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SIRT1 is a NAD+-dependent histone H4K16 deacetylase that controls several different normal physiologic and disease processes. Like most histone deacetylases, SIRT1 also deacetylates nonhistone proteins. Here, we show that two members of the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) acetyltransferase family, hMOF and TIP60, are SIRT1 substrates. SIRT1 deacetylation of the enzymatic domains of hMOF and TIP60 inhibits their acetyltransferase activity and promotes ubiquitination-dependent degradation of these proteins. Importantly, immediately following DNA damage, the binding of SIRT1 to hMOF and TIP60 is transiently interrupted, with corresponding hMOF/TIP60 hyperacetylation. Lysine-to-arginine mutations in SIRT1-targeted lysines on hMOF and TIP60 repress DNA double-strand break repair and inhibit the ability of hMOF/TIP60 to induce apoptosis in response to DNA double-strand break. Together, these findings uncover novel pathways in which SIRT1 dynamically interacts with and regulates hMOF and TIP60 through deacetylation and provide additional mechanistic insights by which SIRT1 regulates DNA damage response.
Silent information regulator 2 (Sir2) was originally identified in a genetic screen for genes involved in controlling expression of silent mating type loci in yeast (58). Initially, the interest in Sir2 was limited to the studies of mating type interconversion or gene silencing in yeast. This situation, however, took a remarkable sharp turn with the intriguing finding that Sir2 promotes longevity in Saccharomyces cerevisiae (34). Subsequent studies by Imai et al. unexpectedly revealed that Sir2 is an NAD+-dependent histone deacetylase (HDAC), opening a new area of Sir2 research (30).
Of the seven human Sir2-like proteins (sirtuins), SIRT1 is most similar to the yeast Sir2 protein, which is the prototypic class III HDAC (3, 23, 25, 46). Although several studies suggest that sirtuins might not directly regulate aging via originally proposed mechanisms (6, 33), it is clear that sirtuins, particularly SIRT1, regulate metabolism, physiological homeostasis, and stress response and suppresses age-associated diseases (26, 27, 60, 72, 86). Most Sirt1-deficient mice die perinatally, and outbred background Sirt1-null animals are sterile with retinal, bone, and cardiac defects, reinforcing the notion of a pivotal function of SIRT1 in physiologic and developmental processes (12, 45). Despite its biological importance and the surge of interest in SIRT1 this past decade, there remain multiple gaps in our knowledge and many questions not yet answered regarding the functions and regulations of this deacetylase.
Consistent with its histone deacetylation function, biochemical studies revealed that SIRT1 deacetylates histone H4K16 and H3K9, interacts with and deacetylates histone H1K26, and mediates heterochromatin formation (75). Also, SIRT1 associates with LSD1, and together they play concerted roles in histones H4K16 deacetylation and H3K4 demethylation to repress gene expression (49). In addition to histones, over 30 nonhistone proteins have been reported to serve as substrates for SIRT1 (60, 72), and many of the effects of SIRT1 are attributed to its role in nonhistone deacetylation. For example, SIRT1 regulates energy metabolism by inducing gluconeogenic while repressing glycolytic gene expression through deacetylation of PGC-1α (59). SIRT1 deacetylates the tumor suppressor protein p53, Hif-1α, Hif-2α, HSF1, FOXO1, FOXO3, and FOXO4 to regulate cell growth, apoptosis, and the stress response (5, 19, 42, 44, 48, 77, 79). Deacetylation of NF-κB, AP1, and Foxp3 by SIRT1 modulates the inflammation and immune system (74, 84, 85, 89). Deacetylation of DNA repair proteins Ku70, NBS1, Werner syndrome protein (WRN), and xeroderma pigmentosum group A protein (XPA) by SIRT1 regulates genomic stability (14, 22, 31, 40, 88). In the current study, we describe the identification of two SIRT1 substrates, hMOF (human ortholog of the Drosophila males-absent-on-the-first) and TIP60 (HIV-1 TAT-interacting protein of 60 kDa), that belong to the MYST family (MOZ, Ybf2/Sas3, Sas2, and TIP60) of histone acetyltransferases (HATs).
MOF, a histone H4K16-specific HAT, was initially found to be a key component of the male-specific lethal (MSL) complex required for dosage compensation in Drosophila (1, 28). hMOF (KAT8 or MYST1) is a 458-amino-acid protein and, like the Drosophila homolog, possesses histone H4K16-specific HAT activity and contains a conserved MYST catalytic domain, a chromodomain, and a C2HC-type zinc finger (50). Through acetylation of H4K16, hMOF regulates chromatin organization and gene transcription. In addition, hMOF plays an important role in DNA damage response (DDR), DNA repair, cell cycle progression, and cell growth (24, 57). Biochemical purification revealed that hMOF exists in at least two multisubunit protein complexes, MSL and MOF-MSL1v1 (7, 32, 41, 63, 80). Both of the hMOF complexes acetylate histone H4K16, but the MOF-MSL1v1 complex acetylates additional nonhistone proteins, including K120 of p53 (p53-K120). hMOF-mediated acetylation of p53-K120 after DNA damage influences the cell's decision to undergo apoptosis instead of a cell cycle arrest (67). hMOF is bound to the oncogenic MLL protein and also partners with MRG15 to activate expression of the B-Myb gene (20, 54). Therefore, like other members of the MYST family, hMOF may have a role in the cause and development of cancer (2, 83).
