Histidine Nτ-methylation identified as a new post-translational modification in histone H2A at His-82 and H3 at His-39

Histone post-translational modifications play critical roles in a variety of eukaryotic cellular processes. In particular, methylation at lysine and arginine residues is an epigenetic mark that determines the chromatin state. In addition, histone "histidine" methylation was initially reported over 50 years ago; however, further studies in this area were not conducted, leaving a gap in our understanding. Here, we aimed to investigate the occurrence of histidine methylation in histone proteins using highly sensitive mass spectrometry (MS). We found that acid hydrolysates of whole histone fraction from calf thymus contained Nτ-methylhistidine, but not Nπ-methylhistidine. Both core and linker histones carried a Nτ-methylhistidine modification and methylation levels were relatively high in histone H3. Furthermore, through MALDI-TOF MS, we identified two histidine methylation sites at His-82 in the structured globular domain of histone H2A and His-39 in the N-terminal tail of histones H3. Importantly, these histidine methylation signals were also detected in histones purified from a human cell line HEK293T. Moreover, we revealed the overall methylation status of histone H3, suggesting that methylation is enriched primarily at lysine residues and to a lesser extent at arginine and histidine residues. Thus, our findings established histidine Nτ-methylation as a new histone modification, which may serve as a chemical flag that mediates the epigenetic mark of adjacent residues of the N-terminal tail and the conformational properties of the globular domain.