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. 2012 Oct;11(10):1048-62.
doi: 10.1074/mcp.M112.019547. Epub 2012 Jul 23.

Quantitative acetylome analysis reveals the roles of SIRT1 in regulating diverse substrates and cellular pathways

Yue Chen  1 , Wenhui Zhao, Jeong Soo Yang, Zhongyi Cheng, Hao Luo, Zhike Lu, Minjia Tan, Wei Gu, Yingming Zhao
Affiliations

Quantitative acetylome analysis reveals the roles of SIRT1 in regulating diverse substrates and cellular pathways

Yue Chen et al. Mol Cell Proteomics. 2012 Oct.

Abstract

Despite of the progress in identifying many Lys acetylation (Kac) proteins, Kac substrates for Kac-regulatory enzymes remain largely unknown, presenting a major knowledge gap in Kac biology. Here we identified and quantified 4623 Kac sites in 1800 Kac proteins in SIRT1(+/+) and SIRT1(-/-) MEF cells, representing the first study to reveal an enzyme-regulated Kac subproteome and the largest Lys acetylome reported to date from a single study. Four hundred eighty-five Kac sites were enhanced by more than 100% after SIRT1 knockout. Our results indicate that SIRT1 regulates the Kac states of diverse cellular pathways. Interestingly, we found that a number of acetyltransferases and major acetyltransferase complexes are targeted by SIRT1. Moreover, we showed that the activities of the acetyltransferases are regulated by SIRT1-mediated deacetylation. Taken together, our results reveal the Lys acetylome in response to SIRT1, provide new insights into mechanisms of SIRT1 function, and offer biomarker candidates for the clinical evaluation of SIRT1-activator compounds.