TIP60 (KAT5 or HTATIP) is an acetyltransferase that is the closest relative of hMOF within the human MYST family (about 50% similarity over 513 amino acids). It is the catalytic subunit of the NuA4 HAT complex, which is involved in the activation of genes by acetylation of histones H4 and H2A (8, 21, 70). Like hMOF, TIP60 has been implicated in cell growth and arrest, apoptosis, and DNA repair (29). Also like hMOF, TIP60 acetylates p53-K120 to modulate the decision between cell cycle arrest and apoptosis (69). At DNA double-strand breaks (DSBs), the MRE11-RAD50-NBS1 (MRN) complex targets TIP60 to histone 3 trimethylated at K9 (H3K9me3). The MRN-TIP60 interaction activates TIP60, which leads to the acetylation and activation of the ATM kinase and initiation of DSB repair (65, 66). The Tip60 of Drosophila (dTip60) catalyzes exchange of phospho-H2Av with unmodified H2Av, the homolog of human histone variant H2A.X, at DNA DSBs (35).
In this work, we found that SIRT1 binds to and deacetylates hMOF and TIP60 in vitro and in vivo. SIRT1-hMOF/TIP60 interaction is dynamically regulated in response to DNA damage. Deacetylation of hMOF/TIP60 has multiple effects. First, deacetylation of hMOF/TIP60's enzymatic domains inhibits their autoacetylation and HAT activity. Second, deacetylation unexpectedly cross talks with ubiquitination and promotes proteosome-mediated degradation of hMOF and TIP60. Third, deacetylation of hMOF/TIP60 inactivates their DDR functions. Together, these results expand the diverse tasks of SIRT1, uncover a novel function of SIRT1 as a negative regulator of HATs in DDR, and furnish a new model of a complex reciprocal network of regulations for HATs and HDACs.
Myc-SIRT1, glutathione S-transferase (GST)–SIRT1, and Myc-tagged SIRT1 with an H363Y mutation [Myc-SIRT1(H363Y)] have previously been described (36). Expression plasmids for Flag-hMOF and Flag-TIP60 were derived from full-length hMOF (NP_115564) and TIP60 (NP_874369) cDNAs, respectively. Flag-hMOF and Flag-TIP60 deletion mutants were generated by subcloning PCR products containing hMOF/TIP60 fragments into the pcDNA3.1-Flag vector. Lysine-to-arginine mutants and the dominant negative (DN) mutants of Flag-hMOF/TIP60 were generated using a QuikChange Multi Site-Directed Mutagenesis Kit from Stratagene. The Flag-p53 expression plasmid was constructed by inserting a full-length human p53 cDNA into the p3XFlag-CMV-7.1 vector.
Mouse anti-Flag, rabbit antihemagglutinin (anti-HA), and mouse anti-β-actin antibodies were purchased from Sigma-Aldrich. Rabbit antiacetyllysine antibodies were purchased from Cell Signaling Technology and from Immunechem. Rabbit polyclonal anti-AcH4K16 (H4 acetylated at K16) antibody and rabbit polyclonal anti-TIP60 for immunoblotting of endogenous TIP60 were purchased from Millipore. Rabbit anti-p53 (FL-393), mouse anti-p53 (DO-1), and rabbit anti-c-Myc antibodies were purchased from Santa Cruz Biotechnology. Mouse anti-acetyl-K120-p53 and mouse monoclonal anti-hMOF for detection of endogenous hMOF were purchased from Abcam. The anti-PUMA (p53 up-regulated modulator of apoptosis) antibody was purchased from Cell Signaling Technology.
Experiments using stable-isotope labeling by amino acids (SILAC) were performed according to protocols previously developed by Mann and colleagues, with minor modifications (52, 53). Reagents including lysine- and arginine-deficient SILAC medium, dialyzed FBS, and heavy and light amino acids were purchased from Thermo Scientific. Briefly, Sirt1+/+ and Sirt1−/− murine embryonic fibroblasts (MEFs) were grown in light (normal l-arginine and l-lysine) and heavy (l-[13C6 15N4]arginine-HCl and l-[13C6]lysine-2HCl) media, respectively, for at least six doubling passages. Cells were harvested, and 1 mg of nuclear extracts was prepared from each cell population, combined, and subjected to immunoprecipitation with 50 μg of a mixture of two different antiacetyllysine antibodies, which were cross-linked onto protein A-agarose by dimethyl adipimidate. After incubation overnight at 4°C, antiacetyllysine agarose was washed five times with NETN buffer (150 mM NaCl, 20 mM Tris-Cl [pH 8.0], 0.5 mM EDTA, 0.5% Nonidet P-40). Immunoprecipitates were resolved on a 4 to 12% Novex gradient gel, followed by Coomassie brilliant blue staining. To reduce complexity, samples were excised from gels and divided into 12 slices before being subjected to trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Peptide identification and protein quantification were done using MaxQuant software (16, 17). Pathway analyses were performed with MetaCore software from GeneGo, and functional classifications were done using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources.
