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Reversible covalent methylation of lysine residues on histone proteins constitutes a principal molecular mechanism that links chromatin states to diverse biological outcomes. Recently, lysine methylation has been observed on non-histone proteins, suggesting broad cellular roles for the enzymes generating and removing methyl moieties. Here we report that the lysine methyltransferase enzyme SET8/PR-Set7 is a novel regulator of the tumor suppressor protein p53. We find that SET8 specifically monomethylates p53 at lysine 382 (p53K382me1). This methylation event robustly suppresses p53-mediated transcription activation of highly responsive target genes, but has little influence on weak targets. Further, depletion of SET8 augments the pro-apoptotic and checkpoint activation functions of p53, and accordingly, SET8 expression is downregulated upon DNA damage. Together, our study identifies a novel p53-modifying enzyme, a new regulatory post-translational modification of p53, and begins to dissect how methylation may contribute to a dynamic post-translational code that modulates distinct p53 functions.
Methylation events at distinct lysine residues within histone proteins are linked to diverse functional outcomes (Jenuwein and Allis, 2001). For example, methylation at histone H3 at lysine 4 (H3K4me) is largely detected at euchromatin and is thought to generally lead to increased DNA accessibility, whereas methylation of histone H3 at lysine 9 (H3K9me) is most commonly associated with heterochromatin and inaccessible DNA (Bannister and Kouzarides, 2004). One mechanism by which lysine methylation aids in the establishment of distinct chromatin states is by mediating modular protein-protein interactions (Daniel et al., 2005). In this regard, the proteins that recognize a methylated lysine within a specific sequence context can define the functional outcome of a lysine methylation event. Further, histone lysines can be mono-, di- or trimethylated, with a unique activity frequently being coupled to the specific state and extent of methylation on the lysine residue. Thus, methylation of lysine residues on a target protein can increase the signaling potential of the modified protein and as such lead to diverse physiologic consequences.
p53 is a transcription regulator that plays a central role in tumor suppression by directing cellular responses to diverse stresses (Laptenko and Prives, 2006; Toledo and Wahl, 2006). The levels and activity of p53 are regulated by a complex network of post-translational modifications that primarily occur within two regions of the protein: an N-terminal region that is phosphorylated at multiple sites and a C-terminal region rich in basic residues (Appella and Anderson, 2001; Toledo and Wahl, 2006). Recent reports indicate that p53 is monomethylated at two different lysine residues within the regulatory C-terminal region (Chuikov et al., 2004; Huang et al., 2006). Akin to how H3K4me and H3K9me are linked to opposing states of chromatin, the two known sites of p53 methylation are coupled to activities that oppose one another. Specifically, SET7/9-mediated monomethylation of p53 at K372 (p53K372me1) activates p53, postulated in part to occur via stabilization of chromatin-associated p53, whereas Smyd2-mediated monomethylation of p53 at K370 (p53K370me1) represents a repressive mark, the generation of which is impeded by p53K372me1 (Chuikov et al., 2004; Huang et al., 2006). In addition to methylation at K370 and K372, the C-terminal region of human p53 harbors several K residues that are subject to modification by acetylation, ubiquitylation, sumoylation and neddylation (reviewed in Toledo and Wahl, 2006). Notably, endogenous p53 protein from two independent mouse models in which these lysines were targeted for mutation did not display an alteration in stability, and the phenotypes of cells derived from the mice were relatively mild (Feng et al., 2005; Krummel et al., 2005). This work argues that in sum, the post-translational modifications (PTMs) on the p53 C-terminal region fine-tune p53 activity. However, as substitution of lysines will prevent all forms of PTMs, including mono-, di- and trimethylation, mutant phenotypes may indicate the elimination of both positive and negative regulatory effects. Thus, identifying and characterizing the enzymes that catalyze p53 modifications is critical for developing a molecular understanding of how p53 PTMs are coordinated to regulate p53 functions.
