Scale matters: the spatial correlation of yeast meiotic DNA breaks with histone H3 trimethylation is driven largely by independent colocalization at promoters

Abstract

During meiosis in many organisms, homologous chromosomes engage in numerous recombination events initiated by DNA double-strand breaks (DSBs) formed by the Spo11 protein. DSBs are distributed nonrandomly, which governs how recombination influences inheritance and genome evolution. The chromosomal features that shape DSB distribution are not well understood. In the budding yeast Saccharomyces cerevisiae, trimethylation of lysine 4 of histone H3 (H3K4me3) has been suggested to play a causal role in targeting Spo11 activity to small regions of preferred DSB formation called hotspots. The link between H3K4me3 and DSBs is supported in part by a genome-wide spatial correlation between the two. However, this correlation has only been evaluated using relatively low-resolution maps of DSBs, H3K4me3 or both. These maps illuminate chromosomal features that influence DSB distributions on a large scale (several kb and greater) but do not adequately resolve features, such as chromatin structure, that act on finer scales (kb and shorter). Using recent nucleotide-resolution maps of DSBs and meiotic chromatin structure, we find that the previously described spatial correlation between H3K4me3 and DSB hotspots is principally attributable to coincident localization of both to gene promoters. Once proximity to the nucleosome-depleted regions in promoters is accounted for, H3K4me3 status has only modest predictive power for determining DSB frequency or location. This analysis provides a cautionary tale about the importance of scale in genome-wide analyses of DSB and recombination patterns.

Figure 1. Resolution of maps around CYS3 DSB hotspot. Top panels show a 60 kb window on chromosome I; bottom panels show a zoomed-in view of the region around CYS3. (A) DSB maps from Spo11 oligo and ssDNA studies. Green bars are hotspot calls from Spo11 oligo mapping. Hotspots from ssDNA mapping are also indicated (red and teal circles, peaks only^,; orange circle and line, peak and region with signal over threshold⁷). (B) Single-nucleosome resolution tag counts and ChIP-chip enrichment ratios for H3K4me3. Gaps in the ChIP-chip plots reflect probes with missing data.

Figure 2. H3K4me3 around DSB hotspots. (A) Mean normalized H3K4me3 enrichment (H3K4me3/H3) around all DSB hotspots, or around hotspots that did or did not overlap with promoters. Data are averaged in 3 bp bins and smoothed using a kernel regression smoother (“ksmooth” function in R). (B) H3K4me3 enrichment at promoter hotspots divided into quintiles by H3K4me3 enrichment. Data are plotted in 3 bp bins and smoothed. Box plots indicate distribution of Spo11 oligo counts (filled boxes) and hotspot widths (open boxes) within each quintile. The thick vertical line in each box is the median, the left and right edges of the boxes are the lower and upper quartiles, and the whiskers indicate the lowest and highest values within 1.5x of the inter-quartile range. Outliers are not shown. (C) H3K4me3 enrichment for promoter hotspots divided into quintiles by Spo11 oligo count. Data are plotted in 3 bp bins and smoothed. Box plots show distribution of Spo11 oligo counts within each quintile. (D) Scatter plot of total Spo11 oligo count vs. H3K4me3 enrichment for promoter hotspots.

Figure 3. Histone marks around transcription start sites of genes. Mean normalized H3K4me3 enrichment at non-regulated genes with upstream genes in divergent orientation (A) or tandem orientation (B) (n = 1668 and 1491, respectively). Data are plotted in 3 bp bins and smoothed. Genes were divided into quintiles by Spo11 oligo count in their promoter. TSS is at 0 bp. Box plots show distribution of Spo11 oligo counts for each quintile.

Figure 4. H3K4me3 enrichment for promoter hotspots grouped by steady-state RNA levels. Non-regulated genes were divided into quartiles by average transcript level, then further divided into thirds based on Spo11 oligo counts within the promoters (see text). Box plots show the distribution of H3K4me3 enrichment for the +1 nucleosomes of the genes in each group. For all quartiles, median H3K4me3 levels for the hottest third were significantly higher than for the coldest third (Wilcoxon rank sum test, p < 6.4 x 10⁻⁵.)