MOLECULAR AND CELLULAR BIOLOGY, May 2010, p. 2170–2180 Vol. 30, No. 90270-7306/10/$12.00 doi:10.1128/MCB.01343-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Functional Interplay between Acetylation and Methylation ofthe RelA Subunit of NF-�B�

Xiao-Dong Yang,1 Emad Tajkhorshid,1,2 and Lin-Feng Chen1,2*Department of Biochemistry1 and College of Medicine,2 University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received 7 October 2009/Returned for modification 5 November 2009/Accepted 8 February 2010

Posttranslational modifications of the RelA subunit of NF-�B, including acetylation and methylation, playa key role in controlling the strength and duration of its nuclear activity. Whether these modifications arefunctionally linked is largely unknown. Here, we show that the acetylation of lysine 310 of RelA impairs theSet9-mediated methylation of lysines 314 and 315, which is important for the ubiquitination and degradationof chromatin-associated RelA. Abolishing the acetylation of lysine 310 either by the deacetylase SIRT1 or bymutating lysine 310 to arginine enhances methylation. Conversely, enhancing the acetylation of lysine 310 bydepleting SIRT1 or by replacing lysine 310 with acetyl-mimetic glutamine inhibits methylation, therebydecreasing ubiquitination, prolonging the stability of chromatin-associated RelA, and enhancing the tran-scriptional activity of NF-�B. The acetylation of lysine 310 of RelA interferes with its interaction with Set9.Based on structural modeling of the SET domain of Set9 with RelA, we propose that the positive charge oflysine 310 is critical for the binding of RelA to a negatively charged “exosite” within the SET domain of Set9.Together, these findings demonstrate for the first time an interplay between RelA acetylation and methylationand also provide a novel mechanism for the regulation of lysine methylation by acetylation.

In several cases an interplay between different posttransla-tional modifications has been identified for histone and non-histone proteins, with one modification either enhancing orinhibiting another modification (11, 37, 42). For example, thephosphorylation of serine 10 of histone H3 interferes with themethylation of lysine 9 and stimulates the acetylation of lysine14 (4, 5, 23, 24). The methylation of lysine 372 of p53 promotesits acetylation (19, 21). These effects can be direct, in whichone modification alters the conformation of the protein,thereby influencing the second modification, or can be indirect,in which the first modification results in the recruitment of aneffector, which alters the other modification. The interplaysbetween these modifications together with the distinct combi-nations of covalent modifications form the basis of the “histonecode” and, probably, the “protein code” hypotheses (20, 35).

The inducible transcription factor NF-�B plays an importantrole in regulating inflammatory responses, apoptosis, cell prolif-eration and differentiation, and tumorigenesis (12). The proto-typical NF-�B complex, a heterodimer of p50 and RelA, is se-questered in the cytoplasm by its assembly with the inhibitorI�B�. Upon stimulation, the I�B kinase (IKK) complex is acti-vated, leading to the phosphorylation and degradation of I�B�,the nuclear translocation of NF-�B, and the activation of its targetgenes (12). Once in the nucleus, the RelA subunit of NF-�Bundergoes a series of stimulus-coupled posttranslational modifi-cations, including phosphorylation, acetylation, and methylation.These modifications impose various effects on nuclear NF-�B,regulating both the strength and duration of NF-�B activity (1,28, 40).

RelA is acetylated at a number of lysine residues by p300/CBP or PCAF. Acetylation at different lysines regulates dis-tinct functions of NF-�B, including its association with I�B�,DNA binding, and transcriptional activation (1). Among all theacetylation sites, lysine 310 is the most well studied, as itsacetylation is required for the full transcriptional potential ofNF-�B and is important for modulating NF-�B-dependent in-flammatory responses (13, 17, 18, 39) and for maintainingconstitutive NF-�B activity in tumors (2, 6, 22). Acetylatedlysine 310 enhances the transcriptional activation of NF-�B bycreating a docking site for the recruitment of the bromodo-main-containing factor Brd4 to activate CDK9 and the RNApolymerase II-mediated transcription of NF-�B target genes(16). The abolishment of the acetylation of lysine 310, either bymutating lysine 310 to arginine or by histone deacetylases(HDACs), significantly inhibits the transactivation of NF-�Band sensitizes cells to tumor necrosis factor alpha (TNF-�)-induced apoptosis (2, 43). Interestingly, cross talk betweenphosphorylation and acetylation is found for RelA. The phos-phorylation of serine 536 in the transactivation domain ofRelA enhances the acetylation of lysine 310 of RelA by in-creasing the recruitment of p300 and the dissociation of thecorepressor SMRT1 and increases the overall transcriptionalactivity of NF-�B (3, 14, 32, 38).

In addition to acetylation, our recent studies demonstratedthat RelA is also subject to lysine methylation. RelA ismonomethylated by the lysine methyltransferase Set9 (alsocalled Set7 or KMT7) at lysines 314 and 315 in vitro and in vivoin response to stimulation (40). The methylation of RelA atthese two residues negatively regulates the functions of NF-�Bby triggering the ubiquitination and proteasome-mediated deg-radation of promoter-associated RelA. The methylation ofRelA serves as a “death” signal for the destruction of DNA-bound activated NF-�B (40). However, it remains unclear howmethylation is regulated and whether a cross-reaction also

* Corresponding author. Mailing address: Department of Biochem-istry, College of Medicine, MC-714, University of Illinois at Urbana-Champaign, Urbana, IL 61801. Phone: (217) 333-7764. Fax: (217)244-5858. E-mail: lfchen@life.uiuc.edu.

� Published ahead of print on 16 February 2010.

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exists for RelA methylation and acetylation, which imposeopposite effects on the transcriptional activation of NF-�B.

In this study, we have explored the possible interplay be-tween the acetylation and methylation of RelA, especiallythe involvement of the acetylation of lysine 310 in regulat-ing the methylation of lysines 314 and 315. We demonstratethat the acetylation of lysine 310 of RelA inhibits the methyl-ation of lysines 314 and 315 and the subsequent ubiquitinationand degradation of promoter-associated RelA. Furthermore,we provide evidence that acetylated lysine 310 interferes withthe assembly of RelA with Set9, likely through destroying di-rect contact between the positively charged lysine 310 and anegatively charged “exosite” of the SET domain of Set9.

MATERIALS AND METHODS

Cell lines, plasmids, and antibodies. Human U2OS, HEK293T, and mouseembryonic fibroblast (MEF) cells were cultured in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% fetal bovine serum (FBS). RelA-deficient MEFs reconstituted with wild-type (WT) RelA or RelA-K314/315R,RelA-K310R, and RelA-K310Q mutants were generated as previously described(40). T7-RelA, Flag-Set9, and Flag-Set9-H279A plasmids and a bacterial expres-sion vector for His-Set9 were used as previously described (40). An expressionvector for SIRT1 small hairpin RNA (shRNA) was kindly provided by K. Pruitt(Louisiana State University Health Sciences Center). RelA point mutations weregenerated by site-directed mutagenesis (Stratagene). Recombinant RelA andHis-Set9 proteins were purified as previously described (40). Anti-RelA (C20and F6), anti-Set9 (s4E5), antiubiquitin, and anti-Flag antibodies were obtainedfrom Santa Cruz Biotechnology Inc.; Flag agarose beads (M2) and anti-SIRT1and anti-tubulin antibodies were obtained from Sigma. Anti-Set9 (07-314) andanti-T7 antibodies were obtained from Upstate and Novagen, respectively. Anti-acetylated lysine 310 antibodies were kindly provided by W. C. Greene (3).Polyclonal antibodies against monomethylated lysine 314/315 RelA (anti-Me-K314/315) were generated by New England Peptide with a synthesized peptidecorresponding to amino acids (aa) 308 to 320 of RelA (NH2-TFKSIMK[Me]K[Me]SPFSGC-COOH) as the antigen.