SET7/9 and Smyd2 were both first reported to function as histone methyltransferases (HMTs), suggesting that other HMTs might have non-histone substrates (Brown et al., 2006; Nishioka et al., 2002a; Wang et al., 2001). SET8/PR-Set7 is an HMT that adds a single methyl moiety to histone H4 tails at lysine 20 (H4K20me1), preferentially to nucleosomal H4 (Fang et al., 2002; Nishioka et al., 2002b). Mutation of the SET8/PR-Set7 gene in Drosophila melanogaster leads to lethality during development (Nishioka et al., 2002b). H4K20me1 generation by SET8 has also been shown to be important for gene silencing and mitotic regulation (Fang et al., 2002; Julien and Herr, 2004; Rice et al., 2002). Here we demonstrate a novel activity for SET8 as a p53 methyltransferase. We find that SET8-mediated methylation of p53 at K382 represses highly responsive p53 target genes to attenuate p53 pro-apoptotic and cell-cycle arrest functions. We propose a model in which SET8-mediated p53 methylation tips the balance of p53 function away from cell elimination towards cell survival.
To screen known HMTs to determine if they might function as p53 methyltransferases, we expressed recombinant SET7/9, Suv39h1, hDOT1L, SET8/PR-Set7 and Suv4-20h1, and performed in vitro methylation assays using full-length GST-p53 and histones as substrates (Figure 1A). As expected, SET7 methylated p53 and histone H3, but not nucleosomes (Figure 1A) (Chuikov et al., 2004). The other enzymes showed methyltransferase activity on their cognate histone substrate, but only SET8 was found to possess methylation activity on p53 (Figure 1A). We performed ms/ms analysis of the methylated p53 protein and mapped K382 as a residue modified by SET8 (Supplementary Figure 1; data not shown). To confirm these results, we generated several single site mutants in the context of full-length p53, and performed in vitro methylation assays with SET8 and SET7. As shown in Figure 1B, substitution of K382 with arginine (p53K382R) abolishes SET8-mediated p53 methylation, but has no effect on SET7-mediated activity. Substitutions at other lysines of p53 had no impact on SET8 methylation (Figure 1B). Based on these data, we conclude that SET8 methylates p53 exclusively at lysine 382.
SET8 is a strict monomethyltransferase for H4K20 (Couture et al., 2005; Xiao et al., 2005). Alignment of the amino acid sequence surrounding the substrate site of SET8 in p53 (K382) and that of histone H4 (K20) reveals significant overlap (Figure 1C). We therefore reasoned that p53 is likewise monomethylated by SET8. To confirm the extent of methylation on p53K382 catalyzed by SET8, peptides bearing amino acids 367–389 of p53 were in vitro methylated by SET8 and analyzed by mass spectrometry. As shown in Figure 1D, SET8 induced a 14Da shift in the p53 peptide, indicating the addition of a single methyl group to K382. Under the conditions of our reaction, virtually all peptide was converted to the monomethyl species, with little to no dimethylation or trimethylation detected. Thus, in vitro, SET8 is a p53 monomethyltransferase.
To confirm the existence of p53K382me1 in vivo, we performed a mass spectrometry analysis of endogenous p53 purified from HeLa nuclear extract (see Experimental Procedures). Two peaks representing unmethylated and monomethylated peptides containing p53K382 are shown in Figure 2A. The mass difference of 14Da indicates the addition of a single methyl moiety. We confirmed by ms/ms that the methyl moiety is specific to K382 and not to other residues present in the peptide fragment (data not shown). These data provide the first definitive confirmation of methylation being present on endogenous p53.
We next raised an antibody against the p53K382me1 epitope (hereafter referred to as αp53K382me1). This antibody specifically recognizes p53 peptides monomethylated at K382 and does not crossreact with unmodified p53, p53K382me2, p53K382me3 peptides or several additional p53 methylated residues (Figure 2B; data not shown). Notably, despite the sequence homology between the SET8 substrate sites of p53K382 and H4K20 (see Figure 1C), our antibody did not detect H4 peptides irrespective of methylation status at K20 (Figure 2B). We also tested the ability of αp53K382me1 to recognize p53 methylated at K382 in the context of the full-length p53 protein. As shown in Figure 2C, αp53K382me1 detects full-length SET8-methylated wild-type (wt) and various K>R mutant derivative p53 proteins (lane 4–7), but not unmethylated p53 (lane 1–3), or the p53K382R mutant (lane 8). Based on these results, we conclude that our αp53K382me1 antibody can be utilized to monitor SET8-dependent monomethylation of p53 at K382 for in vivo studies.