MEF, HEK293T, U2OS, and H1299 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. Stable HeLa cell lines (HeLa-C and HeLa-S5) were cultured under the same conditions with the addition of 0.25 μg/ml of puromycin. Transfections were performed with Lipofectamine 2000 (Invitrogen) or Fugene HD (Roche), according to the manufacturer's instructions. All transfections were normalized for total DNA using vector plasmids.
For immunoprecipitation, cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and protease inhibitor cocktail. Lysates were incubated with primary antibodies overnight at 4°C. Immunoprecipitates were washed three times with the same buffer and resolved by SDS-PAGE. For Western blotting, proteins were transferred onto nitrocellulose membranes, which were then incubated with the antibodies indicated in the figures. Proteins of interest were visualized using a chemiluminescent detection kit (Pierce).
For in vitro deacetylation assays, immunopurified Flag-hMOF/TIP60 was incubated with 1 μg of either GST-SIRT1 or GST in 200 μl of HDAC buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 10% glycerol) in the presence or absence of 50 mM NAD+ for 2 h at 30°C.
HAT assays were performed using a colorimetric HAT assay kit from Millipore according to the manufacturer's instruction. Briefly, 10 μg of nuclear extracts was incubated with biotin-conjugated H4 (residues 1 to 21) peptides and acetyl coenzyme A (acetyl-CoA) in a streptavidin-coated microwell plate for 1 h at 30°C. After samples were washed with Tris-buffered saline (TBS), reaction mixtures were incubated with antiacetyllysine antibodies for 1 h at room temperature, followed by washing with TBS-Tween 20 and then horseradish peroxidase (HRP)-conjugated secondary antibodies for 30 min at room temperature. Acetylated H4 peptides were visualized by tetramethylbenzidine substrate and measured by spectrometry at a wavelength of the optical density at 450 nm (OD450).
After transfection, cells were grown in medium containing 600 μg/ml of G418 for 2 weeks. Stable pools of G418-resistant cells were seeded onto chamber slides (Lab-Tek) and either treated with 50 μM etoposide or 10 Gy of ionizing radiation (IR) or left untreated. Twenty-four hours later, cells were fixed with prechilled methanol and stained with 2 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI) for 20 min. For each condition, at least 500 cells were examined for nuclear morphology on a microscope, and the percentage of the apoptotic cells was calculated.
Neutral comet assays were performed according to the manufacturer's suggestions (Trevigen). Cells were lysed, mixed with low-melting-point agarose gel, spread on slides, and subjected to electrophoresis in neutral Tris-borate-EDTA (TBE) buffer at 21 V for 30 min (Fisher midi-horizontal system FB-SB-1316; 27-cm-wide tank). The nuclei on slides were stained with SYBR green and visualized by microscopy. Images were captured and measured by an automated comet assay analysis system (HCSA 2.3.3; Loats Associates). The extent of DSB damage was represented by the parameter of tail moment, a combination of the amount of DNA and the length in the tail of comet. At least 100 cells were measured for each sample, and each experiment was repeated twice.
Twenty-four hours after transfections, cells were sparsely seeded onto 10-cm dishes in triplicates. After being cultured in selection medium containing 600 μg/ml of G418 for 2 weeks, cell colonies were stained with Giemsa for 30 min and counted. All experiments were repeated at least three times.
A long-term goal of our research is to comprehensively identify proteins whose acetylations are regulated by HDACs. Toward this goal, we began a study to test the feasibility of identifying cellular proteins that become hyperacetylated in the absence of Sirt1. Stable-isotope labeling with amino acids in cell culture (SILAC) was used to identify, characterize, and quantify acetylated proteins in Sirt1+/+ and Sirt1−/− fibroblasts (see Fig. S1A in the supplemental material). In total, 213 proteins were unambiguously identified to be hyperacetylated in the absence of Sirt1 (see Table S1 in the supplemental material). Each potential Sirt1 substrate was assigned to a functional group based on gene ontology molecular functions or biochemical process groups or on previously published literature (see Fig. S1B). Pathway identification and rank analysis, performed using the MetaCore software from GeneGo (Thomson Reuters), suggest that DNA damage response and repair pathways are key Sirt1 targets (see Fig. S1C). Consistent with our previous observations that SIRT1 binds to and deacetylates NBS1 and DNMT1 (56, 88), MRN components and the DNMT1 protein were identified as Sirt1 substrates in this global screen. Other previously reported SIRT1 substrates that were also identified in the present study include p53, p300, and p300/CBP-associated factor (PCAF) (4, 44, 55, 77). However, the majority of proteins identified in this screen were targets that have not yet been reported to be influenced by SIRT1.
