Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome

Abstract

The acetyltransferases CBP and p300 are multifunctional transcriptional co-activators. Here, we combined quantitative proteomics with CBP/p300-specific catalytic inhibitors, bromodomain inhibitor, and gene knockout to reveal a comprehensive map of regulated acetylation sites and their dynamic turnover rates. CBP/p300 acetylates thousands of sites, including signature histone sites and a multitude of sites on signaling effectors and enhancer-associated transcriptional regulators. Time-resolved acetylome analyses identified a subset of CBP/p300-regulated sites with very rapid (<30 min) acetylation turnover, revealing a dynamic balance between acetylation and deacetylation. Quantification of acetylation, mRNA, and protein abundance after CBP/p300 inhibition reveals a kinetically competent network of gene expression that strictly depends on CBP/p300-catalyzed rapid acetylation. Collectively, our in-depth acetylome analyses reveal systems attributes of CBP/p300 targets, and the resource dataset provides a framework for investigating CBP/p300 functions and for understanding the impact of small-molecule inhibitors targeting its catalytic and bromodomain activities.

Keywords: A-485; CBP; acetylation; acetylation kinetics; bromodomain; enhancer; gene transcription; mass spectrometry; p300; proteomics.

Figure 1. A high-confidence map of the CBP/p300 acetylome

(A) Diagram of CBP/p300 domain structure and the interventions (KO, CBP112, Cmpd-R, and A-485) used in this study. (B) Number of quantified acetylation sites and their overlap in the acetylome measurements from the indicated conditions. (C) Distributions of acetylation (Ac) site ratios and summed acetylated peptide intensity (from wild-type or control-treated samples) for the indicated interventions. The number of quantified sites and percent of up- or down-regulated (>2-fold change) sites is indicated. For each intervention, the number of sites (n) indicates the sum of acetylation sites quantified from replicate experiments. (D–F) Correlation and overlap between KO, Cmpd-R and A-485-treated acetylomes. The number of sites analyzed (n), Pearson’s correlation (r), P-value (P), and percent of regulated sites are shown. The Venn diagrams show the overlap between downregulated sites, and the boxplots show the distributions of ratio measurements within each region of the Venn diagram. (G) The fraction (%) of sites that were not quantified (i.e. acetylated peptide detected only in control samples, but not in the KO or the catalytic inhibitor treated samples) in the indicated experiments. (H) Distributions of acetylation (Ac) site ratios for sites that were not quantified in KO cells, but were quantified in the acetylomes from cells treated with the indicated inhibitors. See also Figure S1, Table S1, and Table S2.

Figure 2. Properties of the CBP/p300 acetylome

(A) The subcellular distribution of CBP/p300-regulated acetylation. The analysis was performed using proteins that associated with only one of the indicated UniProt Keywords, (P<1e⁻⁵⁰ for all comparisons, Fisher test). (B) UniProt Keyword enrichment analysis comparing CBP/p300-regulated and unregulated acetylation sites in KO cells (P<5e⁻²⁴, Fisher test). (C) CBP/p300-regulated sites (KO) are significantly more likely to occur in close proximity when compared to random sampling. Error bars indicate the 95^th percentile of the random sampling (*P≤0.05, permutation test). (D) Proteins with CBP/p300-regulated sites interact more frequently than unregulated proteins (**P≤0.001). Protein-protein interaction data were obtained from BioGRID 3.4 (Chatr-Aryamontri et al., 2017). (E) CBP/p300 regulates a majority of detected sites on targeted proteins. (F) Relative acetylation stoichiometry, as determined by abundance-corrected intensity (ACI), of CBP/p300-regulated sites. (P<2e⁻¹⁶ for all comparisons, Wilcoxen test). (G) CBP/p300 regulates a significantly greater proportion of high stoichiometry acetylation sites (top 5% ranked by ACI), as compared to low stoichiometry sites (bottom 95% by ACI) (P<5e⁻⁹⁰, Fisher test). (H) Correlation between in-vitro acetylation by recombinant p300 (n=2) and reduced acetylation in KO cells. The number of sites analyzed (n), Pearson’s correlation (r), P-value (P), and percent of regulated sites are shown. (I) Correlation between increased acetylation in human 293FT cells overexpressing p300 (n=2) and reduced acetylation in CBP/p300 KO MEF cells. See also Figures S2, S3, and S4.

