Dynamic location changes of Bub1-phosphorylated-H2AThr133 with CENH3 nucleosome in maize centromeric regions

The genomic stability of all organisms requires precise cell division with proper chromosome orientation. The Bub1-H2Aph-Sgo1 pathway and spindle assembly checkpoint (SAC) components have been identified in yeast and mammals that are important for sister centromere orientation and chromosome segregation. However, their roles in plants are not clear. Maize meiotic mutants and minichromosomes were used to study the role of H2AThr133 phosphorylation and SAC components in sister centromere orientation and chromosome segregation. Unlike previously reported, SAC protein Bub1-Sgo1 recruitment was independent of Rec8 in maize and did not play a role in centromere protection in meiosis I. Chromatin immunoprecipitation sequencing analysis with immnolocalization results indicate most CENH3 nucleosomes contain phosphorylated H2AThr133 in centromeric regions. H2AThr133ph spreads to encompass centromeric regions including the inner centromeric and pericentromeric regions during (pro) metaphase. The presence and localization of SAC components and H2AThr133ph on maize lines containing sister chromatids separate precociously in anaphase I revealed no direct role of these proteins on centromere orientation in meiosis I. This work sheds light on the relationship between H2AThr133ph and CENH3 nucleosome in plants, and the phosphorylation with dynamic location changes in centomeric regions suggests temporal and spatial regulation roles for H2A phosphorylation in chromosome segregation.