Histone H3 Threonine 11 Phosphorylation Is Catalyzed Directly by the Meiosis-Specific Kinase Mek1 and Provides a Molecular Readout of Mek1 Activity in Vivo

Abstract

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 and Molecular genetic experiments in confirmed that Mek1 is required for H3 T11ph and revealed that phosphorylation is rapidly reversed when Mek1 kinase is no longer active.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 with recombinant proteins demonstrated that Mek1 directly catalyzes H3 T11 phosphorylation. Mutating H3 T11 to nonphosphorylatable residues conferred no detectable defects in otherwise unperturbed meiosis, although the mutations modestly reduced spore viability in certain strains where Rad51 is used for strand exchange in place of Dmc1. H3 T11ph is therefore mostly dispensable for Mek1 function. However, H3 T11ph provides an excellent marker of ongoing Mek1 kinase activity 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.

Homeostatic control of meiotic G2/prophase checkpoint function by Pch2 and Hop1

“…Interestingly, we observed that the pch2Δ- dependent delay was alleviated by reducing the levels of Hop1, underscoring our earlier observation that Hop1 levels influence Pch2-associated phenotypes (Figure 2B and Supplementary Figure 3A). The effects on cell cycle progression were associated with effects on checkpoint activity which was analyzed by monitoring phospho-status of Hop1 via well-documented electrophoretic mobility shift (Hop1 is phosphorylated by Mec1/Tel1 kinases in response to checkpoint-activating conditions [7, 38]), and activation of Mek1, a G2/prophase checkpoint kinase, by querying the phospho-status of Histone H3-threonine (pH3-T11) (a confirmed Mek1 substrate) [39] (Figure 2A). We also observed similar effects between Pch2 and Hop1 when analyzing spore viability (Supplementary Figure 3B).…”

Functional Impact of the H2A.Z Histone Variant During Meiosis in Saccharomyces cerevisiae

“…The htz1 delay in meiotic progression was maintained in the htz1 spo11 and htz1 mek1 double mutants ( Figure 3, A and B, respectively). Moreover, the dynamics of various indicators of checkpoint activity, such as Hop1-T318 phosphorylation (Herruzo et al 2016) and Mek1 activation, as assessed both by Mek1 autophosphorylation (Ontoso et al 2013) and phosphorylation of its H3-T11 target (Cavero et al 2016;Kniewel et al 2017), was similar in wild type and htz1 ( Figure 3C). These results indicate that the lower overall efficiency of meiotic divisions in htz1 does not stem from activation of the MRC, and it is consistent with the observation that CO meiotic recombination does not appear to be significantly affected in the absence of H2A.Z.…”

Regulation of Hed1 and Rad54 binding during maturation of the meiosis‐specific presynaptic complex

“…Rad51 inhibition is achieved through two meiosis-specific regulatory proteins, Hed1 and Mek1 (Niu et al, 2009;Brown & Bishop, 2014). Mek1 is a kinase that phosphorylates a number of proteins during meiosis, including Rad54 (Niu et al, 2009) and histone H3 (Govin et al, 2010;Kniewel et al, 2017), to help suppress sister-directed repair (Liu et al, 2014). Hed1 is a regulatory factor that interferes with Rad54 binding to Rad51 (Busygina et al, 2008(Busygina et al, , 2012.…”