| During meiotic prophase, chromosomes assume looped structures that are anchored at axial sites where the DNA double-strand break (DSB) machinery assembles. A current model of DSB formation posits that chromatin within the loops is recruited to axial sites to incur DSBs. Sites of DSB formation are largely devoid of nucleosomes, yet the chromatin features at axial sites remain an enigma. We identified phosphorylation of histone H3 threonine 11 (H3 T11ph) as a meiosis-specific, DSB-responsive histone modification in Saccharomyces cerevisiae and Schizosaccharomyces pombe. H3 T11ph was directly catalyzed by the meiosis-specific checkpoint-kinase-family kinase, Mek1, as shown by both in vivo inhibition in a strain expressing an inhibitor-sensitive allele of Mek1, as well as two in vitro kinase assays using Mek1 constructs purified from meiotic S. cerevisiae. To address the function of H3 T11ph, we used two different methods to construct strains expressing a series of H3 T11 mutants. However, unphosphorylatable and phosphomimetic substitutions of H3 T11 failed to define a function for this phosphorylation as no overt phenotype was observed in these strains. The role of this modification became more apparent through the determination of its genome-wide enrichment using ChIP-seq. Though its placement is dependent on meiotic DSB formation, H3 T11ph was found to be generally anticorrelated with DSB hotspots. However, we did detect enrichment in nucleosomes immediately flanking DSB hotspots. Additionally, we found that H3 T11ph colocalized both spatially and quantitatively with axial-associated components of chromosomes. This included a strong correlation with the axial components Hop1 and Red1, as well as the DSB protein, Mer2---suggesting that H3 T11ph is present in axis-associated chromatin.;The presence of this single Mek1-dependent histone phosphorylation event in both axial chromatin and DSB sites provides spatial evidence for the interaction between loop DNA and axial sites during DSB formation. Moreover, due to the association of H3 T11ph with chromosomal axes as well as it being an indicator of DSB-dependent Mek1 activity, we conclude that H3 T11ph is an excellent readout for the timing of meiosis-specific DSB formation, Mek1 kinase activity, and the location of axial element structures. |