Peptide separation and detection. Peptides corresponding to amino acids 305to 324 of RelA (TYETFKSIMKKSPFSGPTDP) or aa 308 to 320 of RelA(TFKSIMKKSPFSG), including unmodified, K314/315-monomethylated, orK310-acetylated and K314/315-monomethylated peptides, were chemically syn-thesized by Cambridge Research Biochemicals or New England Peptide, respec-tively. For autoradiography, in vitro-methylated or -acetylated peptides wereboiled in 2� SDS sample buffer and separated by 15% Tris-Tricine PAGE asdescribed previously (33). The dried gel was then exposed to X-ray film. For dotblot analysis, peptides were dissolved and diluted in distilled water to differentconcentrations and spotted onto a nitrocellulose membrane. The air-dried mem-brane was then subjected to immunoblot analysis.

In vitro methylation and acetylation. The in vitro methylation of recombinantRelA and peptides by Set9 was performed as previously described (27, 40).Methylation or acetylation was visualized either by autoradiography or by im-munoblotting with anti-Me-K314/315 or -Ac-K310 antibodies, respectively.

TNF-�-induced methylation and acetylation. Cells were stimulated withTNF-� (20 ng/ml) for the indicated time periods, immediately washed with coldphosphate-buffered saline (PBS), and lysed as described previously (40). Anti-Me-K314/315 or -Ac-K310 antibodies were used to immunoprecipitate methyl-ated or acetylated RelA, respectively. Methylation and acetylation levels wereassessed by immunoblotting of immunoprecipitates with anti-RelA monoclonalantibodies.

ChIP. U2OS cells were left unstimulated or stimulated with TNF-� (20 ng/ml)for 30 or 60 min. A chromatin immunoprecipitation (ChIP) assay using anti-Me-K314/315 RelA antibodies was performed as previously described (3), and thelevels of methylated RelA on interleukin-6 (IL-6) and IL-8 promoters wereassessed by quantitative real-time PCR. The sequence of ChIP primers will beprovided upon request.

RelA stability and ubiquitination. For RelA stability assays, 2 � 106 cells werestimulated with TNF-� (20 ng/ml) for 15 min and then chased in DMEMcontaining cycloheximide (CHX) (10 �g/ml) for various time periods. Aftertreatment, cells were immediately washed with cold 1� PBS buffer, and chro-matin-associated proteins were prepared as described previously (40) and im-munoblotted for RelA. To assess RelA ubiquitination, 2 � 106 cells were stim-

ulated with TNF-� for 15 min, washed with PBS, and then treated with 20 �MMG132 for 4 h. Chromatin-associated proteins were diluted with chromatinextraction buffer without NaCl into a buffer containing 200 mM NaCl andprecleared with protein A beads for 30 min. Finally, the supernatant was immu-noprecipitated with anti-RelA agarose beads and immunoblotted for ubiquitin.

Peptide pulldown. A total of 0.5 �g unmodified or K310-acetylated RelApeptides (aa 305 to 324) was incubated with 1 �g recombinant His-tagged Set9protein in binding buffer (50 mM Tris [pH 9.0], 150 mM NaCl, 0.5 mM dithio-threitol [DTT], 1 mM phenylmethylsulfonyl fluoride) for 30 min at 4°C. Ni-nitrilotriacetic acid (NTA) agarose beads were added for 30 min and washedthree times with the binding buffer. The association of peptides with Set9 wasassessed by Tris-Tricine gel electrophoresis followed by silver staining.

Molecular modeling and simulation. The crystal structures of the complexes ofER� and Set9 (36) and of p53 and Set9 (7) were used to develop a structuralmodel for the RelA/Set9 complex. The sequence of the RelA peptide was thenthreaded over the structure of the peptide substrate in the complex. The ob-tained structure was subjected to energy minimization and structural relaxationin order to remove the overlap between the side chains of the modeled peptideand Set9. The model was then briefly simulated while constraining the side-chainnitrogen atom of lysine 310 to the position of arginine 300 in the ER� peptide.The final structure was then simulated briefly to ensure the conformationalstability of the model. The energy minimization and the molecular dynamicsimulations were performed using the program NAMD2 (30) and theCHARMM22 set of force field parameters (25). During all the optimizationsteps and the performed simulations, all the heavy atoms of Set9 as well as thosein the backbone of the peptide were kept fixed in order to constrain them to theconformations observed in the crystal structure. As such, only the side chains ofthe modeled peptide were conformationally manipulated during these steps.

RESULTS

TNF-� stimulates the methylation of RelA at lysines 314and 315. Our previous studies demonstrated that RelA ismonomethylated by Set9 at lysines 314 and 315 (40). To betterunderstand the methylation of endogenous RelA and its reg-ulation, we generated a site-specific anti-monomethylated ly-sine 314/315 antibody by immunizing rabbits with a RelA pep-tide monomethylated at lysines 314 and 315. First, we used thisantibody to determine if it detects in vitro-methylated RelA bySet9. Immunoblotting revealed a reactivity of this antibodywith wild-type RelA in the presence of Set9 (Fig. 1A, lane 2).However, no methylation signal was observed when lysines 314and 315 were mutated to arginines (RelA-K314/315R) or whenthe catalytically inactive mutant of Set9 (Set9-H297A) wasused (Fig. 1A). Similarly, when we tested the reactivity of theantibody for in vivo-methylated RelA, we found that RelA wasmethylated on lysines 314 and 315 in the presence of wild-typeSet9 but not Set9-H297A (Fig. 1B). Furthermore, RelA-K314/315R displayed no reactivity with this antibody (Fig. 1B).These results confirm that Set9 monomethylates RelA on ly-sines 314 and 315 both in vitro and in vivo.

With this antibody, we next examined the methylation ofendogenous RelA. Immunoprecipitation of methylated RelAwith anti-methylated lysine 314/315 RelA antibodies followedby immunoblotting with an anti-RelA antibody revealed thatendogenous RelA was unmethylated in unstimulated cells, andTNF-� stimulation led to a substantial methylation of RelA(Fig. 1C). The stimulus-dependent RelA methylation requiredendogenous Set9, as the depletion of Set9 by small interferingRNA (siRNA) significantly reduced the TNF-�-induced meth-ylation of RelA (Fig. 1C). More importantly, when the TNF-�-induced methylation of RelA was examined in RelA-defi-cient mouse embryonic fibroblasts (MEFs) reconstituted witheither WT RelA or RelA-K314/315R, the methylation of RelAwas completely abolished in RelA-K314/315R-reconstituted

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MEFs (Fig. 1D). These results further confirm that Set9monomethylates RelA at lysines 314 and 315 in vivo in re-sponse to TNF-�.

