Acetylation dynamics of human nuclear proteins during the ionizing radiation-induced DNA damage response

Abstract

Genotoxic insults, such as ionizing radiation (IR), cause DNA damage that evokes a multifaceted cellular DNA damage response (DDR). DNA damage signaling events that control protein activity, subcellular localization, DNA binding, protein-protein interactions, etc. rely heavily on time-dependent posttranslational modifications (PTMs). To complement our previous analysis of IR-induced temporal dynamics of nuclear phosphoproteome, we now identify a range of human nuclear proteins that are dynamically regulated by acetylation, and predominantly deacetylation, during IR-induced DDR by using mass spectrometry-based proteomic approaches. Apart from cataloging acetylation sites through SILAC proteomic analyses before IR and at 5 and 60 min after IR exposure of U2OS cells, we report that: (1) key components of the transcriptional machinery, such as EP300 and CREBBP, are dynamically acetylated; (2) that nuclear acetyltransferases themselves are regulated, not on the protein abundance level, but by (de)acetylation; and (3) that the recently reported p53 co-activator and methyltransferase MLL3 is acetylated on five lysines during the DDR. For selected examples, protein immunoprecipitation and immunoblotting were used to assess lysine acetylation status and thereby validate the mass spectrometry data. We thus present evidence that nuclear proteins, including those known to regulate cellular functions via epigenetic modifications of histones, are regulated by (de)acetylation in a timely manner upon cell's exposure to genotoxic insults. Overall, these results present a resource of temporal profiles of a spectrum of protein acetylation sites during DDR and provide further insights into the highly dynamic nature of regulatory PTMs that help orchestrate the maintenance of genome integrity.

Keywords: DNA damage response; ionizing radiation; nucleus; protein acetylation; quantitative proteomics.

Figure 2. Acetylation site-specific ratio distribution during the DDR (A) Distribution of log2-transformed ratios of all acetylation sites identified in the nuclear fraction quantified at all three time points. The dashed lines indicate the boundaries for the 1.5-fold change regions. (B) Consensus time profiles of regulated acetylation sites. (C) Heatmap of all acetylation sites, including names and position of modified amino acid. The color code is based on the fold change distribution.

Figure 3. Regulation of nuclear proteins by (de)acetylation during the DDR. Heatmap of acetylation sites regulated more than 1.5-fold compared with the untreated control (0 min). The color code is based on Z-statistic site-specific transformed ratios.

Figure 4. Functional network of proteins dynamically regulated by acetylation or deacetylation during DDR. Manually curated functional network based on hypergeometric testing of significant gene ontology terms and k-means clustering of functional categories and Pfam domains. Acetylation dynamics for each protein are depicted by four color codes: early deacetylation is yellow, late deacetylation is orange, early and late deacetylation is green and early deacetylation followed by late hyperacetylation is blue.

Figure 1. Experimental strategy for determination of protein abundance dynamics in the cytosol. SILAC-encoded cells were irradiated with 6 G and grown in culture for different lengths of time as indicated before harvest (0 min correspond to untreated cells). Cell population in each time series were mixed 1:1:1. Proteins from the nuclear fraction was digested and subjected to affinity enrichment of lysine acetylated peptides by anti-acetyl lysine antibody-conjugated agarose beads. A small part of the digest was separated by isoelectric focusing for protein normalization. All peptides were analyzed by LC-MS/MS and raw data were processed by MaxQuant. Processed data was analyzed using various computational statistics and bioinformatics tools.