A high-confidence interaction map identifies SIRT1 as a mediator of acetylation of USP22 and the SAGA coactivator complex

Abstract

Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex.

Fig 1

Identification and characterization of the SIRT1 interactome. (A) An interaction network for HCIPs (D^N scores > 1) found in SIRT1 IP-MS/MS was created using networking tools in CompPASS. Protein matches for DBC1 and PARP1 did not meet D^N-score cutoffs but were included in the network map. Maps were generated in Cytoscape with attribute files that reflect interactor abundance (thickness of the line) and D^N score (color of the line). STRING database interactions are indicated as dashed lines. (B) Interactor abundance presented as average APSMs for bait proteins in SIRT1 WT and H363Y IP-MS/MS (left) and for selected interacting proteins (right). (C) FLAG-HA-USP22 (TAP) was immunoprecipitated with anti-FLAG-coupled resin and immunoprecipitates, and inputs were probed with antibodies against endogenous SIRT1. (D and E) Antibodies were used to immunoprecipitate endogenous USP22, GCN5, or SIRT1 from HEK 293T cells. Immunoblots were then probed with the indicated antibodies.

Fig 2

Interaction of USP22 with SIRT1 is highly specific and requires the ZnF-UBP domain. (A) C-terminally FLAG-tagged SIRT1 to SIRT7 were overexpressed in HEK 293T cells, immunoprecipitated with FLAG resin, and immunoblotted for endogenous USP22. (B) Various N-terminal FLAG-tagged deubiquitinating enzymes and acetyltransferases were overexpressed, purified as described for panel A, and immunoblotted for endogenous SIRT1. (C and D) Truncations of FLAG-HA-USP22 (C) and FLAG-HA-SIRT1 (D) were generated and tested for their ability to interact with endogenous SIRT1 or USP22, respectively. Domain maps of USP22 (C) and SIRT1 (D) indicate truncations used in interaction studies.

Fig 3

USP22 does not alter steady-state levels or deacetylase activity of SIRT1. siRNA oligonucleotides directed against SIRT1, USP22, or the control were transfected into HEK 293T cells (A) or HCT116 cells (B) for 72 h. Lysates were immunoblotted with the indicated antibodies. (C) NCI-H460 cells were transfected as described for panel A with control or USP22 targeting siRNA oligonucleotides and then treated with or without doxorubicin (0.5 μM) in the presence or absence of the SIRT1 inhibitor EX527 (10 μM). (D) Lysates generated from HEK 293T cells treated for various times with or without EX527 (10 μM) were immunoblotted with the indicated antibodies.

Fig 4

Human USP22 requires complex formation for deubiquitinating activity. (A) (Top) Purified recombinant human USP22 or catalytically inactive USP22-C185S was incubated with either DMSO or 5 μM UbVS. A representative gel stained with Coomassie brilliant blue is shown. (Middle) Reactions were performed as described above with various concentrations of DTT. (Bottom) Kinetic deubiquitinating activity assay of recombinant USP22 or the catalytic core of USP2 (USP2cc) with the substrate Ub-AMC. Data are presented in relative fluorescence units (RFU). (B) Purified intact mononucleosomes were incubated with full-length or catalytic inactive (CS) recombinant human Dubs and immunoblotted with the indicated antibodies. (C) Equal amounts of whole-cell lysates from HEK 293T cells were treated with DMSO or 5 μM UbVS for the indicated times and immunoblotted with the indicated antibodies. (D) (Left) Whole-cell lysates from FLAG-HA-USP22-expressing HEK 293T cells were separated by gel filtration and immunoblotted; (right) fractions from gel filtration samples were pooled on the basis of size, purified by IP, and treated with DMSO or 5 μM UbVS. FPLC, fast-performance liquid chromatography.

Fig 5

SIRT1 associates with the SAGA complex. (A) (Top) The indicated FLAG-HA-tagged proteins were isolated from HEK 293T cells, and Dub activity was analyzed by reactions with UbVS; (bottom) samples were prepared as described above, but wild-type SIRT1 samples were diluted as indicated in assay buffer before adding UbVS. (B) FLAG-HA-tagged proteins were isolated from HEK 293T cells and immunoblotted for the indicated proteins. (C) Control IgG or SIRT1-specific antibodies were used to IP endogenous SIRT1 from DSP-cross-linked HEK 293T cells. Immunoblots were probed with the indicated antibodies.