Since TNF-� stimulates the recruitment of Set9 to the pro-moters of NF-�B target genes (40), we next assessed the meth-ylation of RelA on the promoters of NF-�B target genes bychromatin immunoprecipitation (ChIP) assays with quantita-tion by real-time PCR. In the absence of stimulation, there wasno detectable methylated RelA on the IL-8 or IL-6 promoter(Fig. 1E). However, TNF-� stimulated the methylation ofRelA on both promoters at 30 and 60 min, although the levelsof methylated RelA on the different promoters varied (Fig.1E). These results support the conclusion that lysines 314 and315 of RelA are inducibly methylated following TNF-� stim-ulation and that the Set9-mediated methylation of RelA playsan important role in regulating nuclear NF-�B function (40).

Acetylation of lysine 310 inhibits the methylation of lysines314 and 315 in vitro. To explore the possible interplay betweenthe RelA acetylation of lysine 310 and the methylation oflysines 314 and 315, which are only a few amino acids apart(Fig. 2A), we first examined the effect of the methylation oflysines 314 and 315 on the acetylation of lysine 310. In an invitro acetylation assay using RelA peptides as substrates, meth-ylated peptides were acetylated by p300 to the same extent asunmethylated peptides (Fig. 2B), indicating that methylationdoes not affect the acetylation of lysine 310. However, whenunacetylated and acetylated lysine 310 RelA peptides wereused as substrates in an in vitro methylation assay, there wassubstantial Set9-mediated methylation of unacetylated pep-tides, while the methylation of acetylated peptides was barelydetectable (Fig. 2C), suggesting a direct inhibition of methyl-ation by the acetylation of lysine 310.

Next, we investigated whether the acetylation of lysine 310inhibits the methylation of RelA in the context of the full-length protein. Consistent with the data for the RelA peptides,the prior acetylation of full-length RelA at lysine 310 by p300inhibited the methylation of lysines 314 and 315, visualizedeither by tritium labeling or by immunoblotting with anti-meth-ylated lysine 314/315 RelA antibodies (Fig. 2D). However, theprior methylation of full-length RelA at lysines 314 and 315 bySet9 did not affect the acetylation of lysine 310 (Fig. 2E).

To exclude the possibility that the reduced methylation ofacetylated RelA (Fig. 2D, lane 3) is due to the inability of theantibody to access methylated lysines 314 and 315 when lysine310 is concomitantly acetylated, we synthesized various RelApeptides, including an unmodified peptide, one with only ly-sines 314 and 315 methylated, and one with both lysines 314and 315 methylated and lysine 310 acetylated, and immuno-blotted them with anti-acetylated or anti-methylated antibod-ies. Anti-methylated lysine 314/315 RelA antibodies were ableto recognize methylated lysines 314 and 315 regardless of theacetylation status of lysine 310 (Fig. 2F, top), indicating thatthe acetylation of lysine 310 does not block the access of the

FIG. 1. Set9 methylates lysines 314 and 315 on promoters of NF-�Btarget genes. (A) Recombinant proteins of WT RelA or RelA-K314/315R were incubated with recombinant WT Set9 or Set9-H297A in anin vitro methylation assay. Reaction products were separated by SDS-polyacrylamide gel electrophoresis, and methylation was detected byimmunoblotting (IB) with anti-methylated lysine 314/315 antibodies(top). Levels of RelA, RelA-K314/315R, Set9, and Set9-H297A areshown at the bottom. (B) HEK293T cells were cotransfected withexpression vectors encoding WT RelA or RelA-K314/315R and Set9or Set9-H297A, as indicated. Methylation was detected by immuno-blotting of anti-T7 immunoprecipitates (IP) with anti-methylated ly-sine 314/315 antibodies. Levels of T7-RelA, RelA-K314/315R, Set9,and Set9-H297A are shown at the bottom. (C) U2OS cells were trans-fected with control (Ctr) siRNA or Set9 siRNA and stimulated withTNF-� as indicated. Methylation of RelA was assessed by immuno-blotting the anti-Me-K314/315 RelA immunoprecipitates with RelAantibody. (D) RelA-deficient MEFs reconstituted with empty vector,WT RelA, or RelA-K314/315R were untreated or stimulated withTNF-� (20 ng/ml) for 30 min, and RelA methylation was assessed asdescribed above (C). (E) U2OS cells were stimulated with TNF-� as

indicated. ChIP assays with anti-Me-K314/315 RelA antibody wereperformed, and RelA methylation at the promoters of IL-8 and IL-6was assessed by quantitative real-time PCR. Results from three inde-pendent experiments are shown as means � standard deviations (SD).

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antibodies to methylated lysines 314 and 315. Anti-acetylatedlysine 310 RelA antibodies were also able to recognize acety-lated lysine 310 when lysines 314 and 315 were methylated(Fig. 2F, middle). Together, these data demonstrate a unidi-rectional regulation; namely, the acetylation of lysine 310 in-hibits the methylation of RelA on lysines 314 and 315, butmethylation has no effect on the acetylation of lysine 310.

Acetylation of lysine 310 inhibits the methylation of lysines314 and 315 in vivo. To evaluate the role of acetylation onmethylation in vivo, we first examined the effect of p300 on themethylation of RelA. When wild-type p300 or its histoneacetyltransferase (HAT) domain deletion mutant (p300-

�HAT) was transfected into HEK293T cells and TNF-�-in-duced acetylation and methylation were measured, we foundthat the level of TNF-�-induced acetylation of lysine 310 wasenhanced in the presence of wild-type p300 but was reduced inthe presence of p300-�HAT (Fig. 3A). Conversely, the level ofTNF-�-induced methylation of lysines 314 and 315 was de-creased in the presence of p300 but increased in the presenceof p300-�HAT (Fig. 3A). These data indicate that lysine 310acetylation of RelA might inhibit the methylation of lysines 314and 315 in vivo.

