9-dependent sites and correlates with PRDM9 occupancy.

(A) Average read enrichment and heatmaps showing 5hmC enrichment in the B6 (blue) and RJ2 (red) strains. Signal was calculated in a +/- 2 kb window around hotspot centers (determined by DMC1-SSDS). 5hmC enrichment was calculated from pooled replicates within 50bp bins and normalized by reads per million (RPM) and input. The sites on the heatmaps are ranked by decreasing DMC1-SSDS signal intensity from top to bottom. (B) Read distribution from DMC1 and 5hmC ChIP-seq experiments in the B6 (blue) and RJ2 (red) strains at representative DMC1 PRDM9Dom2 (B6) and PRDM9Cst (RJ2) specific sites on chromosome 1. Read distribution was calculated from pooled replicates within 50bp bins and normalized by library size and input, except for the DMC1 ChIP experiments. (C) Average read enrichment showing 5hmC enrichment (left y axis) and PRDM9 read enrichment (right y axis) in the B6 (blue) and RJ2 (red) strains centered in a +/- 2 kb window around DMC1 B6 and DMC1 RJ2 sites, respectively. Read distribution was calculated from pooled replicates within 50bp bins and normalized by library size and input. (D) 5hmC signal at hotspots is HELLS-dependent. Average read enrichment showing 5hmC in the B6 (blue) and Hells cKO (orange) strains centered in a +/- 2 kb window around the hotspot centers (DMC1-SSDS B6 sites). 5hmC enrichment was calculated from pooled replicates within 50bp bins and normalized by read per million (RPM) and input. (E) 5hmC signal at hotspots is dependent on PRDM9 methyltransferase activity. Average read enrichment showing 5hmC in the B6 (blue), RJ2 (red) and B6-Tg(YF)(magenta) strains centered in a +/- 2 kb window around the hotspot centers (DMC1-SSDS B6 and RJ2 sites). 5hmC enrichment was calculated from pooled replicates within 50bp bins and normalized by read per million (RPM) and input. (F) 5hmC signal at hotspots is independent of DSB formation. Average read enrichment showing 5hmC enrichment in the B6 (blue) and Spo11 KO (green) strain centered in a +/- 2 kb window around the hotspot centers (DMC1-SSDS B6 sites). 5hmC enrichment was calculated form pooled replicates within 50bp bins and normalized by read per million (RPM) and input. The duplicate analysis for all genotypes is shown in Figure 4—figure supplement 2.

Figure 4.

Figure 4—figure supplement 1. Similar distributions of 5hmC and PRDM9 enrichments.

Figure 4—figure supplement 1.

(A) Average read enrichment and heatmaps showing 5hmC enrichment in B6 (blue) and RJ2 (red) strains centered in a +/- 2 kb window around the PRDM9 B6 and RJ2 sites, respectively. 5hmC enrichment was calculated from pooled replicates within 50 bp bins and normalized by read per million and input. The sites on the heatmaps are ranked by decreasing PRDM9 signal intensity from top to bottom. (B) Average read enrichment showing 5hmC enrichment (left y axis) and H3K4me3 or DMC1 read enrichment (right y axis) in the B6 (blue) and RJ2 (red) strains centered in a +/- 2 kb window around the DMC1 B6 and DMC1 RJ2 sites, respectively. Read distribution was calculated from pooled replicates within 50 bp bins, and normalized by library size and input, except for the DMC1 ChIP experiments. (C) Top panels: Scatter plots of 5hmC enrichment versus PRDM9 and H3K4me3 enrichment in the B6 (blue) and RJ2 (red) strain at the DMC1 B6 and DMC1 RJ2 sites, respectively (only sites featuring the strongest 5hmC signals, containing at least 6 CpGs in a window within +/- 250 bp around hotspots were considered, see Figure 5 and Materials and methods). Read enrichment was calculated in a window within +/- 250 bp around hotspots (DMC1-SSDS) normalized by library size and input. Bottom panels: Scatter plot of 5hmC read enrichment versus SPO11-oligos peak strength (data available only for B6; Lange et al., 2016) and DMC1 peak strength. Rho is the Spearman correlation coefficient.
Figure 4—figure supplement 2. Reproducibility of 5hmC enrichment.

Figure 4—figure supplement 2.

Reproducibility of 5hmC enrichment in B6, RJ2, Hells cKO, B6-Tg(YF), and Spo11 KO samples. Scatter plots of 5hmC read enrichment (RPM) in replicate 1 versus replicate 2. Blue: B6, Red: RJ2, Orange: Hells cKO, Purple: B6-Tg(YF), Green: Spo11 KO. Read enrichment was calculated in a +/- 250 bp window around the center of DMC1 sites normalized by library size and input. 5hmC enrichment was calculated at DMC1 B6 sites for the B6, Hells cKO and Spo11 KO strains, and at DMC1 RJ2 sites for the RJ2 strain.