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Figures

Fig. 1.
Fig. 1.
The experimental strategy for identification and quantification of Lys acetylation sites in mouse embryonic fibroblast (MEF) cells. A, MEF SIRT1+/+ (WT) and SIRT1−/− (KO) cells were grown in SILAC media with U-13C6-Lys and U-12C6-Lys respectively. Cells were sequentially lysed, generating two fractions: an NETN-soluble fraction and an NETN-pellet fraction. For each fraction, equal amounts of proteins from the two pools of cells were mixed. Proteins from NETN-soluble fractions were further resolved by ion-exchange chromatography, followed by tryptic digestion and immunoprecipitation using anti-Kac pan-antibodies. The combined proteins from NETN-pellet fractions were digested by trypsin. The resulting peptides were immunoprecipitated by anti-Kac pan-antibodies followed by OFFGel fractionation. The enriched Kac-containing peptides in each fraction were subjected to HPLC/MS/MS analysis. B, Western blot analysis of protein whole-cell lysates from MEF SIRT1+/+ and SIRT1−/− cells. C, Clustering analysis of Lysine acetylation motifs among different quantiles. Lys acetylation motifs were identified using Motif-x for all Lys acetylation sites and for sites in each quintile with Bonferroni corrected p < 0.05. HyperG test was performed for the enrichment test of each motif in all quantiles and p value was transformed into Z-score for hierarchical clustering analysis.
Fig. 2.
Fig. 2.
Enrichment and clustering analysis of the Lys acetylation data sets based on Gene Ontology annotations. Genes were classified by Gene Ontology annotation based on three categories: A, biological process, B, cellular compartment, and C, molecular function. In each category, quantification ratios of all Kac sites for each gene were divided into five quantiles, four of which were based on accumulative normal distribution (0–15%, 15–50%, 50–85%, 85–100%). The fifth quantile consisted of genes with Kac sites identified only in SIRT1−/− cells. An enrichment analysis was performed using the HyperG test with Benjamini-Hochberg adjustment. The p values were transformed into z-scores prior to hierarchical clustering analysis.
Fig. 3.
Fig. 3.
Enrichment and clustering analysis of Kac substrate proteins based on protein domains, cellular pathways, and protein complexes. Genes were annotated based on (A) the PFAM domain database, (B) the KEGG pathway database, and (C) the CORUM protein complex database. The analysis was carried out as described in Fig. 2. In addition, k-means clustering was used to identify 20 SIRT1-regulated mouse protein complexes.
Fig. 4.
Fig. 4.
Impact of SIRT1 knockout on selected cellular pathways and protein complexes. SIRT1 modulates acetylation levels on (A) the subunits of the long-patch base excision repair (BER) complex in the DNA repair pathway, (B) Notch co-activator and corepressor complexes in the Notch signaling pathway, (C) the RNA spliceosome in the RNA splicing pathway, (D) the Brd4-PTEFb complex, (E) the NuA4-Kat5 (Tip60) complex, and (F) the Kat7 (Hbo1) protein complex. Each gene is color-coded based on the highest SILAC KO/WT ratios for the gene.
Fig. 5.
Fig. 5.
SIRT1 deacetylates histone acetyltransferases (HATs). A, Schematic representation of HATs. The acetylated lysine residues identified through SILAC are indicated by arrows. Yellow arrows indicate a site quantification ratio (SIRT1 KO/WT) greater than 2, red arrows indicate sites only identified in SIRT1 knockout cells, and blue arrows indicate all other sites. B, C, and D, MEF cells (SIRT1 KO and WT) were treated with 1 μm trichostatin A (TSA) for 16 h to prevent the interferences from class I and II HDACs. The protein lysate containing 1 μm TSA and 10 mm nicotinamide was immunoprecipitated using corresponding antibodies, and immunoblotted with an anti-acetyllysine antibody. E, MEF cells were treated with 1 μm TSA for 16 h. The protein lysate was immunoprecipitated using anti-acetyllysine antibody and immunoblotted with antibodies of interest. F, G, and H, Protein extract from whole transfected 293 cells was immunoprecipitated and immunoblotted with antibodies of interest. I, Protein extracts from whole Flag-SIRT1/H1299 cells and H1299 parental cells were immunoprecipitated with M2 beads and immunoblotted.
Fig. 6.
Fig. 6.
SIRT1 deacetylates histone acetyltransferases (HATs) and impacts their acetylation activity. A, 293 cells were transfected with Flag-Kat8 (Myst1) and SIRT1, and treated with 1 μm Trichostatin A (TSA) for 8 h. Kat8 (Myst1) proteins were purified from cell lysates. Protein levels and their acetylation status were evaluated by Western blot. B, Flag-Kat8 (Myst1) was purified from transfected 293 cell lysates, untreated or treated with 1 μm TSA and 5 mm nicotinamide for 16 h. Kat8 (Myst1) (purified from nontreated cells) and Ac-Kat8 (Myst1) (purified from treated cells) were incubated with mono-nucleosomes for the indicated times. Protein levels and their acetylation status were evaluated by Western blot. C, Schematic representation of Kat8 (Myst1) wild type and KR mutant. D, 293 cells were transfected with Flag-Kat8 (Myst1) or Flag-Kat8 (Myst1) KR mutant, and treated with 1 μm TSA and 5 mm nicotinamide for 16 h. The tagged proteins were purified from cell lysates, and protein levels and acetylation status were evaluated by Western blot. E, 293 cells were transfected with a Flag-tagged protein of interest, and treated with 1 μm TSA and 5 mm nicotinamide for 16 h. The protein of interest was purified from cell lysates. Reactions similar to those described in b were carried out for 20 min; besides in lane 6 and lane 7, Flag- Kat8 (Myst1) were preincubated with Sirt1 with NAD for 30 min. F, The 293 cells were transfected with Flag-Kat7 (Myst2), with or without SIRT1, and treated with 1 μm TSA for 8 h. The Flag-Kat7 (Myst2) protein was purified from cell lysates. The protein levels and their acetylation status were evaluated by Western blot. G, The Flag-Kat7 (Myst2) was purified from transfected 293 cell lysates, untreated or treated with 1 μm TSA and 5 mm nicotinamide for 16 h. Kat7 (Myst2) (purified from nontreated cells) and Ac-Kat7 (Myst2) (purified from treated cells) were incubated with mono-nucleosomes for 20 min; besides in lane 4 and lane 5, Flag- Kat7 (Myst2) were preincubated with Sirt1 with NAD for 30 min. Protein levels and their acetylation status were evaluated by Western blot. H, Schematic representation of SIRT1 regulation of KATs through deacetylation and potentially further regulate cellular functions.
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