Since SIRT1 specifically deacetylates lysine 310 of RelA(43), we next determined the effect of the SIRT1-mediated

FIG. 2. Acetylation of RelA at lysine 310 inhibits methylation of RelA at lysines 314 and 315 in vitro. (A) Schematic domain structure of RelAand the sequences surrounding the acetylation site (lysine 310) and methylation sites (lysines 314 and 315). NLS, nuclear localization signal. RHD,Rel homology domain; TA, transactivation domain. (B) RelA peptides (aa 308 to 320) unmodified (UM) or methylated (Me) at lysines 314 and315 were in vitro acetylated by p300 in the presence of [14C]acetyl coenzyme A (CoA). Samples were separated in a Tris-Tricine gel, and acetylationlevels and peptide loading amounts were assessed by autoradiography and silver staining, respectively. (C) RelA peptides (aa 305 to 324)unmodified (UA) or acetylated (Ac) at lysine 310 were in vitro methylated by Set9 in the presence of [3H]S-adenosine-methionine (SAM). Sampleswere separated in a Tris-Tricine gel, and methylation levels and peptide loading amounts were assessed as described above (B). (D) Full-lengthRelA was in vitro acetylated by p300 in the absence or presence of acetyl-CoA and then subjected to a Set9-mediated in vitro methylation assayin the presence of [3H]SAM. Samples were separated by SDS-PAGE, and levels of methylated RelA were assessed by either autoradiography orimmunoblotting with anti-methylated lysine 314/315 antibodies. Levels of acetylated RelA and total RelA are shown at the bottom. (E) Unmodifiedor in vitro-methylated recombinant full-length RelA was subjected to a p300-mediated in vitro acetylation assay. Samples were separated bySDS-PAGE; levels of acetylated RelA, methylated RelA, or total RelA were assessed by immunoblotting as indicated. (F) A series of dilutionsof RelA peptides (aa 308 to 320), including unmodified, monomethylated at lysines 314 and 315 (Me-K314/315), or acetylated at lysine 310 andmonomethylated at lysines 314 and 315 (Ac-K310/Me-K314/315), were spotted onto a nitrocellulose membrane. Dot blot analysis was performedwith antibodies as indicated. The Ponceau S staining of the same membrane served as the peptide loading control.

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deacetylation of lysine 310 on the methylation of RelA. TheTNF-�-induced acetylation of lysine 310 was impaired whenSIRT1 was expressed in HEK293T cells (Fig. 3B), confirmingthat SIRT1 deacetylates lysine 310 in vivo. Consistent with thenotion that the acetylation of lysine 310 inhibits the methyl-ation of lysines 314 and 315, the TNF-�-induced methylationof RelA at lysines 314 and 315 was enhanced (Fig. 3B). Incontrast, when the enzymatically inactive form of SIRT1(H363Y), which likely functions as a dominant negative toinhibit the activity of endogenous SIRT1, was expressed in thecells, the TNF-�-induced acetylation of lysine 310 was en-hanced, and as a result, the level of methylation of lysines 314and 315 was decreased (Fig. 3B). To confirm the effect ofSIRT1 on RelA methylation, we knocked down the expressionof SIRT1 in HEK293T cells with an shRNA against SIRT1(31). The depletion of SIRT1 by shRNA enhanced the TNF-�-induced acetylation of lysine 310 (Fig. 3C) and consistentlydecreased the methylation of lysines 314 and 315 (Fig. 3C).Collectively, these data indicate that the acetylation of lysine310 interferes with the methylation of lysines 314 and 315 invivo.

To explore the potential functional consequence of the in-terference, we examined the transcriptional activity of NF-�Bin SIRT1-knocked-down cells using a �B-luciferase reporterassay. Compared to cells transfected with control shRNA, cellstransfected with SIRT1 shRNA displayed a higher level ofTNF-�-induced NF-�B activity (Fig. 3D), suggesting that in-terfering with the methylation of lysines 314 and 315 by acety-lated lysine 310 might enhance the transcriptional potential ofNF-�B.

Replacement of lysine 310 with acetyl-mimetic glutamineinhibits the methylation of lysines 314 and 315 in vivo. Toassess whether the effects from SIRT1 and p300 are deriveddirectly from the acetylation of lysine 310, we generated RelAmutants with lysine 310 replaced with arginine or with theacetyl-mimetic glutamine (designated RelA-K310R and RelA-K310Q, respectively). When the Set9-mediated methylationsof these different RelA mutants were examined in vivo, wefound that RelA-K310Q displayed a much lower methylationsignal, whereas RelA-K310R displayed a higher methylationlevel (Fig. 4A), consistent with the findings that the acetylationof lysine 310 of RelA inhibits the methylation of lysines 314and 315 (Fig. 2 and 3). Interestingly, the methylation levels ofRelA mutants correlated with their abilities to interact withSet9. While RelA-K310Q reduced its binding to Set9, RelA-K310R increased its binding to Set9 (Fig. 4A), indicating thatthe acetylation status of RelA might regulate its associationwith Set9.

To investigate the effect of different lysine 310 mutations onthe TNF-�-induced methylation of RelA in vivo, we tested the

FIG. 3. Acetylation at lysine 310 inhibits methylation of RelA invivo. (A) HEK293T cells were transfected with empty vector or ex-pression vectors for p300 or the p300-�HAT mutant. At 30 h post-transfection, cells were treated with TNF-� for the indicated timeperiods. Levels of methylated or acetylated RelA were assessed byimmunoblotting anti-methylated lysine 314/315 or anti-acetylated ly-sine 310 immunoprecipitates, respectively, with RelA antibodies. Thequantitation of methylated or acetylated RelA (fold increase or de-crease relative to levels of RelA shown at the bottom) is indicatedbelow each sample. (B) HEK293T cells were transfected with an emptyvector or expression vectors for Flag-SIRT1 or the Flag-SIRT1(H363Y) mutant. At 30 h posttransfection, cells were treated withTNF-� for the indicated time periods. Levels of methylated or acety-lated RelA were assessed as described above (A). (C) HEK293T cellswere transfected with an empty vector or expression vectors for SIRT1shRNA. TNF-�-induced RelA methylation at K314/315 and acetyla-tion at K310 were assessed as described above (A). Levels of RelA and

SIRT1 are shown. (D) HEK293T cells were transfected with an emptyvector or expression vectors for SIRT1 shRNA; 24 h posttransfection,cells were retransfected with 5� �B and Renilla luciferase reporterplasmids. Another 24 h later, cells were treated with TNF-� (20 ng/ml)for 5 h, and luciferase activity was measured. Results represent theaverages of data from three independent experiments � SD. **, P �0.01. Expression levels of SIRT1 and tubulin are shown at the bottom.

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methylation of RelA in RelA-deficient MEFs reconstitutedwith these different RelA mutants. TNF-� stimulated themethylation of RelA in WT RelA-reconstituted MEFs (Fig.4B). However, RelA methylation was significantly impaired inRelA-K310Q-reconstituted MEFs (Fig. 4B) but enhanced inRelA-K310R-reconstituted MEFs. These data further supportthe conclusion that the acetylation of lysine 310 inhibits the

methylation of lysines 314 and 315 under physiological condi-tions in response to a stimulus.

Since the transcriptional potential of NF-�B appears to beincreased when the acetylation of RelA interferes with itsmethylation (Fig. 3D), we next examined TNF-�-inducedNF-�B activation in various reconstituted MEFs. Similar to thefindings shown in Fig. 3D, the transcriptional activity of eachmutant inversely correlated with its methylation level. TheTNF-�-stimulated activation of NF-�B was enhanced in RelA-K310Q-reconstituted MEFs (Fig. 4C), which displayed lowerlevels of TNF-�-induced RelA methylation (Fig. 4B). How-ever, in RelA-K310R-reconstituted MEFs, which displayedhigher levels of TNF-�-induced RelA methylation (Fig. 4B),the level of TNF-�-induced activation of NF-�B was decreased(Fig. 4C). These data, together with the findings shown in Fig.3D, suggest that the inhibition of the methylation of RelA byacetylation enhances the transcriptional potential of NF-�B.

