The phosphorylation of heterochromatin protein 1 (HP1) has been previously 
described in studies of mammals, but the biological implications of this 
modification remain largely elusive. Here, we show that the N-terminal 
phosphorylation of HP1α plays a central role in its targeting to chromatin. 
Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger 
binding affinity for K9-methylated histone H3 (H3K9me) than that produced in 
Escherichia coli. Biochemical analyses revealed that HP1α was multiply 
phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells 
and that this phosphorylation enhanced HP1α's affinity for H3K9me. Importantly, 
the N-terminal phosphorylation appeared to facilitate the initial binding of 
HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 
tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α 
exhibited severe heterochromatin localization defects in vivo, and its prolonged 
expression led to increased chromosomal instability. Our results suggest that 
HP1α's N-terminal phosphorylation is essential for its proper targeting to 
heterochromatin and that its binding to the methylated histone tail is achieved 
by the cooperative action of the chromodomain and neighboring posttranslational 
modifications.