Developing Chemical Approaches to Examine Histone Posttranslational Modifications

Abstract

Diverse posttranslational modifications (e.g. acetylation, methylation and phosphorylation) of histones are known to regulate DNA-templated processes, such as gene transcription, DNA replication and DNA damage repair. Histone modifications can serve as a signaling platform that would be recognized (or ‘read’) by specific binding proteins, which would then, in turn, exert effects on chromatin structure and function. While various modifications have been detected at more than 100 different sites of histones, the progress on finding proteins that recognize these modifications have largely lagged behind. As a result, it remains poorly understood how histone modifications are recognized and translated into meaningful biological processes. Here, we report an approach that combines a photo-crosslinking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein-protein interactions. We applied this approach to examine the interactions mediated by PTMs of the unstructured N-terminal ‘tail’ of histone, including histone H3 trimethylation at lysine 4 (H3K4Me3) or at lysine 9 (H3K9Me3), symmetrical dimethylation at arginine 2 (H3R2Me2s) and phosphorylation at threonine 3 (H3T3-Phos). Our chemical approach was also used to study the interplay between these proximal histone PTMs. Together, our data indicate that our cross-linking-assisted and SILAC-based protein identification (CLASPI) approach can be generally applied to examine protein-protein interactions mediated by PTMs.