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

Area of Science:

• Epigenetics and Chromatin Biology
• Molecular Cell Biology
• Protein Post-translational Modifications

Background:

• Heterochromatin Protein 1 (HP1) is a key protein in heterochromatin formation, recognized by its binding to methylated histone H3 lysine 9 (H3K9me).
• The biological significance of HP1 phosphorylation, a known modification, has remained largely unclear.
• Understanding HP1's interaction with chromatin is crucial for deciphering epigenetic regulation.

Purpose of the Study:

• To investigate the functional consequences of HP1 phosphorylation on its nucleosome binding properties.
• To identify the specific kinase responsible for HP1 phosphorylation and its effect on H3K9me recognition.
• To determine if the observed effects of phosphorylation are conserved across different organisms.

Main Methods:

• In vitro phosphorylation assays using casein kinase II (CK2) and purified human HP1α.
• Pull-down assays with in vitro-reconstituted nucleosomes to assess binding affinities.
• Electrophoretic mobility shift assays (EMSAs) to evaluate DNA binding properties.

Main Results:

• Casein kinase II (CK2) was identified as the primary kinase phosphorylating the N-terminus of human HP1α.
• Phosphorylated HP1α exhibited significantly enhanced specificity for H3K9me3-modified nucleosomes compared to unmodified HP1α.
• CK2-mediated phosphorylation reduced HP1α's intrinsic DNA binding, influencing its nucleosome interaction.
• Similar effects on nucleosome-binding specificity were observed for fly HP1a and S. pombe Swi6, indicating evolutionary conservation.

Conclusions:

• HP1 phosphorylation by CK2 critically modulates its nucleosome binding properties.
• Phosphorylation enhances HP1's specificity for H3K9me3-marked nucleosomes, refining heterochromatin recognition.
• This phosphorylation-dependent mechanism for recognizing H3K9me marks is evolutionarily conserved.