Next, to determine whether p53 is methylated by SET8 in vivo, U2OS cells were transfected with SET8 and either Flag-tagged wt-p53, p53K382R or p53K372R. As shown in Figure 3A, Western analysis detected p53K382me1 in whole cell extracts (WCE) and Flag-immunoprecipitates (IPs) from cells transfected with wt-p53 and the p53K372R mutant, but not with the p53K382R mutant. Moreover, SET8, but not a catalytically inactive mutant (SET8D338A), methylated endogenous p53 at lysine 382 in WCE and in αp53 IPs (Supplementary Figure 2; see Figure 3C; Couture et al., 2005). Thus, ectopic SET8 methylates endogenous p53 in vivo.
We next investigated whether SET8 is physiologically responsible for the monomethylation of p53K382. To this end, endogenous SET8 protein levels were knocked-down by RNA interference (RNAi) in the p53+ U2OS cell line, and levels of p53K382me1 determined (Figure 3B). The RNAi treatment did not alter p53 levels in whole cell extracts (WCE), but levels of p53 detected in αp53K382me1 IPs were specifically reduced by SET8 RNAi relative to control RNAi treatment, denoting a decrease of endogenous p53K382me1 levels in SET8 knock-down cells (Figure 3B). Three sequential transfections of SET8 RNAi are reported to result in a reduction of H4K20me1 levels (Botuyan et al., 2006); however, under our experimental conditions of a single round of SET8 RNAi treatment, a decrease in p53K382me1 levels is observed with no reduction in H4K20me1 levels detected (Figure 3B). The manifestation of p53K382me1 depletion without reduction of H4K20me1 upon acute SET8 knock-down is likely a consequence of (i) p53 protein levels being orders of magnitude lower than H4, (ii) the rapid kinetics of p53 protein turnover and (iii) the reported high stability of the H4K20me1 mark (Karachentsev et al., 2005). Taken together, we conclude that SET8 is required for maintenance of p53K382me1 levels in vivo.
DNA damage was reported to increase monomethylation at p53K372, suggesting the possibility that p53K382me1 might likewise be regulated (Chuikov et al., 2004). We therefore examined p53K382me1 levels in U2OS cells in response to the radiomimetic drug neocarzinostatin (NCS), in the presence of ectopic SET8 expression (Figure 3C) and upon knock-down of endogenous SET8 protein (Figure 3D). As expected, total p53 levels (and acetylation of p53 at K382 (p53K382ac)) increased in response to DNA damage induced by NCS treatment (Figure 3C and 3D). In contrast, levels of p53K382me1 decreased in response to DNA damage compared to control treatment (Figure 3C, compare lanes 3 and 4; Figure 3D, compare lanes 1 and 2). Consistent with these findings, SET8 mRNA and protein expression levels in U2OS cells are markedly repressed in response to NCS treatment, whereas Smyd2 expression does not change with DNA damage (Figure 3E and 3F). These data suggest the hypothesis that the generation of p53K382me1 by SET8 represses p53 functions, an activity that is itself curbed during the physiologic DNA damage response (see discussion). Finally, SET8 monomethylation at p53K382 impaired DNA damage-induced acetylation at the same residue (p53K382ac) (Figure 3C and 3D). The observations that p53K382me1 generation negatively correlates with DNA damage and that SET8 inhibits the formation of p53K382ac, a modification linked to DNA damage responses, raises the possibility that monomethylation at K382 inhibits p53-mediated DNA damage responses (Appella and Anderson, 2001; Toledo and Wahl, 2006).
