Background: Meiotic recombination hotspots control the frequency and distribution of Spo11 (Rec12)-initiated recombination in the genome. Recombination occurs within and is regulated in part by chromatin structure, but relatively few of the many chromatin remodeling factors and histone posttranslational modifications (PTMs) have been interrogated for a role in the process.
Results: We developed a chromatin affinity purification and mass spectrometry-based approach to identify proteins and histone PTMs that regulate recombination hotspots. Small (4.2 kbp) minichromosomes (MiniCs) bearing the fission yeast ade6-M26 hotspot or a basal recombination control were purified approximately 100,000-fold under native conditions from meiosis; then, associated proteins and histone PTMs were identified by mass spectrometry. Proteins and PTMs enriched at the hotspot included known regulators (Atf1, Pcr1, Mst2, Snf22, H3K14ac), validating the approach. The abundance of individual histones varied dynamically during meiotic progression in hotspot versus basal control MiniCs, as did a subset of 34 different histone PTMs, implicating these as potential regulators. Measurements of basal and hotspot recombination in null mutants confirmed that additional, hotspot-enriched proteins are bona fide regulators of hotspot activation within the genome. These chromatin-mediated regulators include histone H2A-H2B and H3-H4 chaperones (Nap1, Hip1/Hir1), subunits of the Ino80 complex (Arp5, Arp8), a DNA helicase/E3 ubiquitin ligase (Rrp2), components of a Swi2/Snf2 family remodeling complex (Swr1, Swc2), and a nucleosome evictor (Fft3/Fun30).
Conclusions: Overall, our findings indicate that a remarkably diverse collection of chromatin remodeling factors and histone PTMs participate in designating where meiotic recombination occurs in the genome, and they provide new insight into molecular mechanisms of the process.
Keywords: Chromatin remodeling; Histones; Homologous recombination; Mass spectrometry; Meiosis; Proteomics; Schizosaccharomyces pombe.