Ultradeep Lysine Crotonylome Reveals the Crotonylation Enhancement on Both Histones and Nonhistone Proteins by SAHA Treatment

Lysine crotonylation is a newly discovered protein post-translational modification and was reported to share transferases and deacylases with lysine acetylation. The acetyltransferase p300 was reported to also contain crotonyltransferase activity, and class I histone deacetylases were demonstrated to be the major histone decrotonylases. However, the decrotonylases for nonhistone proteins are unclear. Moreover, because of the lack of high-quality pan-antibodies, large-scale analysis of crotonylome still remains a challenge. In this work, we comprehensively studied lysine crotonylome on both histones and nonhistone proteins upon SAHA treatment and dramatically identified 10 163 lysine crotonylation sites in A549 cells. This is the first identification of tens of thousands of lysine crotonylation sites and also the largest lysine crotonylome data set up to now. Moreover, a parallel-reaction-monitoring-based experiment was performed for validation, which presented highly consistent results with the SILAC experiments. By intensive bioinformatic analysis, it was found that lysine crotonylation participates in a wide range of biological functions and processes. More importantly, it was revealed that both the crotonylation and acetylation levels of most core histones sites and a number of nonhistone proteins as well as some known substrates of class Ha and lib HDACs were up-regulated after SAHA treatment. These results suggest that SAHA may have decrotonylation inhibitory activities on both histones and nonhistone proteins by inhibiting HDACs.