To investigate the biological consequences for SET8-mediated methylation of p53, the ability of SET8 to regulate transcription of the p53 target genes p21 and PUMA was determined. For these experiments, we used H1299 cells, which lack endogenous p53 and can therefore be complemented with either wild-type or mutant p53, allowing for the selective investigation of the role of K382 (Knights et al., 2006; Lokshin et al., 2005). p53 reconstitution in H1299 cells resulted in marked induction of p21 mRNA (Figure 4A) and p21 protein (Figure 4B) levels relative to cells transfected with vector alone. The p53K382R mutant likewise triggered a strong induction of the p53 target genes, though the phenotype was slightly less pronounced than wild-type p53, indicating that K382 residue per se is not required for p53 transactivation activity (Figures 4A and 4B; Supplementary Figure 3A). Notably, co-expression of SET8 with p53, which did not affect p53 protein expression, largely abolished the induction of p21 mRNA and p21 protein elicited by wt-p53, yet did not impinge on the activity of the p53K382R mutant (Figures 4A and 4B). Equivalent results were obtained for PUMA, a second p53 target gene (Supplementary Figure 3). Additionally, we observed that in chromatin immunoprecipitation (ChIP) assays, SET8 co-expression reduced occupancy of p53 at the p21 and PUMA promoters (Figure 4C; Supplementary Figure 3B), but notably had no effect on H4K20me1 levels at these promoters (Figure 4D; Supplementary Figure 3C). p53K382me1 was not detected at the p21 and PUMA promoters, though global distribution of this species is grossly similar to that of total p53 (Supplementary Figure 4). Finally, the SET8 catalytic mutant SET8D338A, which fails to methylate p53 in vivo, likewise failed to inhibit p53 transactivation activity (Figure 4E; Supplementary Figure 3D). Taken together, these data argue for a model in which it is the direct addition of the methyl moiety to K382 by SET8, rather than alterations of H4K20me1 levels by SET8 or the failure to otherwise modify p53K382, which acts to inhibit p53 transcriptional activity.
To test this hypothesis in a more physiologic setting, endogenous SET8 protein levels were knocked-down in the p53+ U2OS cell line, and p53-dependent responses to NCS treatment were determined. Consistent with the overexpression data, knock-down of SET8 led to an increase of p21 mRNA levels and p21 protein expression induced by the genotoxic agent relative to the control siRNA treatment (Figure 5A and 5B). For these experiments, two independent siRNA pools were utilized to exclude off-target effects (Figure 5A and 5B). Like p21, the expression of PUMA was upregulated by SET8 RNAi treatment relative to control treatment (Supplementary Figure 5A).
It is possible that the altered regulation of p21 and PUMA in response to DNA damage by SET8 knock-down is due to modulation of H4K20 methylation rather than p53 methylation. However, this possibility is highly unlikely for a number of reasons: (1) irrespective of the different treatments of our experiments, no SET8-dependent changes in H4K20me1 levels were observed globally (Figure 5B) or at specific promoters (Figure 5G; Supplementary Figure 5D); (2) the increase in p21 and PUMA induction upon DNA damage observed with SET8 knock-down is dependent on p53 protein, as co-knock-down of p53 (Figure 5C and 5D) eliminated any effects of SET8 (Figure 5E; Supplementary Figure 5B); (3) the SET8 knock-down mediated increase in gene expression of p21 and PUMA correlates with increased p53 occupancy at the cognate promoters, indicating SET8 directly alters p53 behavior (Figure 5F; Supplementary Figure 5C); (4) the regulation of highly responsive p53 target genes is specific, as expression of a constitutively active non-p53 regulated gene (actin) did not change upon SET8 knockdown, as might be expected were the phenotype due to global loss of H4K20me1 (Supplementary Figure 6); (5) knock-down of SET8 did not impact the expression of a number of weak p53 targets such as Bax and NOXA, and suppressed mRNA induction of the DNA repair factor GADD45, the inverse of what would be expected were the phenotype due to H4K20me1 depletion (Supplementary Figure 7; data not shown). Together, these observations strongly argue for direct methylation by SET8 at lysine 382 of p53 – and not lysine 20 of histone H4 – in the regulation of p53 responses.
