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

Gohei Nishibuchi,Yoshifumi Nishimura,Shinichi Machida,Hideaki Tagami,Jun-Ichi Nakayama,Hiromu Murakoshi,Kyoko Hiragami-Hamada,Wolfgang Fischle,Reiko Nakagawa,Akihisa Osakabe,Hitoshi Kurumizaka

doi:10.1093/nar/gku995

Abstract

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 N-terminus of human HP1α. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1α bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1α showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1α's intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1's recognition of H3K9me-marked nucleosomes.

Highlights

Heterochromatin, a distinctive structure in the nucleus of eukaryotic cells, plays an important role in chromosomal function and epigenetic gene regulation
We previously showed that mouse heterochromatin protein 1 (HP1)␣ is constitutively phosphorylated at its N-terminal serine residues (S11–14), and that this phosphorylation enhances HP1␣’s binding to H3K9me3 peptides [29]
The unphosphorylated form of HP1␣ was barely detected in any examined cells, suggesting that HP1␣ was constitutively phosphorylated in human cells

Summary

Introduction

Heterochromatin, a distinctive structure in the nucleus of eukaryotic cells, plays an important role in chromosomal function and epigenetic gene regulation. The cytological distribution of each isoform and the phenotypes of mice deficient for each of the isoforms suggest that they have distinct functions [11,12,13,14,15], their underlying molecular mechanisms have yet to be determined. While these HP1 isoforms are widely recognized to play primary roles in heterochromatin assembly, recent studies have revealed functions for them in cell-cycle control, transcriptional activation, DNA repair and other biological processes [16,17,18,19]

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Conclusion