 50% of all detectable Rec12-DNA linkages (DSBs). Thus, this genomic feature is highly predictive. We presume that one or more nucleotide sequences, currently unknown but restricted to the IGRs with DSBs, are essential for DSB formation. These sequences might be collections of transcription factor binding sites, exemplified by M26, the binding site for Atf1-Pcr1. The S. cerevisiae transcription factor Bas1 and its binding site also account for a small fraction of the meiotic DSB hotspots observed in that distantly related yeast . We think it likely that transcription factors other than Atf1-Pcr1 also influence DSB formation in S. pombe. Transcription factor binding sites are concentrated in IGRs and collectively may account for a significant fraction of meiotic DSBs. Other sequences, such as those affecting nucleosome positioning, may determine additional DSB hotspots. The data reported here provide a framework for identifying such determinants.
Ludin and colleagues also noted an enrichment of Rec12 in IGRs in their genome-wide analysis of Rec12 crosslinked with formaldehyde to meiotic DNA (K. Ludin, J. Mata, J. Bahler, and J. Kohli, personal communication). In their analysis Rec12 was more uniformly distributed across the genome than in our analysis, perhaps a reflection of only a fraction of chromatin-bound Rec12 being active in making DSBs and becoming crosslinked without an exogenous agent.
We observed that DSB hotspots are over-represented in IGRs whose flanking genes are divergently transcribed, compared to their frequency in all IGRs (p < 0.001; contingency chi-squared test). However, the orientation of transcription of adjacent genes is itself correlated with the size of the IGR , and no enrichment for DSB hotspots was seen in divergent IGRs when the size of the IGR was controlled for (unpublished data). We know of no clear evidence that transcription per se influences meiotic DSB formation (e.g., ).
Relation of Meiotic DSBs and Other Genomic Features
We searched for other features of the S. pombe genome that might correlate with DSB hotspots and thus be important factors for their determination. The meiosis-specific cohesin subunit Rec8 is required for most DSB formation in S. pombe . Rec8 is thought to be part of the chromosomal core that forms the basis of the axial elements (linear elements in S. pombe) and from which chromatin loops emanate . Studies in S. cerevisiae have led to the conclusion that DSB hotspots preferentially occur in the loops, where Rec8 density is low . S. pombe Rec8 is distributed nonuniformly, at least over the portion of the genome surveyed (Chromosome II and part of Chromosome III) . We observed a weak negative correlation between Rec8 binding and Rec12-DNA linkage. For example, 83% of the 69 prominent Rec12 peaks on Chromosome II are centered on an 11-probe window whose Rec8-binding ratio is below the median for Chromosome II (ignoring the centromere), significantly different from the 50% expected (p < 0.001; contingency chi-squared test). Among the 55 weak Rec12 peaks, this bias falls to 64%, not significantly different from 50%. Conversely, the quarter of probes on Chromosome II with the highest Rec8 enrichment ratios contain only 9% of the prominent Rec12 peaks, less than the 25% expected, and the quarter with the lowest Rec8 enrichment ratios contain 55% of the prominent Rec12 peaks, more than the 25% expected. Therefore, in S. pombe as in S. cerevisiae, DSBs are associated with regions of low Rec8 localization, but neither distribution is very useful for predicting the other.
As noted above, Atf1-Pcr1 and perhaps other transcription factors are required for some DSB hotspots. We therefore looked for a correlation between DSB hotspot positions and the promoters of genes induced during meiosis. Among such genes , we found no significant enrichment of Rec12 peaks 5′ of the corresponding open reading frame. We then looked among all genes with Rec12-peaks in their presumptive promoters for enrichment of related functions, as indicated by related gene ontology (GO) terms. Using the GO