Of the proteins whose acetylation levels were increased in the absence of Sirt1 in three independent experiments, histone modification enzymes such as HATs and their interacting proteins are highly represented (see Fig. S1B in the supplemental material). A large number of proteins that are involved in DDR or DNA repair also appear to be potential SIRT1 substrates. Two of these proteins are members of the MYST acetyltransferase family, hMOF and TIP60. Given the important role of hMOF, TIP60, and SIRT1 in DDR and chromatin modification, we focused our analysis on the regulation of hMOF/TIP60 by SIRT1.
To confirm that hMOF and TIP60 are SIRT1 substrates, we expressed Flag-hMOF and Flag-TIP60 in 293T cells, immunoprecipitated the two proteins, and analyzed the acetylation and protein abundance by Western blotting. As shown in Fig. 1A, overexpression of SIRT1 resulted in a small reduction of Flag-hMOF (about 3-fold decrease) and Flag-TIP60 (less than 2-fold decrease) proteins. Acetylation of Flag-hMOF and Flag-TIP60, however, was significantly decreased (more than 10-fold on average). Treatment of cells with a class III HDAC inhibitor, nicotinamide, resulted in hyperacetylation of hMOF and TIP60, further supporting the notion that SIRT1 is responsible for deacetylation of hMOF and TIP60 (Fig. 1B). In vitro, GST-SIRT1, but not GST alone, efficiently deacetylated hMOF and TIP60 in the presence of NAD+ and in the absence of nicotinamide (Fig. 1C). Additionally, endogenous hMOF became hyperacetylated in a HeLa cell line depleted of SIRT1 (56) compared to control cells (Fig. 1D). Taken together, these results confirm that hMOF and TIP60 are SIRT1 substrates.
Like many nonhistone HDAC substrates that interact with HDACs and consistent with the observation that hMOF and TIP60 are SIRT1 substrates, Myc-SIRT1 coimmunoprecipitated with Flag-hMOF and Flag-TIP60 (Fig. 1E). Endogenous SIRT1 also coimmunoprecipitated with endogenous hMOF and TIP60 (Fig. 1E). Further, TIP60 coprecipitated with endogenous SIRT1 as determined by mass spectrometry analysis (L. Peng, B. Fang, and J. Koomen, unpublished data).
To identify the acetylated lysines in hMOF and TIP60, Flag-tagged hMOF and TIP60 were expressed in 293T cells and treated with HDAC inhibitors. Immunopurified Flag-hMOF and Flag-TIP60 were analyzed by mass spectrometry. Our results indicate that both hMOF and TIP60 are abundantly acetylated, with 18 sites on hMOF and 15 sites on TIP60 distributed throughout the two proteins (Fig. 1F).
To elucidate the region of hMOF/TIP60 that interacts with SIRT1 and to narrow down the area of deacetylation, truncation mutants were generated, and the acetylation levels of these mutants and their interactions with SIRT1 were examined. As shown in Fig. 1G, SIRT1 interacts with the middle (residues 121 to 232 of hMOF; residues 75 to 285 of TIP60) and the C-terminal (residues 232 to 458 of hMOF; residues 285 to 513 of TIP60) regions of both proteins. Both the middle and C-terminal regions contain parts of the enzymatic MYST domain. SIRT1 did not interact with the N-terminal region of either protein containing the chromodomain (residues 1 to 121 of hMOF; residues 1 to 75 of TIP60). Although mass spectrometry analysis of intact hMOF/TIP60 revealed that acetylated lysines exist outside the C-terminal region, acetylation in the N-terminal and middle regions was undetectable in the absence of the C-terminal regions, most likely because of the requirement of the C terminus (containing hMOF-K274 and TIP60-K327) for autoacetylation (87). Also, acetylation of the C-terminal regions was completely erased by SIRT1. Our results, therefore, indicate that SIRT1 preferentially interacts with and deacetylates the enzymatic MYST domains of hMOF and TIP60 although at this time we cannot rule out the possibility that SIRT1 might also act on the chromodomains of these two proteins. Our finding is also consistent with the idea that autoacetylation of hMOF/TIP60 requires the MYST domains.
To assess the consequences of hMOF/TIP60 deacetylation, a series of lysine-to-arginine mutations was generated at the sites of acetylation (see Table S2 in the supplemental material). Mutations of acetylated lysines in the chromodomains of hMOF or TIP60 (C mutants) did not result in a decrease of acetylation in either protein (Fig. 2A). In contrast, acetylation of hMOF and TIP60 was severely compromised with mutations of acetylated lysines on the MYST enzymatic domains of hMOF or TIP60 (E mutants [hMOF-E-mut or TIP60-E-mut, respectively]). These results, together with the observation that autoacetylation of hMOF/TIP60 requires the MYST domains (Fig. 1G), suggest that deacetylation of the enzymatic domain of hMOF/TIP60 inhibits their autoacetylation and HAT activity. As a further confirmation, we found that E mutants of hMOF and TIP60 completely lost their abilities to acetylate histone H4 in vitro (Fig. 2B). Also, compared to wild-type hMOF/TIP60, E mutants had significantly less H4K16 acetyltransferase activity in vivo (Fig. 2C). Consistent with an earlier report that SIRT1 binds strongly to TIP60 only when TIP60 is acetylated (78), the hMOF E mutant does not interact with SIRT1 (Fig. 2D).
