Deciphering histone H4 lysine acetylation and methylation via sortase-mediated semisynthesis

The post-translational modifications of histone H4 play significant roles in regulation of epigenetic status. Reconstituted nucleosomes that mimic the native chromatin are valuable for histone modification research, which requires site-specifically modified histone. Sortase-mediated ligation (SML) has advantages of easy preparation of substrates and robust kinetics, and recent nucleosome-based deacylation research of histone H3 and H2B has benefitted from SML. Here we report a novel semisynthesis method of histone H4 via SML, which allowed us to prepare nucleosomes containing multivalently modified histone H4 for investigations of histone deacetylation by sirtuins. In addition, we identified a subfamily of PWWP domain-containing proteins as novel H4K20me3 readers by photo-crosslinking from multifunctional nucleosome probes. The studies revealed new mechanisms that might not be available using histone peptides. As a result, the new method is expected to accelerate our understanding of histone H4 modifications, and the identified new readers will expand our understanding of H4K20 methylation.