Acetylation of lysine 310 enhances the stability of RelA. Ourprevious studies demonstrated that the methylation of RelA bySet9 triggers the ubiquitination and degradation of chromatin-associated RelA (40). We next determined whether an en-hanced acetylation of RelA might decrease the ubiquitinationof RelA and therefore enhance its stability. To explore thispossibility, we first examined the ubiquitination of RelA inSIRT1-deficient MEFs. TNF-� stimulated the ubiquitination ofchromatin-associated RelA in WT MEFs (Fig. 5A). However, inSIRT1-deficient MEFs, the ubiquitination of RelA was reduced(Fig. 5A, lane 2). Furthermore, when the TNF-�-induced ubiq-uitination of RelA in RelA-K310Q- or RelA-K310R-reconsti-tuted MEFs was examined, we observed that the ubiquitination ofRelA was remarkably reduced in RelA-K310Q-reconstitutedMEFs but was slightly enhanced in RelA-K310R-reconstitutedMEFs (Fig. 5B).

Next, we investigated the stability of chromatin-associatedRelA in SIRT1-deficient MEFs. SIRT1-deficient MEFs werefirst pulse stimulated with TNF-� for 15 min, and cyclohexi-mide (CHX) was then added to the cells to prevent the resyn-thesis of I�B�, since resynthesized I�B� is able to bind NF-�Band remove it from DNA (12). When WT MEFs were pulsestimulated with TNF-� and the level of chromatin-associatedRelA was examined, we found that chromatin-associated RelAwas gradually degraded (Fig. 5C). In contrast, chromatin-as-sociated RelA was more stable in SIRT1-deficient MEFs.These data are consistent with the finding that the inhibition ofSIRT1 reduces the ubiquitination of chromatin-associatedRelA (Fig. 5A).

We also examined the stability of various RelA lysine 310mutants in reconstituted MEFs. Correlating with their ubiq-uitination levels, the stability of chromatin-bound RelA-K310Q was enhanced after pulse stimulation with TNF-� inthe presence of CHX, and the stability of RelA-K310R wasslightly reduced (Fig. 5D). Together, these data demonstratethat the acetylation of lysine 310 interferes with the methyl-ation of lysines 314 and 315, increasing the stability of RelA byreducing its ubiquitination.

Acetylation of lysine 310 impairs the association of Set9 withRelA. The decreased or increased interaction of RelA-K310Qor RelA-K310R, respectively, with Set9 (Fig. 4A) prompted usto investigate whether the acetylation status of lysine 310 reg-ulated the assembly of the complex of RelA and Set9. First, we

FIG. 4. Mutation of lysine 310 regulates methylation of RelA invivo. (A) HEK293T cells were transfected with expression vectors forT7-tagged WT RelA, RelA-K310Q, or RelA-K310R together withgreen fluorescent protein (GFP)-Set9. The methylation of RelA wasassessed as described in the legend of Fig. 1B. RelA-associated Set9was detected by immunoblotting of the RelA immunoprecipitates withanti-GFP antibodies. Levels of RelA and Set9 are shown in the bottomtwo panels. The quantitation of methylated RelA (fold increase ordecrease relative to levels of RelA shown at the bottom) is indicatedbelow each sample. (B) RelA-deficient MEFs reconstituted with WTRelA, RelA-K310Q, or RelA-K310R were stimulated with TNF-� asindicated. Levels of methylated RelA were determined as described inthe legend of Fig. 1C. The quantitation of methylated RelA (foldincrease or decrease relative to levels of RelA shown at the bottom) isindicated below each sample. (C) RelA-deficient MEFs reconstitutedwith WT RelA, RelA-K310Q, or RelA-K310R were transfected with5� �B and Renilla luciferase reporter plasmids and stimulated withTNF-� 24 h after transfection. Luciferase activity was measured 5 hafter stimulation. Results represent the averages of data from threeindependent experiments � SD. *, P � 0.05.

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examined the binding of Set9 to the unacetylated or acetylatedlysine 310 RelA peptides. For these assays, recombinant His-tagged Set9 was incubated with RelA peptides with or withoutacetylated lysine 310. When Set9-associated peptides were vi-sualized by silver staining, significant amounts of unmodifiedRelA peptides were pulled down by Set9 (Fig. 6A), whereasfew acetylated RelA peptides were pulled down by Set9. Thesefindings further support the notion that the acetylation of ly-sine 310 impairs the interaction of RelA with Set9.

The effect of the acetylation of lysine 310 on the recruitmentof Set9 was also evaluated with coimmunoprecipitation exper-iments with RelA-deficient MEFs reconstituted with WTRelA, RelA-K310Q, or RelA-K310R. After treatment withTNF-�, RelA interacted with Set9 in WT RelA-reconstitutedMEFs (Fig. 6B). However, the TNF-�-induced association be-tween RelA and Set9 was reduced in RelA-K310Q-reconsti-tuted MEFs and increased in RelA-K310R-reconstitutedMEFs (Fig. 6B). Taken together, these results support a model

in which the acetylation of lysine 310 interferes with the effec-tive assembly of RelA with Set9.

Positively charged lysine 310 interacts with an “exosite” ofthe SET domain of Set9. To understand the detailed molecularmechanism underlying the inhibitory effect of the acetylationof lysine 310 on the assembly of RelA with Set9, we employedcomputational modeling to examine the interaction betweenRelA and Set9. Several crystal structures of the SET domain ofSet9 in complex with its substrates have been determined thusfar (7, 36). Taking advantage of these existing structures (thecomplex of ER� and Set9 and that of p53 and Set9), wedeveloped a molecular model for Set9 in complex with theRelA peptide (Fig. 6C). In this model, as expected, the meth-ylation site (lysine 314 or 315) in the peptide is in close contactwith the active site of Set9 (Fig. 6C). In addition to the meth-ylation site, we identified a close contact between the enzymeand the RelA substrate involving lysine 310. The contact at thissite is furnished by a salt bridge interaction between the basic

FIG. 5. Acetylation regulates the ubiquitination and stability of RelA. (A) WT or SIRT1-deficient MEFs were pulse stimulated with TNF-� for15 min, followed by treatment with the proteasome inhibitor MG132 for 4 h. Levels of ubiquitinated RelA were assessed by immunoblotting theRelA immunoprecipitates prepared from chromatin-associated proteins with antiubiquitin (Ubn) antibodies. Levels of RelA, SIRT1, and tubulinare shown at the bottom. The quantitation of ubiquitinated RelA (fold increase or decrease relative to levels of RelA shown at the bottom) isindicated below each sample. (B) TNF-�-induced ubiquitination of RelA in RelA-deficient MEFs reconstituted with WT RelA, RelA-K310Q, orRelA-K310R was measured and quantitated as described above (A). Levels of RelA from different cells are shown at the bottom. (C) WT orSIRT1-deficient MEFs were pulse stimulated with TNF-� for 15 min, followed by a chase in the presence of 10 �g/ml cycloheximide (CHX). Cellswere harvested at the indicated time points, and chromatin-associated proteins were extracted and immunoblotted for RelA. A typical result isshown at the top. The quantification of the results from three independent experiments are shown as means � SD at the bottom. (D) Stability ofRelA from RelA-deficient MEFs reconstituted with WT RelA, RelA-K310Q, or RelA-K310R was assessed as described above (C).