Additional lysine-to-arginine mutants were generated to determine precisely which lysine(s) within the MYST domains is critical for hMOF/TIP60 autoacetylation and HAT activities. Consistent with recent reports that K274 of hMOF is a major autoacetylation site and functionally important (32, 43, 64, 87), mutation of K274 to arginine alone (K274R) was sufficient to abrogate hMOF autoacetylation and enzymatic activity (Fig. 2E and andG).G). Similarly, a deacetylation mimic of K327 in TIP60, corresponding to hMOF-K274 (see Fig. S2 in the supplemental material), inhibited TIP60 acetylation and enzymatic activity (Fig. 2F and andG).G). Surprisingly, lysine-to-arginine mutation at residue 357 of TIP60 behaved identically to the K327R mutation, with a complete loss of TIP60 acetylation and HAT activity. Although at this time we do not know if the corresponding lysine hMOF-K304 undergoes acetylation, a K304R mutant also abrogates hMOF acetylation (Fig. 2E). Deacetylation of other acetylated lysines on MYST domains, including hMOF-K351, TIP60-K296, and TIP60-K404, had no effect on hMOF/TIP60 acetylation and HAT activity (Fig. 2F and andG).G). Therefore, in addition to previous reports that hMOF activity is regulated by autoacetylation of K274 (43, 64, 87), our data indicate that hMOF-K304, TIP60-K327, and TIP60-K357 on the MYST domains also control the acetylation of hMOF/TIP60 and HAT activity.
TIP60 protein is regulated by ubiquitination and proteasome-dependent degradation via interaction with MDM2 (15, 38). Whether the stability of hMOF is regulated in a similar fashion is unknown at this time. During the course of our study, we noticed that mutations of acetylated lysines mimicking a deacetylated state in the enzymatic domains of hMOF and TIP60 (E mutants) resulted in reduced protein levels compared to wild-type levels or mutations of acetylated lysines in the chromodomains (C mutants) (see Fig. S3A and B in the supplemental material). The amount of protein decrease was more apparent with an increase in the number of lysines mutated in the MYST domain of hMOF and TIP60. Further, DN mutants of hMOF and TIP60 (hMOF-DN-mut and TIP60-DN-mut, respectively), which do not possess autoacetylation activity and are not acetylated, behaved similarly to hMOF-E-mut and TIP60-E-mut, further suggesting that deacetylation of hMOF/TIP60 is important in the regulation of hMOF/TIP60 protein abundance.
To extend and confirm that deacetylation in the enzymatic domain of hMOF/TIP60 affects ubiquitination-mediated degradation, protein levels were determined for wild-type and hMOF/TIP60 mutants in the presence of the protein synthesis inhibitor cycloheximide. In the absence of protein synthesis, E mutants and DN mutants of hMOF and TIP60 turned over much more rapidly than the wild type and C mutants, and the increased protein degradation was reversed with the proteasome inhibitor MG132 (Fig. 3A and andB).B). In the presence of cycloheximide, the amount of protein degradation was directly proportional to the number of lysines mutated in the MYST domain of hMOF. For example, a mutant with seven lysines replaced by arginines (7KR) was degraded slightly faster than a 3KR and K274R mutant but much more slowly than the E mutant (Fig. 3C).
Overexpression of SIRT1 reduces hMOF and TIP60 protein levels (Fig. 1A and and3D;3D; see also Fig. S3C in the supplemental material). The diminution of protein as a result of SIRT1 is more pronounced in hMOF than in TIP60. Protein levels of hMOF-E-mut were not affected by SIRT1 (Fig. 3D), suggesting that deacetylation of the MYST domain is a prerequisite for hMOF/TIP60 degradation. Also, overexpression of SIRT1 had no effect on the expression of Flag-hMOF-E-mut or a green fluorescent protein (GFP) under the control of the same cytomegalovirus (CMV) promoter used to express hMOF/TIP60 (Fig. 3D) (L. Peng, unpublished data), ruling out the possibility that SIRT1 represses transcription from promoters used to drive expression of hMOF/TIP60.
To provide further support that deacetylation of hMOF/TIP60 promotes protein degradation and that the effect is mediated by SIRT1, we assessed endogenous hMOF protein levels in HeLa-S5 cells (56). Compared to control cells (HeLa-C), endogenous hMOF is much more abundant in the HeLa cell line depleted of SIRT1 (HeLa-S5) (Fig. 3E). Also, we repeated the SIRT1 overexpression experiments in the presence of cycloheximide and nicotinamide. As shown in Fig. 3F and andG,G, the effects of SIRT1-mediated decrease in hMOF/TIP60 protein levels were more pronounced in the presence of cycloheximide, and the effects could be reversed with the SIRT1 inhibitor nicotinamide. These results suggest that SIRT1-mediated deacetylation reduces hMOF and TIP60 protein levels in the absence of new protein synthesis and causes a degradation of these two proteins.
