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 IIa and IIb 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.