N-terminal phosphorylation of HP1α increases its nucleosome-binding specificity

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin. Although HP1 phosphorylation has been described in several organisms, the biological implications of this modification remain largely elusive. Here we show that HP1’s phosphorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation assays and conventional chromatography, we demonstrated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the Nterminus of human HP1. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1 bound H3K9-methylated and H3K9unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1 showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1’s intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombeSwi6.TheseresultssuggestedthatHP1phosphorylation has an evolutionarily conserved role in HP1’s recognition of H3K9me-marked nucleosomes.