Fig 6

Acetylation of USP22 regulates protein-protein interaction. (A) FLAG-HA-USP22 was purified from HEK 293T cells treated with DMSO or a combination of TSA (400 nM) and nicotinamide (20 mM) and immunoblotted with panacetyllysine (Ac-K) antibodies 9441 and 9681. (B) Map of identified acetyllysine residues on USP22. (C) Cell lysates from HEK 293T cells stably expressing FLAG-HA-USP22 wild type, K129R, or K129Q were treated with 5 μM UbVS and then immunoblotted for HA. (D) FLAG-HA-USP22 WT or K129R was purified from HEK 293T cells treated with DMSO or EX527 (10 μM) and immunoblotted with the indicated antibodies. (E) The indicated FLAG-HA-USP22 mutants were purified from HEK 293T cells and subjected to DMSO or 5 μM UbVS and immunoblotted as indicated. Densitometry was performed using the ImageJ program (NIH).

Fig 7

SAGA components are acetylated in response to SIRT1 inhibition. (A) FLAG-HA-USP22 purified from HEK 293T cells treated with DMSO, TSA (400 nM), NAM (20 mM), or EX527 (10 μM) was immunoblotted with the indicated antibodies. The lower Ac-K 9441 blot is a shorter exposure displaying only the main band found between 50 and 75 kDa. (B) FLAG-HA-USP22 was purified as described for panel A from HEK 293T cells treated with DMSO, EX527 (10 μM), or FK866 (10 nM). (C) Whole-cell lysates from HEK 293T cells stably expressing FLAG-HA-USP22 treated with DMSO or EX527 (10 μM) were separated by gel filtration, pooled by size, and further purified by FLAG IP. Eluted complex pools were immunoblotted with the indicated antibodies. (D) Several FLAG-HA-tagged SAGA components or a FLAG-HA-GFP control were purified from HEK 293T cells treated with DMSO or EX527 (10 μM) and immunoblotted for the indicated proteins. (E) GCN5-FLAG was purified from HEK 293T cells treated with DMSO or EX527 (10 μM) and immunoblotted with the indicated antibodies.

Fig 8

Quantitative mass spectrometry reveals a SIRT1-regulated SAGA acetylome. (A) Outline of the work flow used to analyze acetylation of USP22-associated proteins in response to SIRT1 inhibition. (B) Dot plot of individual peptides representing the sum of the signal to noise for a given peptide plotted against its fold change versus the results for the control. Both unmodified (black) and acetylated (colored) peptides were recovered following antiacetyllysine IP. Quantitative measurements of peptide-level changes following acetyllysine IP were normalized to pre-IP total protein measurements. Changes observed in the subset of unmodified peptides were assumed to represent a distribution of measurements unaffected by SIRT1 inhibition. The standard deviation (SD) of this subset was used to approximate the significance of changes in levels of acetylated peptides. Acetylated peptides representing SAGA components with changes greater than 1 standard deviation are indicated by colored squares. The dot color represents the standard deviation with respect to the unmodified peptide distribution. (C) Quantification of individual acetylation sites is displayed as the fold change versus the results for the control. Bar colors represent standard deviations, as described for panel B. (D) Domain map of SAGA components displaying acetylation sites discovered. Acetylation site colors represent standard deviations, as described for panel B. Orange acetylation sites were identified but not quantified. UCH, ubiquitin carboxyl-terminal hydrolase; FATC, FRAP, ATM, TRRAP C terminal; FAT, FRAP, ATM, TRRAP; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase.

Fig 9

DUBm complex formation and SAGA interaction are regulated by SIRT1-mediated deacetylation. (A) FLAG-HA-USP22 was purified from HEK 293T cells transfected with siRNA against SIRT1 or a control siRNA treated with and without EX527 (10 μM) and immunoblotted with the indicated antibodies. (B) Eluted FLAG-HA-USP22 complexes from EX527 (10 μM)-treated cell lysates (performed as described for panel A) were incubated with or without recombinant human GST-tagged SIRT1 in the presence or absence of NAD⁺. Samples were immunoblotted with the indicated antibodies. (C) FLAG-HA-GFP or FLAG-HA-USP22 was purified from HEK 293T cells treated with DMSO or EX527 (10 μM). Samples were immunoblotted with the indicated antibodies. Densitometry on TAP-USP22 IP lanes was performed using the ImageJ program (NIH). (D) Samples were prepared as described for panel C from HEK 293T cells expressing FLAG-HA-tagged CCDC101. Densitometry on TAP-CCDC101 IP lanes was performed as described for panel C.