2176 YANG ET AL. MOL. CELL. BIOL.

side chain of lysine 310 and a clamp-like region, which is richin acidic (negative) residues, including aspartic acids 256, 259,and 338 and glutamic acid 348 (shown as red patches in Fig.6C). This negatively charged clamp likely acts as an “exosite”in the enzyme, increasing the binding affinity of the substrate.According to this model, any modification of the positivecharge carried by lysine 310 in RelA will have an effect on itsbinding to Set9 and, thus, on its methylation. This model suc-cessfully accounts for the inhibitory effect of the acetylation oflysine 310 on methylation, as the main effect of acetylation isthe neutralization of the lysine side chain. Lysine 310 is theonly positively charged amino acid available in RelA in theregion close to the exosite in the enzyme, so the replacement oflysine 310 with glutamine has a similar neutralization effect.

Conversely, not only is an arginine side chain replacing lysine310 capable of establishing similar salt bridge interactions withthe exosite, it likely does so more extensively due to its largerterminal group. Since the clamp-like exosite region in the en-zyme is large enough to accommodate either lysine or arginineside chains, steric hindrance is not a concern. Therefore, acet-ylation of lysine 310 is predicted to interfere with RelA’s as-sociation with the exosite of Set9, thus resulting in the de-creased methylation of RelA.

DISCUSSION

In this study, we demonstrate a functional interplay betweenRelA acetylation and RelA methylation. By reducing the bind-

FIG. 6. Acetylation of lysine 310 of RelA inhibits its binding to Set9. (A) RelA peptides (aa 308 to 324) that were unmodified (unacetylated[UA])or acetylated at lysine 310 were incubated with recombinant His-tagged Set9 for 30 min. Set9 was recovered with Ni-NTA agarose beads andassessed by immunoblotting. Set9-associated peptides were separated in a Tris-Tricine gel, followed by silver staining. (B) WT RelA-, RelA-K310Q-, or RelA-K310R-reconstituted MEFs were left untreated or stimulated with TNF-� for 30 min. Set9 immunoprecipitates prepared fromwhole-cell lysates were immunoblotted for RelA. Levels of RelA and Set9 are shown in the bottom two panels. (C) Structural model of the complexof the RelA peptide and Set9 constructed using complexes of Set9 with p53 and Set9 with ER�. (Left) The enzyme is shown in a surfacerepresentation with the residues in red (acidic), blue (basic), green (polar, noncharged), and white (nonpolar). The backbone of the peptide(common for RelA and “the other peptide”) and the side chains of key residues in the model are shown explicitly and labeled. The methylatedlysines (lysines 314 and 315 in RelA and lysine 302 in ER�) are shown in a stick representation and are colored based on atom types (carbon incyan and nitrogen in blue). The model shows how Lys310 in RelA can occupy the same position as Arg300 in ER� (shown using a transparentstick representation), which is clamped by several negatively charged residues (red patches) from the Set9 enzyme. (Top right) Slightly rotated viewof the two peptides (at left) with the enzyme removed in order to clearly demonstrate the occupancy of the same location by the positively chargedmoieties of ER�/Arg300 and RelA/Lys310. (Bottom right) Sequence alignment of the p53, ER�, and RelA peptides, with the lysine pair in greenand the basic residues that interact with the negative clamp of the enzyme in magenta. (D) Sequence alignment of peptides from six Set9 substrates,with the methylation sites in green and the basic residues that might interact with the negative clamp of Set9 in red. The methylation site in eachsubstrate is assigned a position of 0.

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ing of RelA to Set9, the acetylation of RelA on lysine 310inhibits the methylation of lysines 314 and 315 and the subse-quent methylation-triggered ubiquitination and degradation ofchromatin-associated RelA (Fig. 7). By modeling the structureof the SET domain of Set9 in complex with a RelA peptide, wealso provide a mechanistic explanation for this interplay. Ourstudies reveal new insights into the regulation of NF-�B func-tion through cross talk between acetylation and methylation.

Supporting the idea that RelA undergoes stimulus-coupledlysine methylation (40), we used site-specific anti-methylatedlysine 314/315 RelA antibodies and found that endogenousRelA was methylated by Set9 at lysines 314 and 315 in re-sponse to TNF-� (Fig. 1). Furthermore, promoter-associatedRelA was effectively methylated at lysines 314 and 315 in vivoin a stimulus-coupled manner (Fig. 1). Although the exactkinetics and stoichiometry of methylation are not preciselyclear, the finding of a stimulus-coupled methylation of endog-enous RelA in various cell lines, including U2OS cells (Fig.1C), MEFs (Fig. 1D), HEK293T cells (Fig. 3), and macro-phages (data not shown), strengthens the notion that the meth-ylation of lysines 314 and 315 plays a role in regulating thefunction of NF-�B (40). Also note that the level of TNF-�-induced methylation of RelA decreased at later time points(Fig. 1C and E). This decreased methylation, similar to otherposttranslational modifications including phosphorylation andacetylation, might reflect oscillatory NF-�B activation, in whichthe amount of nuclear NF-�B decreases due to the resynthesisof I�B� (15). It is also possible that an unidentified demeth-ylase contributes to the decreased signal as well, since meth-ylation, like phosphorylation and acetylation, is also a revers-ible event (34).

The acetylation of lysine 310 is required for the full tran-scriptional activation of NF-�B (2). Consistently, the depletionof SIRT1 by shRNA or the replacement of lysine 310 withacetyl-mimetic glutamine enhances the transcriptional activityof NF-�B (Fig. 3D and 4C). Interfering with methylation andenhancing the stability of RelA would certainly account for theacetylation-dependent transcriptional activation of NF-�B.Notably, the acetylation of lysine 310 also creates a docking sitefor the binding of the bromodomains of Brd4, which further

recruits and activates CDK9 for the RNA polymerase II-me-diated transcription of NF-�B target genes (16). Therefore, themechanistic impact of the acetylation of lysine 310 is multifac-eted. The ubiquitination and degradation of promoter-associ-ated RelA play an important role in the termination of NF-�Bsignaling (26), and the methylation of lysines 314 and 315 bySet9 is critical for triggering this process, since the methylationof RelA by Set9 enhances the ubiquitination of RelA (40) andthe depletion of Set9 decreases the TNF-�-induced ubiquiti-nation of chromatin-associated RelA (data not shown). Theacetylation of lysine 310 inhibits the methylation of lysines 314and 315 (Fig. 2, 3, and 4); therefore, acetylated RelA needs tobe deacetylated by an HDAC in order for methylation tooccur. The deacetylation of lysine 310 by SIRT1 enhances themethylation of RelA and promotes the ubiquitination and deg-radation of RelA (Fig. 3 and 5). Conversely, the inactivation ofSIRT1 inhibits methylation and stabilizes RelA (Fig. 3, 5, and7). It appears that the deacetylation of RelA lysine 310 bySIRT1 or by other deacetylases functions as a cellular signal todetermine the “on” or “off” state of the transcriptional activityof NF-�B. However, the exact signal triggering the activationof HDACs and the deacetylation of lysine 310 remains unclear.

