Abstract
Meiotic recombination is initiated by Spo11-catalyzed DNA double-strand breaks (DSBs) that are promoted by histone modifications and histone modifying enzymes. Herein we investigated the role of histone H3 lysine 56 acetylation (H3K56ac) located near the entry/exit points of the DNA in the globular H3 domain. We generated a series of mutant cells (asf1Δ, rtt109Δ, hst3/4Δ, and H3K56A) in which the endogenous level of H3K56ac was manipulated and tracked during meiotic growth. We show that complete loss or increased abundance of H3K56ac in these mutants allows timely entry into meiosis and sporulation and does not impair S phase progression, first and second meiotic cell divisions, and spore viability. In the asf1Δ, rtt109Δ, hst3/4Δ mutants, DSBs and crossovers form normal levels with a short (60-min) delay at the HIS4-LEU2 artificial recombination hotspot, however, DSB formation shows a ∼threefold decrease in the H3K56A mutant at the natural BUD23-ARE1 hotspot. The latter DSB phenotype, showing significant DSB reduction in the H3K56A mutant, was also observed at DSB sites using genome-wide mapping of Rfa1-coated single-stranded DNA flanking DSBs (RPA ChIP). Parallel mapping of H3K56-acetylated histones in wild type cells revealed strong depletion of the H3K56ac ChIP signal over Spo11-oligo DSBs, albeit most H3K56-acetylated histones were enriched adjacent to the identified RPA ChIP binding sites. Taken together, these associations demonstrate a prominent role of H3 lysine 56 acetylation in the formation of DNA breaks within recombination hotspot regions.
Highlights
Meiosis is a cellular differentiation process which is accompanied by high levels of recombination between the homologous chromosomes, initiated by DNA double-strand breaks (DSBs) catalyzed by Spo11 and accessory factors (Keeney et al, 1997; Székvölgyi and Nicolas, 2010; Székvölgyi et al, 2015)
This Venn diagram analysis shows that 65.8% of RPA peaks were eliminated by the H3K56A mutation (661 sites out of 1004), whereas 343 RPA sites were not affected or the signal was even increased (182 peaks). The latter RPA binding sites may represent unscheduled DSBs that are not related to H3K56 acetylation, which is clearly apparent from the increased relative distance between H3K56ac histones and RPA ChIP peaks detected in the H3K56A mutant (Figure 4H, RPA-H3K56A)
The above functional results highlight the association of H3K56 acetylation and meiotic DSB formation, suggesting that H3K56acetylated histones are required to produce normal levels of DSBs within recombination hotspot regions
Summary
Meiosis is a cellular differentiation process which is accompanied by high levels of recombination between the homologous chromosomes, initiated by DNA double-strand breaks (DSBs) catalyzed by Spo and accessory factors (Keeney et al, 1997; Székvölgyi and Nicolas, 2010; Székvölgyi et al, 2015). In S. cerevisiae, meiotic DSBs are controlled by the elements of chromatin structure This involves a complex interplay between DNA sequence composition, local chromatin status, nucleosome occupancy, and transcription factor binding (Pan et al, 2011). The widely localized H3K4me mark has the virtue of initiating recombination at numerous places in the genome, other histone modifications are needed to keep recombination flexible for the diversity of recombinant haplotypes (Szekvolgyi and Nicolas, 2010). These “alternative” pathways remain to be clarified to better understand the plasticity of crossover patterning. Our results demonstrate that H3K56ac is necessary for formation of normal levels of DSBs within recombination hotspot regions
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