We next addressed the role of SET8 in p53-dependent cell-cycle arrest and apoptosis. In the absence of DNA damage and consistent with a previous report (Julien and Herr, 2004), SET8 knock-down cells behave like control RNAi cells with respect to cell-cycle progression (Figure 6A). In contrast, upon DNA damage, SET8 knock-down renders cells more prone to apoptosis relative to control cells (9.9% Sub-G1 fraction in SET8 RNAi cells versus 3.1% for control cells; Figure 6A). Further, SET8 RNAi cells exhibit a higher fraction of cells in G1 than control cells (56.5% versus 38%, respectively; Figure 6A). These effects are strictly dependent on p53, as co-knock-down of p53 (see Figure 5D) eliminates the DNA damage-dependent phenotype of SET8 knock-down cells (Figure 6B). To further test whether the increased sensitivity of SET8 knock-down cells to DNA damage is mediated by p53, cell death induced by the DNA damage agent doxorubicin was also determined in the absence or presence of both SET8 and p53 knock-down. As shown in Figure 6C, whereas SET8 RNAi alone increases sensitivity of cells to DNA damage relative to control, this effect is abolished in a p53 knock-down background.
Here we have shown that the HMT enzyme SET8/PR-Set7 specifically monomethylates p53 at lysine 382 in vitro and in vivo. We have also provided the first mass spectrometry evidence that endogenous p53 is methylated (see Figure 2A). Ectopic expression of SET8 suppresses p53 transactivation activity and knock-down of endogenous SET8 by RNAi augments a number of p53 activities, including induction of highly responsive target genes, and increased apoptosis and cell-cycle arrest (see model, Figure 7). Numerous experiments indicate that these phenotypes are a consequence of SET8 methylation of p53, and not an indirect effect of H4K20 methylation by SET8. For example, in H1299 cells, SET8 represses ectopic p53, but fails to do so if p53 harbors a mutation at K382 (p53K382R) (Figure 4A and 4B). If SET8 repression of p53 target genes occurred via H4K20 methylation rather than p53K382 methylation, then the transactivation activity of wild-type and mutant p53K382R proteins should both be equally affected by SET8. As we do observe a requirement for K382 to be intact, we conclude that availability of this residue for methylation by SET8 is needed for SET8 to repress p53. Further evidence for a direct regulation of p53 by SET8 is that all SET8 RNAi phenotypes associated with DNA damage (e.g. increased p21 induction and cell death) are absolutely dependent on the presence of p53 protein. Thus, SET8, like a number of other p53-regulatory enzymes, such as TIP60, SIRT1, SET7/9 and HDAC1, utilizes both p53 and histones as substrates – with distinct functions specific to the different substrates (Sykes et al., 2006; Tang et al., 2006) (Chuikov et al., 2004; Luo et al., 2001; Luo et al., 2000; Vaquero et al., 2004; Vaziri et al., 2001; Wang et al., 2001).
We observe that p53K382me1 levels decrease with DNA damage. This observation, in conjunction with our functional characterization, argues that SET8 is a negative regulator of p53 activity. However, we do observe a SET8-dependent up-regulation of the DNA repair gene GADD45, indicating that p53K382 methylation might have more complex regulatory roles than simply contributing to on/off functions. In this regard, under normal conditions, monomethylation at K382 might render p53 largely inert but predisposed to respond to specific cellular stresses (Figure 7). Alternatively, this methylation event might dampen p53 as a mechanism for measured responses to mild insults, allowing for p53-dependent repair of DNA without the induction of cell death.
At the molecular level, how might K382 monomethylation be coupled to regulation of p53 functions? We postulate that the biological consequences of p53K382me1, alone, or in the context of additional modifications, might be dictated by distinct protein binding partners; different proteins can therefore define and channel p53 towards divergent activities. In this context, the MBT domain is a protein module found commonly on nuclear proteins and that has been shown to bind monomethyllysines (Kim et al., 2006), raising the possibility that a modular interaction between an MBT domain-containing protein and p53K382me1 acts to repress p53. There are several other methyllysine-binding domains that preferentially recognize higher methyl states than monomethyllysine (Daniel et al., 2005; Ruthenburg et al., 2007; Zhang, 2006). This raises the question of whether SET8 monomethylation of p53 at K382 leaves p53 poised to be further methylated in response to a specific signal, and that the higher methyl forms are linked to additional p53 functions (Figure 7). We have not detected methyltransferase activity on p53K382 by Suv4-20h1 and Suv4-20h2, two well-characterized H4K20 HMTs (X. Shi and O. Gozani, unpublished observations) (Schotta et al., 2004). In addition to these two enzymes, there are other putative H4K20 HMTs, such as ASH1 and NSD1, as well as numerous uncharacterized SET domain containing proteins, and perhaps one of these functions as a di- or trimethyltransferase at p53K382 (Beisel et al., 2002; Rayasam et al., 2003). In summary, this study reveals novel functions for the SET8 HMT in regulating the non-histone protein p53, and supports the hypothesis that protein lysine methylation likely modulates diverse cellular processes (Zhang and Dent, 2005).
