Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation

Histone H3 (H3) phosphorylation at Ser¹⁰ occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr⁷, we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser²⁸ phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Set²⁸. This antibody specifically recognized the phosphorylation of H3 Ser²⁸ but not that of glial fibrillary acidic protein Thr⁷. Immunocytochemical studies with HTA28 revealed that Ser²⁸ phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using ³²P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser²⁸ during early mitosis. In addition, we found that H3 is phosphorylated at Ser²⁸ as well as Ser¹⁰ when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Set²⁸, together with Ser¹⁰, is a conserved event and is likely to be involved in mitotic chromosome condensation.