It is well known that, on histones, one specific posttransla-tional modification can determine the existence of anothermodification in a cis or trans manner either by creating analtered protein surface or by recruiting an effector protein (10,42, 44). Such a cross-reaction between different modificationscan also be observed for nonhistone proteins (42). Here weshow that the acetylation of lysine 310 of RelA clearly inhibitsthe methylation of lysines 314 and 315 in vitro and in vivo (Fig.2, 3, and 4). Interestingly, peptides with acetylated lysine 310are poor substrates for methylation (Fig. 2C), suggesting thatacetylation alone, without the recruitment of any effector pro-teins, is sufficient to inhibit methylation. In fact, acetylationneutralizes the positive charge of the side chain of the lysineresidue, which is required for its interaction with Set9, therebyinhibiting the Set9-mediated methylation of RelA (Fig. 6). Theinterplay between lysine acetylation and methylation has alsobeen observed for p53 (19, 21). Different from the interplay ofRelA, p53 acetylation does not regulate its methylation. In-stead, methylation enhances p53 acetylation, and the regula-tion appears to be an indirect effect. The Set9-mediated meth-ylation of p53 (at human K372 or mouse K369) serves as adocking site to recruit Tip60 and/or other acetyltransferasesfor the subsequent acetylation of p53 (19, 21). The uniqueregulation for RelA and p53 suggests that acetylation andmethylation cross-react in distinct manners to regulate thefunctions of different proteins.

Although the acetylation of lysine 310 inhibits the methyl-ation of lysines 314 and 315 (Fig. 2, 3, and 4), both modifica-tions could be detected at the same time point after TNF-�stimulation (Fig. 3). The detectable acetylated or methylatedsignals likely represent signals from different RelA molecules,since it is clear from our in vitro experiments that the methyl-ation of lysines 314 and 315 is inhibited when all the RelAmolecules are acetylated at lysine 310 (Fig. 2C). The interplaybetween RelA acetylation and methylation is unidirectional,since a prior methylation of lysines 314 and 315 has no effect onthe acetylation of lysine 310 (Fig. 2) and the depletion of Set9does not affect the TNF-�-induced acetylation of lysine 310

FIG. 7. Schematic model for the inhibition of RelA methylation byacetylation. The p300-mediated acetylation of lysine 310, which neu-tralizes the positive charge of this residue, inhibits the binding of Set9to RelA, thereby impairing the Set9-mediated methylation of lysines314 and 315. The deacetylation of RelA by SIRT1 restores the positivecharge of lysine 310, which facilitates the recruitment of Set9 to RelAvia a salt bridge interaction between the basic side chain of lysine 310and the negatively charged exosite of Set9 and consequently leads tothe methylation of lysines 314 and 315.

2178 YANG ET AL. MOL. CELL. BIOL.

(data not shown). In addition to the inhibition of RelA meth-ylation by acetylation, the phosphorylation of RelA at serines276 and 536 was previously shown to enhance the acetylationof lysine 310 by increasing RelA binding to p300 (3, 14). Thesedifferent modifications appear to form a regulatory cascade,with one modification affecting the next. Note that acetylationand methylation occur within a small region (aa 310 to 315)(Fig. 2A) where other modifications also occur. For example,serine 311 is phosphorylated by PKC�, which enhances tran-scription activation by NF-�B (9). Whether there is also a crosstalk between phosphorylation and acetylation or phosphoryla-tion and methylation remains to be determined.

Examination of the crystal structures of Set9 in complex withits substrates and our structural model of Set9 in complex withRelA reveals that an exosite exists for the SET domain of Set9and that this exosite region is consistently bound to a basicresidue from the substrates in all the structure models (Fig.6C) (7, 36). In the crystal structures of ER�/Set9 and p53/Set9(7, 36), the basic residues engaged with the exosite region(arginine 300 in ER� and lysine 370 in p53) are separated fromthe methylation site by only one residue. RelA lacks a basicresidue at this position (position 312); however, it can establisha salt bridge interaction with the exosite region through lysine310, which can bind to exactly the same site as the basic resi-dues in p53 and ER� (Fig. 6C). We note that basic side chains(lysines and arginines) are very long and can easily reach theexosite region by slight variations in their orientation despitetheir slightly different positions in these substrates. As such,any basic residue in the region corresponding to residues 310to 312 in RelA would be sufficient to interact with the proposedexosite region, resulting in the increased binding affinity andmethylation level of the substrate. In contrast, the neutraliza-tion of the positive charge of lysine 310 via acetylation ormutation to glutamine is expected to reduce the binding affinityand, thus, the rate of methylation, in accordance with ourexperimental results (Fig. 6B). Note that in our model (Fig.6C), only the configuration in which lysine 314 is inserted intothe methylation active site has been captured, and this modelis the only structural model that we have to work with in orderto investigate the interaction between the peptide and theenzyme, as we do not have any structural models for the con-figuration in which the other lysine residue (lysine 315 in thiscase) is being methylated. Future crystal structures of Set9 incomplex with two methylated lysines would provide insight intothe mode of interaction involved in the methylation of lysine315 as well.

Previous studies by Couture et al. suggested that Set9 rec-ognizes a consensus motif of its substrates for methylation:K/R--K-X (where is a small residue, K is the methylationsite, and X is any residue) (8). This motif corresponds with theP2 to P�1 positions of the methylation region in each sub-strate, and the basic residue in the P2 position is required formethylation by Set9 (8). Our studies demonstrate that thelysine (residue 310) in the P4 position in RelA plays a rolesimilar to that of K/R in the P2 position in the proposedconsensus motif (Fig. 6D), suggesting that the positivelycharged residue is not necessarily limited to the P2 position.In fact, K/R in any of the P2 to P4 position could meet therequirement of a basic residue for Set9 recognition. This ex-tended consensus motif can accommodate all known Set9 sub-

strates and could help predict and identify new Set9 substrates(Fig. 6D).

RelA undergoes several posttranslational modifications (1,29). By analogy to the histone code, a “protein code” or“NF-�B code” may also exist. First, different RelA modifica-tions might regulate each other, as demonstrated in the currentstudy. Second, various posttranslational modifications of RelAmight create specific marks for the recruitment of differenteffectors to control the temporal and spatial activation ofNF-�B (1, 29). Supporting this, the acetylation of lysine 310 isspecifically recognized by the bromodomains of Brd4, enhanc-ing transactivation by NF-�B (16). Similarly, methylated ly-sines 314 and 315 are likely recognized by some domain-con-taining E3 ligase for the ubiquitination and degradation ofNF-�B (40, 41). Therefore, modifications of RelA by methyl-ation or acetylation at specific sites could, in fact, dictate spe-cific biological responses, reflecting the gain or loss of selectivecofactors whose association with RelA is regulated by its stateof modifications.