SET8 cDNA was cloned into pcDNA3.1. p53 cDNA was cloned into pCAG-Flag and pGEX6p. SET8 and p53 mutants were generated by site-direct mutagenesis (Stratagene). Primer sequences are available upon request. αp53K382me1 antibody was generated in rabbits immunized with the monomethylated peptide: 377-TSRHKK(me)LMFKT-387. Other antibodies used in this study are: HRP-p53 (R&D systems); p53 (DO1) and p21 (EA10) (Calbiochem); p53K382ac (Cell Signaling); SET8, H4K20me1 and H3 (Abcam); p53(FL-393) and GST (E5) (Santa Cruz); Flag (M2) and tubulin (Sigma). p53 and histone peptides bearing different modifications were synthesized at the W.M. Keck Facility at Yale.
U2OS, H1299 and 293T were maintained in DMEM medium supplemented with 10% fetal bovine serum. Cells were transfected with plasmids or siRNA duplexes by TransIT-LT1 (Mirus) or DharmaFECT (Dharmacon), respectively, according to the manufacturers’ protocols.
Endogenous p53 or ectopically expressed Flag-tagged p53 were IPed with agarose conjugated p53 or Flag antibodies from whole cell extracts in cell lysis buffer (50 mM TrisHCl pH7.4, 250 mM NaCl, 0.5% Triton X100, 10% glycerol, 1 mM DTT, 1mM PMSF and protease inhibitors). After incubation at 4°C for overnight, the beads were washed 3X with the same buffer, and boiled in 2xLaemmli buffers. The IPed p53 was resolved on SDS-PAGE gel and detected by antibodies against state-specific p53 antibodies or HRP-αp53 to avoid crossreactivity with IgG heavy chain.
Methyltransferase assays were performed as previously described (Shi et al., 2006). Briefly, 2 μg of GST-p53, bulk histones, nucleosomes, or 1 μg of p53 or H4 peptides were incubated with 1μg of recombinant HMT and 0.1 mM S-adenosyl-methionine (SAM, Sigma), or 2 μCi 3H-SAM Amersham) in reaction buffer containing 50mM Tris-HCl pH 8.0, 10% glycerol, 20mM KCl, 5 mM MgCl2, 1 mM DTT and 1 mM PMSF, at 30°C for 30 min to 2hrs. The reaction mixtures were then subject to electrophoresis on SDS-PAGE, followed by either radiography or Westerns. The reactions with peptides were subject to mass spectrometry analysis.