Because the stability of many proteins is determined by whether they become ubiquitinated, we sought to determine if lysine deacetylation in the enzymatic domain of hMOF/TIP60 promotes ubiquitination and subsequent proteasome-dependent degradation. As shown in Fig. 4A and andB,B, wild-type hMOF and TIP60 were readily ubiquitinated, and the ubiquitination was enhanced in the presence of MG132. Consistent with the observation that E mutants (MYST domain deacetylation mimics) of hMOF and TIP60 are less stable than wild-type hMOF/TIP60, the ubiquitination of hMOF and TIP60 E-mutants is much greater than that of wild-type hMOF/TIP60. Also consistent with the premise that the amount of hMOF/TIP60 degradation is increased corresponding to an increase of lysines deacetylated in the MYST domains, ubiquitination of the hMOF-E mutant is much greater than that of hMOF-K274R, which in turn is greater than that of wild-type hMOF (Fig. 4C).
To confirm that SIRT1 deacetylase enzymatic activity is necessary and sufficient to promote ubiquitination of hMOF and TIP60, ubiquitination of hMOF and TIP60 was assessed in the presence of either overexpressed SIRT1 or a SIRT1 with a single point mutation (H363Y) that renders the enzyme inactive. As shown in Fig. 4D, wild-type SIRT1 promotes hMOF/TIP60 ubiquitination much more efficiently than catalytic-deficient SIRT1. Further, endogenous hMOF is less ubiquitinated in Sirt1 knockout MEFs than in wild-type MEFs (Fig. 4E).
Our results that lysine-to-arginine substitutions in the MYST domains of hMOF/TIP60 increased ubiquitination indicate that different lysines are targets for deacetylation and ubiquitination. Mass spectrometry-based proteomics identified two sites of ubiquitination, K432 and K444, on hMOF, and one site, K282, on TIP60 (see Fig. S4A, B, and C in the supplemental material). Treatment of cells with the sirtuin inhibitor nicotinamide, but not treatment with the class I and II HDAC inhibitor trichostatin A (TSA), resulted in a decrease of ubiquitination at all three sites (see Fig. S4D), confirming again that deacetylation by class III HDAC(s) promotes ubiquitination of hMOF and TIP60.
Next, to further confirm hMOF/TIP60 ubiquitination target sites, hMOF-E-mut and TIP60-E-mut were modified to contain additional mutations at the identified ubiquitination sites (see Table S2 in the supplemental material). As shown in Fig. 5A and andB,B, hMOF-E-mut-K432R and hMOF-E-mut-K444R (hMOF-E-mut with additional lysine-to-arginine substitutions at K432 and at K444, respectively) are less ubiquitinated and more stable than hMOF-E-mut. Similarly, TIP60-E-mut-K282R is less ubiquitinated and more stable than TIP60-E-mut (Fig. 5C and andD).D). Collectively, these data provide strong evidence that SIRT1 deacetylation of enzymatic domains of hMOF/TIP60 accelerates the degradation of hMOF and TIP60 by upregulating the ubiquitination of these proteins.
Because both hMOF and TIP60 participate in the DDR and have critical roles in DNA DSB repair, we determined the interaction between SIRT1 and hMOF/TIP60 during the DDR. Interestingly, DNA DSB damage induced by gamma irradiation or etoposide dissociates the SIRT1-hMOF/TIP60 complexes transiently. Reassociation of SIRT1 with hMOF/TIP60 begins between 2 and 4 h after initiation of DNA damage, and interactions are completely restored by 6 h after initiation of DNA damage (Fig. 6A). Corresponding to the initial decrease in SIRT1-hMOF/TIP60 interaction, hMOF/TIP60 became transiently hyperacetylated upon exposure to etoposide (Fig. 6B). These data argue that SIRT1-hMOF/TIP60 interaction and the acetylation status of hMOF/TIP60 are tightly controlled by DNA damage signals.
Both hMOF and TIP60 acetylate K120 of p53 to influence the cell's decision to undergo apoptosis instead of cell cycle arrest (10, 67, 69). A p53-K120R mutation selectively blocks the transcription of proapoptotic target genes such as PUMA, and depletion of hMOF and/or TIP60 inhibits the ability of p53 to activate PUMA transcription (67). To examine the effects of deacetylation of hMOF/TIP60 on p53 signaling, hMOF/TIP60 E mutants were compared to wild-type hMOF/TIP60 for their ability to acetylate p53-K120 and to regulate PUMA expression. Under ionizing radiation, p53-K120 acetylation and PUMA expression were lowered in hMOF-E-mut and TIP60-E-mut compared to wild-type hMOF and TIP60, respectively, in U2OS (Fig. 7A) and H1299 (see Fig. S5 in the supplemental material) cells. Consistent with this finding, deacetylation mimics of hMOF/TIP60 (hMOF-E-mut/TIP60-E-mut) no longer promote apoptosis as effectively as wild-type hMOF/TIP60 after gamma irradiation or treatment with etoposide (Fig. 7B).
