Mitotic phosphorylation of HP1a regulates its cell cycle-dependent chromatin binding

Abstract

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that plays a crucial role in heterochromatin-mediated gene silencing. We previously showed that mammalian HP1a is constitutively phosphorylated at its N-terminal serine residues by casein kinase II (CK2), and that this phosphorylation enhances HP1a's binding specificity for nucleosomes containing lysine 9-methylated histone H3 (H3K9me). Although the presence of additional HP1a phosphorylation during mitosis was reported more than a decade ago, its biological significance remains largely elusive. Here we found that mitosis-specific HP1a phosphorylation affected HP1a's ability to bind chromatin. Using biochemical and mutational analyses, we showed that HP1a's mitotic phosphorylation was located in its hinge region and was reversibly regulated by Aurora B kinase and serine/threonine phosphatases. In addition, chromatin fractionation and electrophoretic mobility shift assays revealed that hinge region-phosphorylated HP1a was preferentially dissociated from mitotic chromatin and exhibited a reduced DNA-binding activity. Although HP1's mitotic behaviour was previously linked to H3 serine 10 phosphorylation, which blocks the binding of HP1's chromodomain to H3K9me3, our findings suggest that mitotic phosphorylation in HP1a's hinge region also contributes to changes in HP1a's association with mitotic chromatin.