Histone H3 threonine 11 phosphorylation is catalyzed directly by the meiosis-specific kinase Mek1 and provides a molecular readout of Mek1 activity in vivo

Saccharomyces cerevisiae Mek1 is a CHK2/Rad53-family kinase that regulates meiotic recombination and progression upon its activation in response to DNA double-strand breaks (DSBs). The full catalog of direct Mek1 phosphorylation targets remains unknown. Here, we show that phosphorylation of histone H3 on threonine 11 (H3 T11ph) is induced by meiotic DSBs in S. cerevisiae and Schizosaccharomyces pombe. Molecular genetic experiments in S. cerevisiae confirmed that Mek1 is required for H3 T11ph and revealed that phosphorylation is rapidly reversed when Mek1 kinase is no longer active. Reconstituting histone phosphorylation in vitro with recombinant proteins demonstrated that Mek1 directly catalyzes H3 T11 phosphorylation. Mutating H3 T11 to nonphosphorylatable residues conferred no detectable meiotic defects, indicating that H3 T11ph is dispensable for Mek1 functions in controlling recombination. However, H3 T11ph provides an excellent marker of ongoing Mek1 kinase activity in vivo. Anti-H3 T11ph chromatin immunoprecipitation followed by deep sequencing demonstrated that H3 T11ph was highly enriched at presumed sites of attachment of chromatin to chromosome axes, gave a more modest signal along chromatin loops, and was present at still lower levels immediately adjacent to DSB hotspots. These localization patterns closely tracked the distribution of Red1 and Hop1, axis proteins required for Mek1 activation. These findings provide insight into the spatial disposition of Mek1 kinase activity and the higher order organization of recombining meiotic chromosomes.酿酒酵母（Saccharomyces cerevisiae）中的Mek1是CHK2/Rad53家族激酶，可在响应DNA双链断裂（double-strand breaks, DSBs）并激活后，调控减数分裂重组与进程。目前，Mek1直接磷酸化靶点的完整目录仍未明确。本研究证实，在酿酒酵母与粟酒裂殖酵母（Schizosaccharomyces pombe）中，减数分裂DSBs可诱导组蛋白H3苏氨酸11位点发生磷酸化修饰（H3 T11ph）。在酿酒酵母中开展的分子遗传学实验证实，Mek1是介导H3 T11ph的必需因子，同时研究发现，当Mek1激酶活性消失时，该磷酸化修饰会被快速逆转。通过重组蛋白在体外重建组蛋白磷酸化体系的实验证明，Mek1可直接催化H3 T11位点的磷酸化。将H3 T11突变为无法被磷酸化的氨基酸残基后，未检测到明显的减数分裂缺陷，这表明H3 T11ph对于Mek1调控重组的功能并非必需。然而，H3 T11ph可作为体内持续激活的Mek1激酶活性的优良标记物。采用抗H3 T11ph染色质免疫沉淀联合高通量测序技术分析显示，H3 T11ph在染色质与染色体轴的假定附着位点处高度富集，在染色质环区域的信号相对较弱，而在DSB热点紧邻区域的信号水平更低。该定位模式与Red1和Hop1这两种Mek1激活所必需的轴蛋白的分布高度吻合。上述研究结果为解析Mek1激酶活性的空间分布以及重组减数分裂染色体的高级结构组织提供了新的认识。