Knockdown of hMOF/TIP60 by siRNA or overexpression of hMOF/TIP60 dominant negative mutants (DN mutants) impairs DNA DSB repair and inhibits cell proliferation (24, 63). Comet assays were performed to assess the ramifications of hMOF/TIP60 deacetylation on DNA damage and repair. As shown in Fig. 7C, following ionizing radiation, U2OS cells overexpressing E mutants of hMOF and TIP60 had a slower DNA repair response than cells overexpressing wild-type hMOF/TIP60. Additionally, without DNA damage or exogenous stress, overexpression of hMOF/TIP60 E-mutants resulted in significantly less colony formation (Fig. 7D) than in cells expressing wild-type hMOF/TIP60, consistent with the premise that deacetylation of the MYST domains of hMOF/TIP60 negatively affects cell growth.
Recent studies suggest that hMOF is autoacetylated at K274, and this autoacetylation is required for HAT activity and protein substrate binding (43, 64, 87). Lu et al. propose that SIRT1 modulates hMOF autoacetylation and regulates hMOF recruitment to chromatin (43). Likewise, Wang and Chen showed that SIRT1 regulates autoacetylation and HAT activity of TIP60 though the site(s) of acetylation was not identified (78). In addition, Yamagata and Kitabayashi have shown that SIRT1 interacts with TIP60 (81). In this study, we confirm that hMOF and TIP60 are SIRT1 substrates. We discovered multiple novel acetylated lysines on hMOF and TIP60, and mutations of hMOF-K274 or hMOF-K304 and TIP60-K327 or TIP60-K357 affect acetylation and HAT activities of these proteins. SIRT1 binds to and deacetylates the MYST domains of hMOF and TIP60. Most important, we found that SIRT1 negatively regulates the activities, functions, and stabilities of hMOF and TIP60.
A previous study suggests that HATs and HDACs interact to mutually balance their activities toward histones in vivo (82). It was proposed that by physically forming complexes, HATs and HDACs execute rapid cycles of coordinated histone acetylation and deacetylation in the same regions of chromatin. Our findings that hMOF and TIP60 plus five other HATs (p300, CBP, PCAF, GCN5, and TAF1) are SIRT1 substrates, together with a previous report that SIRT1 deacetylates p300 and PCAF (4, 55), further support the idea that HATs and HDACs do not act independently but, rather, that their activities and functions are interdependent on each other. Unlike the previous study, however, we propose here an alternative, although nonmutually exclusive, model in which HDACs, and SIRT1 in particular, modify the activities and functions of HATs by directly deacetylating HATs. This model is reminiscent of many kinase proteins, whose activities are activated or repressed by phosphorylation/dephosphorylation in signaling cascades.
Unlike other HATs, which usually target multiple lysines on different histones, a remarkable feature of hMOF is its specificity for histone H4K16 (1, 63, 71). In parallel, although there are reports that SIRT1 can deacetylate all four core histones in vitro, SIRT1's chief target site on histones is H4K16 (75, 76). Therefore, SIRT1 does not only deacetylate acetylated H4K16 but also prevents hMOF from acetylating nonacetylated H4K16. Given the importance of H4K16 deacetylation in higher-order chromatin organization, transcription repression, cellular life span, and X inactivation in flies and mammals (18, 61, 62, 76), it is reasonable to predict that SIRT1 might control these processes both directly by deacetylation of H4K16 and indirectly by inhibiting hMOF.
The ε-amino group of lysine is accessible to many different modifications including acetylation, methylation, ubiquitination, sumoylation, neddylation, biotinylation, propionylation, butyrylation, and crotonylation (11, 68). One type of modification could exclude another modification on the same lysine residue within a protein. Lysine acetylation often inhibits ubiquitination-dependent, proteasome-mediated protein degradation by this principle (9). The stability of a number of proteins has been shown to increase after acetylation as a result of lysine site competition preventing ubiquitination. In these situations, by converting acetylated lysines to nonacetylated lysines, HDACs accelerate protein degradations by exposing the same lysines for ubiquitination. One of the most intriguing results from our study is that SIRT1 deacetylation of hMOF and TIP60 regulates their degradation in a proteosome-dependent manner. Ubiquitination and degradation of hMOF/TIP60 were dependent on both deacetylation of particular lysine(s) in the MYST domains and the total number of lysines that are deacetylated. However, unlike proteins whose stability is regulated by competition between acetylation and ubiquitination of the same lysine residues, acetyl-acceptor sites on hMOF and TIP60 are distinct from the sites of ubiquitination. Mutations of acetylated lysines to arginines in the MYST domains or deacetylation of hMOF/TIP60 by SIRT1 does not simply provide access of previously acetylated lysines for ubiquitination. Rather, deacetylation of hMOF/TIP60 more likely induces protein conformational change, alters protein subcellular localization, or changes interaction of hMOF/TIP60 with other cellular proteins, which ultimately favors ubiquitination on nonacetylation targets.
