Maize centromeres are composed of CentC tandem repeat arrays, centromeric retrotransposons (CRs), and a variety of other repeats. One particularly well-conserved CR element, CRM, occurs primarily as complete and uninterrupted elements and is interspersed thoroughly with CentC at the light microscopic level. To determine if these major centromeric DNAs are part of the functional centromere/kinetochore complex, we generated antiserum to maize centromeric histone H3 (CENH3). CENH3, a highly conserved protein that replaces histone H3 in centromeres, is thought to recruit many of the proteins required for chromosome movement. CENH3 is present throughout the cell cycle and colocalizes with the kinetochore protein CENPC in meiotic cells. Chromatin immunoprecipitation demonstrates that CentC and CRM interact specifically with CENH3, whereas knob repeats and Tekay retroelements do not. Approximately 38 and 33% of CentC and CRM are precipitated in the chromatin immunoprecipitation assay, consistent with data showing that much, but not all, of CENH3 colocalizes with CentC.

We have identified and characterized a 17-to 18-kD Ser50-phosphorylated form of maize (Zea mays) CENTROMERIC HISTONE H3 (phCENH3-Ser50). Immunostaining in both mitosis and meiosis indicates that CENH3-Ser50 phosphorylation begins in prophase/diplotene, increases to a maximum at prometaphase-metaphase, and drops during anaphase. Dephosphorylation is precipitous (approximately sixfold) at the metaphase-anaphase transition, suggesting a role in the spindle checkpoint. Although phCENH3-Ser50 lies within a region that lacks homology to any other known histone, its closest counterpart is the phospho-Ser28 residue of histone H3 (phH3-Ser28). CENH3-Ser50 and H3-Ser28 are phosphorylated with nearly identical kinetics, but the former is restricted to centromeres and the latter to pericentromeres. Opposing centromeres separate in prometaphase, whereas the phH3-Ser28-marked pericentromeres remain attached and coalesce into a well-defined tether that binds the centromeres together. We propose that a centromere-initiated wave of histone phosphorylation is an early step in defining the two major structural domains required for chromosome segregation: centromere (alignment, motility) and pericentromere (cohesion).