Figure 3. Acetylation of diverse signaling effectors and enhancer-associated regulators

(A) Key signaling effectors of the indicated signaling pathways that have reduced acetylation in KO, Cmpd-R, or A-485-treated cells. (B) CBP/p300-regulated acetylation on proteins present in the experimentally defined Wnt enhanceosome (van Tienen et al., 2017). (C) Proteins and protein complexes associated with enhancer-driven gene expression are highly acetylated by CBP/p300. The data show maximum downregulated acetylation site ratio from KO, Cmpd-R, or A-485-treated cells. See also Figure S5.

Figure 4. Kinetic analysis of the CBP/p300 acetylome

(A) Reduced acetylation over time in A-485-treated MEF cells, sites were ordered based on acetylation half-life. (B) The fraction of CBP/p300-regulated acetylation sites and the magnitude of regulation at the indicated time points after A-485 treatment. (C) Correlation of acetylation (Ac) changes in A-485-treated (16 hours) MEFs to the same cells treated with A-485 for different time periods. The Pearson’s correlation (r) is shown. (D) Acetylation changes over time in A-485-treated MEFs. Sites were clustered by a fuzzy c-means method into three categories, as indicated. (E) The distribution of acetylation half-lives for 619 sites with a median value of 93.6 minutes. (F) The distribution of turnover rates (half-life) for acetylation (Ac sites) and the corresponding proteins. Protein half-lives were determined by (McShane et al., 2016). (G) The distributions of relative acetylation stoichiometry as determined by abundance corrected intensity (ACI) for sites with long (t_1/2 >30 minutes (m)) and short (t_1/2 <30 minutes (m)) half-lives. Significance (P) was calculated by the Wilcoxen test. (H) Deacetylation half-life (t_1/2) for the indicated proteins and acetylation sites. Red dots indicate outlier data points that were excluded for calculating half-life, dashed lines indicate variance. See also Figure S6.

Figure 5. Histone acetylation by CBP/p300

(A) The heatmaps show the log2 SILAC ratios for acetylation sites on the indicated histones, treatment conditions, and cell lines. The log2 SILAC ratio is the median of sites conserved in different isoforms; CBP/p300-regulated sites are color coded as indicated. (B) Half-lives of the indicated histone acetylation sites from A-485-treated MEFs are shown. (C) The data show reduced acetylation at histone H2B sites following treatment with A-485 for different time points. (D) The rank plot shows the summed acetylated peptide intensity (SILAC light, control-treated) of the indicated core histone acetylation sites in untreated wild-type MEFs. Sites are ordered from the least to the most intense and colored according to their regulation in KO cells (Log2 Ac ratio KO/Ctrl). See also Figure S6.

Figure 6. CBP/p300-catalyzed acetylation drives rapid transcriptional regulation

(A–C) Correlation between transcriptome changes in KO, Cmpd-R, and A-485-treated MEFs. The number of transcripts analyzed (n), Pearson’s correlation (r), and P-value (P) are shown. (D) Quantification of protein abundance changes in KO, Cmpd-R, A-485 and CBP112-treated MEFs. (E) Proteome (protein) and transcriptome (RNA) changes are well-correlated in KO cells. The number of transcripts and proteins analyzed (n), Pearson’s correlation (r), and P-value (P) are shown. (F) Cyp1a1 transcript induction in 6-Formylindolo(3,2-b)carbazole (FICZ)-treated cells, relative to untreated control (Ctrl) cells in the indicated cell types. Cmpd-R and CBP112 were added at the same time as FICZ. (G) FICZ-induced Cyp1a1 transcript expression relative to time = 0. For Cmpd-R-treated samples, the inhibitor was added at the 1h time point and Cyp1a1 expression was determined at the 2h and 3h time points.

Figure 7. P300DB: a database of CBP/p300-regulated acetylome, proteome and transcriptome

A snapshot of the P300DB web resource database. Using Mef2a as an example, the figure shows functional and sub-cellular localization annotation of the protein from the UniProt database, positions of acetylated lysine in the protein, site-specific regulation of acetylation in different perturbations, acetylation site kinetics, network of Mef2a interacting proteins with CBP/p300-regulated acetylation, and impact of CBP/p300 perturbations on expression of Mef2a protein and transcript levels. A more detailed description of the database is available at http://p300db.choudharylab.org.