Our findings that deacetylation regulates hMOF and TIP60 add to the growing list of posttranslational modifications that control the activities and functions of hMOF and TIP60. In addition to acetylation and ubiquitination, TIP60 undergoes sumoylation at K430 and K451, and sumoylation promotes TIP60 HAT activity in response to UV irradiation (13). Also, phosphorylation of S86 and S90 of TIP60 has been shown to facilitate TIP60 activation (10, 39). To the best of our knowledge, for hMOF, phosphorylation or sumoylation modification has not yet been reported, and our work here is the first demonstration of ubiquitination. Using mass spectrometry analysis, we discovered that S86, S90, S155, S199, Y158, and Y401 of TIP60 and Y45 and Y418 of hMOF are phosphorylated, suggesting additional modifications that can regulate hMOF/TIP60 (Peng et al., unpublished). As increasingly sophisticated and sensitive techniques to identify posttranslational modifications become available, we predict there will probably be even more modifications that will be discovered for hMOF and TIP60. The next step in furthering our understanding of the mechanisms and cellular functions of hMOF/TIP60 is to determine which modifications are physiologically relevant and how different modifications work together with acetylation/deacetylation to extend the functions of hMOF and TIP60.
A proper DNA damage response (DDR) is critical for genome stability. Sir2 enhances DNA repair through several different mechanisms in yeast (37, 73). Similarly, SIRT1 plays a positive role in promoting DNA repair (22, 31, 40, 51, 88). hMOF/TIP60 also have previously been implicated in DDR, and, in this study, we found that they operate in a common pathway with SIRT1. Without exogenous stress, SIRT1 binds to and deacetylates hMOF/TIP60, maintaining hMOF/TIP60 in a low physiological concentration and in an inactive state. Upon DNA damage, hMOF and TIP60 are transiently released from SIRT1 and then become acetylated and activated. After a burst of hMOF/TIP60 activity, SIRT1 reassociates with hMOF/TIP60, inhibits them from activating p53 and expressing proapoptotic genes, and prevents excessive apoptosis. Thus, SIRT1 not only directly enhances DNA repair but also functions indirectly in DNA repair by maintaining a critical level of active hMOF and TIP60 during DDR.
hMOF and TIP60 are closely related in both sequence homology and in some biological functions, and results in this study suggest that SIRT1 regulates both proteins via similar mechanisms. However, there are also clear differences between hMOF and TIP60. For example, while hMOF selectively acetylates H4K16 within histones, the histone substrate specificity for TIP60 is less restrictive. hMOF and TIP60 exist as distinct multiprotein complexes in vivo. Interestingly, in this study, not all of the acetylated lysines in hMOF and TIP60 identified by mass spectrometry are conserved between hMOF and TIP60. For example, while four acetylated TIP60 residues, K296, K327, K357, and K404, are conserved in hMOF (corresponding to K243, K274, K395, and K341, respectively), we did not find acetylation of K243 or K395 in hMOF. Reciprocally, several lysines that are acetylated in hMOF are not conserved in TIP60. This, then, suggests that the regulation of hMOF and TIP60 by HATs and HDACs could be much more complicated than we anticipate. Further experiments are necessary to understand how SIRT1 deacetylation might regulate both similar and different biological functions of these two proteins.
By deacetylation of histones, changing chromatin conformation, or altering histone-cellular protein interactions, HDACs are commonly believed to play a key role in the repression of gene transcription. This is indeed the case for Sir2 in yeast, especially at telomeric sequences, the MAT loci, and the ribosomal DNA locus (47). Unexpectedly, a study found no evidence of failure of gene silencing in Sirt1 null animals, suggesting either that SIRT1 has a different role in mammals than it does in Saccharomyces cerevisiae or that its role in gene silencing is confined to a limited subset of mammalian genes (45). In our study here, a casual inspection revealed no significant difference in protein expression from cells derived from Sirt1 knockout animals compared to the wild type (L. Peng, and J. Koomen, unpublished data), confirming that SIRT1 probably does not induce generalized global transcription repression. Rather, SIRT1 most likely carries out many of its functions by targeting nonhistone substrates such as hMOF and TIP60. Current work in our laboratory is focused on understanding the biological and physiological relevance of other potential SIRT1 substrates identified in this study.
We thank Tony Kouzarides for plasmids and John Neveu, Bogdan Budnik, Renee Robinson, Vicki Izumi, Umut Oguz, Xiaotao Qu, and the Moffitt Cancer Center Core Facility for their technical support. We are grateful to Xiang-Jiao Yang (McGill University), Yali Dou (University of Michigan), Jacques Cote (Centre hospitalier universitaire de Québec), and John Lucchesi (Emory University) for advice.
This work was supported by grants to E.S. from the NIH (R01GM081650) and the Kaul Foundation and by a fellowship to L.P. from the American Heart Association.
Published ahead of print 14 May 2012
Supplemental material for this article may be found at http://mcb.asm.org.