Quantitative Crotonylome Analysis Expands the Roles of p300 in the Regulation of Lysine Crotonylation Pathway

Abstract

Lysine crotonylation (Kcr) is a recently identified post-translational modification (PTM) that is regulated by an acetyltransferase, p300. The p300-catalyzed histone Kcr is able to stimulate transcription to a greater degree than the well-studied histone lysine acetylation (Kac). Despite these progresses, the global Kcr substrates regulated by p300 remain largely unknown, hindering efforts to establish mechanistic links between Kcr and p300-mediated phenotypes. Here, a quantitative proteomics study to characterize the p300-regulated lysine crotonylome is reported. A total of 816 unique endogenous crotonylation sites are identified across 392 proteins, with 88 sites from 69 proteins being decreased by more than 0.7-fold (log2 < 0.5) and 31 sites from 17 proteins being increased by more than 1.4-fold (log2 > 0.5) in response to p300 knockout (KO). The most downregulated crotonylome alterations under p300 deficiency concern components of the nonsense-mediated decay, infectious disease, and viral/eukaryotic translation pathways. Moreover, some p300-targeted Kcr substrates are potentially linked to diseases such as cancer. Taken together, this study reveals the lysine crotonylome in response to p300, which sheds light on the role for lysine crotonylation in regulation of diverse cellular processes and provides new insights into mechanisms of p300 functions.

Keywords: acetyltransferase; lysine crotonylation; p300; post-translational modification; quantitative proteomics.

Figure 1.

Systematic Profiling of Kcr proteome. A) Validation of the p300 depletion by Western blot. B) Schematic representation of experimental workflow for the identification and quantification of Kcr in WT and p300 KO cells. C) Pie chart shows experimental reproducibility of three biological replicates. D) Distribution of the number of Kcr sites per protein. E) Consensus sequence logo shows a representative sequence for all Kcr sites. F) Venn diagram shows cellular compartment distribution of Kcr proteins.

Figure 2.

A) Scatter plot shows the quantification of Kcr sites in relation to peptide intensities. The Kcr sites that could only be detected in WT cells are not shown in this scatter plot. B) Box plot shows representative proteins with p300-regulated Kcr sites. C) Validation of the Kcr level change in response to p300 KO by Western blot. D) Graph indicates the abundance distribution of Kcr modified proteins and all proteins in either “light” or “heavy” labeled cells.

Figure 3.

Bar graphs showing representative biological process and pathway annotations enriched with total and p300-regulated Kcr proteome, respectively.

Figure 4.

Interaction network of p300-regulated Kcr proteome based on STRING database (v10). The network is visualized in Cytoscape, and the size of the node is positively corresponded to the number of Kcr sites per protein. The proteins with median ratios (KO/WT) lower than 1 are marked in red (darker red color represents lower median ratio).