Hela nuclear extracts were prepared as previously described (Gozani et al., 1994). To IP endogenous p53 proteins, ~10 mg of nuclear extracts were incubated with 50 μl of DO1-conjugate agarose in buffer containing 20 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.01% SDS, 1% Trition X 100, 1 mM EDTA and protease inhibitors with gentle rotation at 4°C for overnight. The beads were washed 2X with the same buffer, 2X with high salt buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 0.1% SDS, 1% Trition X 100, 2 mM EDTA), once with LiCl buffer (20 mM Tris, pH 8.0, 500 mM LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA), and once with TE buffer (10 mM TrisHCl 8.0, 1 mM EDTA). The p53 protein bound to the beads was subject to SDS-PAGE, excised from the gels, and incubated with trypsin overnight at 37 C. Pooled supernatants containing extracted peptides were dried and resuspended in 30% acetonitrile and 0.1% TFA prior to mass spectrometry analysis. Samples were analyzed on a reflectron time-of-flight mass spectrometer, MALDI-TOF instrument (Ultraflex, Bruker Daltonics, Billerica, MA), equipped with a 337 nm nitrogen laser and delayed ion extraction capability (delay times: 30–50 ns). Ion structure information was obtained by post source decay (PSD), using the mass gate feature, to select specific m/z window for fragmentation. The mass gate resolution was 1% of the precursor mass. Data was recorded in both positive and negative ion modes at 20kV acceleration, and mass analysis of ions determined using a dual micro-channel plate detector. Detector output was collected with a 1 GHz digitizer and displayed directly on a Windows NT based computer. Ten positive ion reflectron TOF mass spectra of 1000 laser shots were accumulated and externally calibrated with commercial peptide mix (Bruker Daltonics, Billerica, MA). For analysis of in vitro methylated synthetic peptides, the synthetic peptides untreated and treated with SET8 were equilibrated with 0.1% trifluoroacetic acid (TFA), and 50% acetonitrile with 0.1% TFA, and applied to the MALDI target plate with equal volumes of the matrix α-cyano-4-hydroxycinnamic acid (CHCA) (Sigma).
The knockdown of SET8 was performed by transfection of U2OS cells for 48 hrs with two sets of Dharmacon on-target plus siRNA duplex targeting human SET8 (5′-AGUCAAAGAUCUAUUCCUAUU-3′/5′-GCAACUAGAGAGACAAAUCUU-3′) or (5′-GGAAACCAUUAGCCGGAAUUU-3′/5′-GUACGGAGCGCCAUGAAGUUU-3′) respectively, or with on-target plus SMARTpool SET8 siRNA, by using DharmaFECT according to the manufacturer’s protocol. p53 knockdown was carried out with Dharmacon on-target plus p53 siRNAs (5′-GAAAUUUGCGUGUGGAGUAUU-3′/GUGCAGCUGUGGGUUGAUUUU-3′). On-target plus siControl siRNA (5′-UGGUUUACAUGUCGACUAA-3′, Dharmacon) or on-target plus SMARTpool siControl siRNA were used as controls.
RT-PCR and real-time PCR and ChIP assays were performed as previously described (Shi et al., 2006). Cells were treated with NCS (0.5 μg/ml, Sigma) for 2 to 4 hrs. mRNA was prepared using RNeasy plus kit (Qiagen) and reverse-transcribed using First Strand Synthesis kit (Invitrogen). Quantitative Real-time RT-PCR was performed on the ABI PRISM 7700 Sequence Detection System using Taqman Gene Expression Assay primer/probe sets (Applied Biosystems). Gene expressions were calculated following normalization to GAPDH levels by the comparative Ct (Cycle threshold) method. Primer sequences used for ChIP analyses are: p21 promoter: 5′-GTGGCTCTGATTGGCTTT-CTG-3′/5′-CTGAAAACAGGCAGCCCAAG-3′. PUMA promoter: 5′-GCGAGACTGTGGCCTTGTGT-3′/5′-CGTTCCAGGGTCCACAAAGT-3′. Other primer sequences are available upon request.
For flow cytometry, U2OS cells were collected and fixed with 70% ethanol at 4°C for 4 hrs to overnight. Cells were then washed with PBS for 3 times and stained with 10 μg/ml propidium iodide and 100 μg/ml RNase. Cell death assays were performed as previously described (Shi et al., 2006; Vaziri et al., 2001). Briefly, following treatment, cells were stained with trypan blue and the fraction of cells uptaking the dye was determined utilizing a hemocytometer.
We thank Y. Zhang for GST-SET7 construct, T. Jenuwein for Suv4-20h1 and Suv4-20h2 GST constructs and M. Bedford for SET8, G9a, Suv39h1, hDOT1L GST constructs and E. Michishita for Flag-p53 construct. This work was supported in part by grants from the NIH to O.G and by the Intramural Research Program of the NIH (E.A., H.Y.). O.G. is a recipient of a Burroughs Wellcome Career Award in Biomedical Sciences and a Kimmel Scholar Award.
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