CR signals were observed at 4, 5, and 6 h after meiotic induction (Figure S1), in parallel with the accumulation of DSBs at these times in rad50S cells as measured by Southern blot hybridizations . This meiosis-specific signal was dependent on the FLAG antibody (unpublished data), the FLAG tag on Rec12, and intracellular Rec10 protein, which is also essential for recombination and DSB formation . Approximately 30 times more signal was observed with primers specific to ade6, which contains a DSB hotspot, than with primers specific to ura1, which lacks detectable DSBs. As expected, approximately equal yields of product using the whole cell extract (WCE) (nonimmunoprecipitated) DNA were obtained at the ade6–3049, mbs1, and ura1 sites. Since no crosslinking agent such as formaldehyde was used, we infer that the observed PCR signals using the IPs reflect covalent linkage or very tight binding of Rec12 specifically to DNA at meiotic hotspots.
Genome-Wide Analysis of Rec12-DNA Linkages
To extend this analysis to the entire genome, we analyzed DNA in IPs of chromatin prepared from cells harvested before and 5 h after meiotic induction, using tiling microarrays of oligonucleotides (60-mers; “probes”) spaced on average every 300 bp across the S. pombe genome . These arrays have been used to analyze the genome-wide distribution of heterochromatin proteins and histone modifications . We have analyzed three meiotic inductions, two with a haploid strain (Dataset S1) and one with a diploid (Dataset S2). These strains are pat1–114 (Ts) mutants, which initiate meiosis synchronously when the temperature is raised, even from the haploid state ; where tested, haploids and diploids give DSB patterns indistinguishable by Southern blot analysis .
Using the tiling microarrays, we compared the Rec12-DNA signals obtained using the IPs to those obtained using WCEs. The ratio of the IP:WCE signals at each probe reflects the enrichment of DNA linked to Rec12. This ratio was normalized using the median values of the IP and WCE signals for all of the ∼41,000 probes . Because the chromatin DNA was sheared to ∼0.5–1 kb before immunoprecipitation, and because Spo11 (and likely Rec12) becomes linked to both 5′ ends at a DSB , we expect genuine Rec12-DNA signals to hybridize to at least three adjacent probes on the array. For our subsequent analysis we thus excluded isolated single or double probes (“spikes”) with high values (IP:WCE ratios >2), as done similarly by Cam et al. . High points in the induced (5 h) data corresponding to spuriously high uninduced (0 h) points were also removed. The effect of this filtering and its comparison with other noise-reducing methods are shown in Figure S2. High Rec12 enrichment ratios in the 0-h data are restricted to isolated spikes, whereas in the 5-h data high enrichment ratios are also seen in contiguous groups of high ratio probes, indicative of genuine Rec12 enrichment (Figure S10). Filtering removed the spikes (Figure S11). As expected , the filtered data from the 0-h samples were approximately normally distributed, whereas the data from the 5-h samples were approximately normally distributed but with an additional, substantial non-normal distribution of high values (i.e., sites of Rec12-DNA linkage) (Figures S3 and S4). Greater variance in the diploid 0-h background distribution meant that filtering of spikes was somewhat less effective in that dataset than in the others.
We first focused our attention on a 0.5-Mb region near the left end of Chromosome I, defined by NotI restriction fragment J, which we have previously analyzed extensively for meiotic recombination and DSBs . Our microarray analysis reveals a consistent distribution of Rec12-DNA linkages that closely parallels the distribution of DSBs determined by Southern blot hybridization (see next section). Strong Rec12-DNA signals (IP:WCE ratios >2 and up to ∼40) were observed at ∼20 consecutive probes at the position of the previously mapped mbs1 and mbs2 DSB hotspots of NotI fragment J in each of the three inductions (5-h data; Figures 1 and S5). Additi