Lysine methylation occurs on both histone and nonhistone proteins. However, our knowledge on the prevalence and function of nonhistone protein methylation is poor. We describe an approach that combine Show more
Lysine methylation occurs on both histone and nonhistone proteins. However, our knowledge on the prevalence and function of nonhistone protein methylation is poor. We describe an approach that combines peptide array, bioinformatics, and mass spectrometry to systematically identify lysine methylation sites and map methyllysine-driven protein-protein interactions. Using this approach, we identified a high-confidence and high-resolution interactome of the heterochromatin protein 1β (HP1β) and uncovered, simultaneously, numerous methyllysine sites on nonhistone proteins. We found that HP1β binds to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and regulates its localization to double-strand breaks (DSBs) during DNA damage response (DDR). Mutation of the methylation sites in DNA-PKcs or depletion of HP1β in cells caused defects in DDR. Furthermore, we showed that the methylation of DNA-PKcs and many other proteins in the HP1β interactome undergoes large changes in response to DNA damage, indicating that Lys methylation is a highly dynamic posttranslational modification. Show less
An important issue in epigenetic research is to understand how the numerous methylation marks associated with histone and certain nonhistone proteins are recognized and interpreted by the hundreds of Show more
An important issue in epigenetic research is to understand how the numerous methylation marks associated with histone and certain nonhistone proteins are recognized and interpreted by the hundreds of chromatin-binding modules (CBMs) in a cell to control chromatin state, gene expression, and other cellular functions. We have assembled a peptide chip that represents known and putative lysine methylation marks on histones and p53 and probed the chip for binding to a group of CBMs to obtain a comprehensive interaction network mediated by lysine methylation. Interactions revealed by the peptide array screening were validated by in-solution binding assays. This study not only recapitulated known interactions but also uncovered new ones. A novel heterochromatin protein 1 beta (HP1β) chromodomain-binding site on histone H3, H3K23me, was discovered from the peptide array screen and subsequently verified by mass spectrometry. Data from peptide pull-down and colocalization in cells suggest that, besides the H3K9me mark, H3K23me may play a role in facilitating the recruitment of HP1β to the heterochromatin. Extending the peptide array and mass spectrometric approach presented here to more histone marks and CBMs would eventually afford a comprehensive specificity and interaction map to aid epigenetic studies. Show less