Developing diazirine-based chemical probes to identify histone modification 'readers' and 'erasers'

Post translational modifications (PTMs, e.g., phosphorylation, acetylation and methylation) of histone play important roles in regulating many fundamental cellular processes such as gene transcription, DNA replication and damage repair. While 'writer' and 'eraser' enzymes modify histones by catalyzing the addition and removal of histone PTMs, 'reader' proteins recognize these modified histones and 'translate' the PTMs by executing distinct cellular programs. Therefore, identification of the regulating enzymes and binding partners of histone PTMs is essential for understanding their regulatory mechanisms and cellular functions. Here we report the development of diazirine-based photoaffinity probes for identification of 'readers' and 'erasers' of histone PTMs. When compared with previously described benzophenone-based photoaffinity probes, the present probes demonstrate significantly improved photo-cross-linking rates, yields and specificities for capturing proteins that recognize a trimethylation mark on histone H3 lysine 4 (H3K4Me₃). Furthermore, we show that the diazirine-based probes can also be used to identify enzymes that catalyse the removal of histone lysine acetylation and malonylation. This study provides new chemical tools for examining PTM-mediated protein-protein interactions and broadens the scope of our photo-cross-linking strategy from finding histone PTM 'readers' to identifying dynamic and transient interactions between PTMs and their 'erasers'.