A High-Resolution View Of Meiotic Recombination Initiation In Saccharomyces Cerevisiae

Abstract

Meiotic recombination initiates with DNA double-strand breaks (DSBs) made by Spo11. In Saccharomyces cerevisiae, many DSBs occur in “hotspots” coinciding with nucleosome-depleted gene promoters. Transcription factors (TFs) stimulate DSB formation in some hotspots, but TF roles are complex and variable between locations. Until now, available data for TF effects on global DSB patterns were of low spatial resolution and confined to a single TF. Here, I examine at high resolution the contributions of two TFs to genome-wide DSB distributions: Bas1, which was known to regulate DSB activity at some loci, and Ino4, for which some binding sites were known to be within strong DSB hotspots. I examined fine-scale DSB distributions in TF mutant strains by deep sequencing oligonucleotides that remain covalently bound to Spo11 as a byproduct of DSB formation, mapped Bas1 and Ino4 binding sites in meiotic cells, and evaluated chromatin structure around DSB hotspots. Our findings definitively support the hypothesis that TF control of DSB numbers is context-dependent and frequently indirect. TFs often affected the fine-scale distributions of DSBs within hotspots, and when seen, these effects paralleled effects on local chromatin structure. In contrast, changes in DSB frequencies in hotspots showed no obvious correlation with quantitative measures of chromatin accessibility or of histone H3 lysine 4 trimethylation levels. I also ruled out hotspot competition as a major source of indirect TF effects on DSB distributions. Thus, counter to prevailing models, roles of these TFs on DSB hotspot strength cannot be simply explained via chromatin “openness”, histone modification, or compensatory interactions between adjacent hotspots. In addition to TFs, meiotic DSB formation is regulated by factors involved in chromatin structure and modifications. The effect of some of these factors on DSB landscape has been examined by hybridizing DSB-associated DNAs on microarrays. However, the DSB maps generated by microarrays usually have relatively low resolution and high background. To overcome these limitations, I generated high-resolution meiotic DSB maps from mutants of PCH2, SIR2 and SET1. Analysis of these maps further supports the view that the global DSB landscape is shaped by a hierarchical combination of factors.