Fig. 3

Affinity-seq binding site activities measured in vitro influence hotspot detection in vivo. a PRDM9 affinity to DNA and chromatin features determine hotspot activation qualitatively and quantitatively. Density distribution of Affinity-seq binding sites in hotspots detected in vivo (blue) or not detected (in vitro only, red) in spermatocytes compared to all sites (green). Active hotspots contain Affinity-seq binding sites with higher average activity compared to the sites detected in vitro only. b Closed chromatin state defined by H3K9me2/3 marks impacts lower affinity hotspots more strongly than higher affinity hotspots. We ranked hotspots by their affinity for PRDM9 determined by Affinity-seq and divided them into five equal quintiles. For each group, we then calculated the ratio of hotspot number to total number of Affinity-seq sites in regions of closed chromatin (H3K9me2 or 3 positive) to regions of open chromatin (lacking H3K9me2 or me3). A ratio of 1 indicates no effect of heterochromatin. These data indicate that the strength of binding can increasingly overcome the effects of heterochromatin