Overall, our data uncover a novel interplay between RelAacetylation and methylation and further support the “NF-�Bcode” hypothesis. Methylation is contingent upon the priordeacetylation of RelA at lysine 310 (Fig. 2 and 3). The acety-lation of lysine 310 interferes with the interaction betweenRelA and the exosite of the SET domain of Set9 by neutral-izing the positively charged side chain. Our studies also providea better understanding of the regulation of methylation byacetylation. Since many nonhistone proteins are modified byacetylation as well as Set9-mediated methylation, it will be ofgreat interest to explore whether an interplay and mechanismsimilar to those identified with RelA would apply to the reg-ulation of other proteins.

ACKNOWLEDGMENTS

We thank X. L. Lin and M. McBurney for providing the SIRT1-deficient MEFs, A. Beg for the RelA-deficient MEFs, W. C. Greenefor reagents, D. J. Shapiro for critical reading of the manuscript, andmembers of the Chen laboratory for discussion.

This work is supported in part by ICR provided by the University ofIllinois at Urbana-Champaign and a biomedical research grant fromthe American Lung Association.

REFERENCES

1. Chen, L. F., and W. C. Greene. 2004. Shaping the nuclear action of NF-�B.Nat. Rev. Mol. Cell Biol. 5:392–401.

2. Chen, L. F., Y. Mu, and W. C. Greene. 2002. Acetylation of RelA at discretesites regulates distinct nuclear functions of NF-�B. EMBO J. 21:6539–6548.

3. Chen, L. F., S. A. Williams, Y. Mu, H. Nakano, J. M. Duerr, L. Buckbinder,and W. C. Greene. 2005. NF-kappaB RelA phosphorylation regulates RelAacetylation. Mol. Cell. Biol. 25:7966–7975.

4. Cheung, P., C. D. Allis, and P. Sassone-Corsi. 2000. Signaling to chromatinthrough histone modifications. Cell 103:263–271.

5. Cheung, P., K. G. Tanner, W. L. Cheung, P. Sassone-Corsi, J. M. Denu, andC. D. Allis. 2000. Synergistic coupling of histone H3 phosphorylation andacetylation in response to epidermal growth factor stimulation. Mol. Cell5:905–915.

6. Choi, K. C., M. G. Jung, Y. H. Lee, J. C. Yoon, S. H. Kwon, H. B. Kang, M. J.Kim, J. H. Cha, Y. J. Kim, W. J. Jun, J. M. Lee, and H. G. Yoon. 2009.Epigallocatechin-3-gallate, a histone acetyltransferase inhibitor, inhibitsEBV-induced B lymphocyte transformation via suppression of RelA acety-lation. Cancer Res. 69:583–592.

7. Chuikov, S., J. K. Kurash, J. R. Wilson, B. Xiao, N. Justin, G. S. Ivanov, K.McKinney, P. Tempst, C. Prives, S. J. Gamblin, N. A. Barlev, and D. Rein-berg. 2004. Regulation of p53 activity through lysine methylation. Nature432:353–360.

8. Couture, J. F., E. Collazo, G. Hauk, and R. C. Trievel. 2006. Structural basisfor the methylation site specificity of SET7/9. Nat. Struct. Mol. Biol. 13:140–146.

VOL. 30, 2010 ACETYLATION INHIBITS METHYLATION OF RelA 2179

9. Duran, A., M. T. Diaz-Meco, and J. Moscat. 2003. Essential role of RelASer311 phosphorylation by zetaPKC in NF-kappaB transcriptional activa-tion. EMBO J. 22:3910–3918.

10. Fingerman, I. M., H. N. Du, and S. D. Briggs. 2008. Controlling histonemethylation via trans-histone pathways. Epigenetics 3:237–242.

11. Fischle, W., Y. Wang, and C. D. Allis. 2003. Histone and chromatin cross-talk. Curr. Opin. Cell Biol. 15:172–183.

12. Ghosh, S., and M. Karin. 2002. Missing pieces in the NF-kappaB puzzle. Cell109(Suppl.):S81–S96.

13. Gringhuis, S. I., J. den Dunnen, M. Litjens, B. van Het Hof, Y. van Kooyk,and T. B. Geijtenbeek. 2007. C-type lectin DC-SIGN modulates Toll-likereceptor signaling via Raf-1 kinase-dependent acetylation of transcriptionfactor NF-kappaB. Immunity 26:605–616.

14. Hoberg, J. E., A. E. Popko, C. S. Ramsey, and M. W. Mayo. 2006. IkappaBkinase alpha-mediated derepression of SMRT potentiates acetylation ofRelA/p65 by p300. Mol. Cell. Biol. 26:457–471.

15. Hoffmann, A., A. Levchenko, M. L. Scott, and D. Baltimore. 2002. TheIkappaB-NF-kappaB signaling module: temporal control and selective geneactivation. Science 298:1241–1245.

16. Huang, B., X. D. Yang, M. M. Zhou, K. Ozato, and L. F. Chen. 2009. Brd4coactivates transcriptional activation of NF-kappaB via specific binding toacetylated RelA. Mol. Cell. Biol. 29:1375–1387.

17. Ishinaga, H., H. Jono, J. H. Lim, S. M. Kweon, H. Xu, U. H. Ha, T. Koga, C.Yan, X. H. Feng, L. F. Chen, and J. D. Li. 2007. TGF-beta induces p65acetylation to enhance bacteria-induced NF-kappaB activation. EMBO J.26:1150–1162.

18. Ito, K., C. E. Charron, and I. M. Adcock. 2007. Impact of protein acetylationin inflammatory lung diseases. Pharmacol. Ther. 116:249–265.

19. Ivanov, G. S., T. Ivanova, J. Kurash, A. Ivanov, S. Chuikov, F. Gizatullin,E. M. Herrera-Medina, F. Rauscher III, D. Reinberg, and N. A. Barlev. 2007.Methylation-acetylation interplay activates p53 in response to DNA damage.Mol. Cell. Biol. 27:6756–6769.

20. Jenuwein, T., and C. D. Allis. 2001. Translating the histone code. Science293:1074–1080.

21. Kurash, J. K., H. Lei, Q. Shen, W. L. Marston, B. W. Granda, H. Fan, D.Wall, E. Li, and F. Gaudet. 2008. Methylation of p53 by Set7/9 mediates p53acetylation and activity in vivo. Mol. Cell 29:392–400.

22. Lee, H., A. Herrmann, J. H. Deng, M. Kujawski, G. Niu, Z. Li, S. Forman,R. Jove, D. M. Pardoll, and H. Yu. 2009. Persistently activated Stat3 main-tains constitutive NF-kappaB activity in tumors. Cancer Cell 15:283–293.

23. Lo, W. S., L. Duggan, N. C. Emre, R. Belotserkovskya, W. S. Lane, R.Shiekhattar, and S. L. Berger. 2001. Snf1—a histone kinase that works inconcert with the histone acetyltransferase Gcn5 to regulate transcription.Science 293:1142–1146.

24. Lo, W. S., R. C. Trievel, J. R. Rojas, L. Duggan, J. Y. Hsu, C. D. Allis, R.Marmorstein, and S. L. Berger. 2000. Phosphorylation of serine 10 in histoneH3 is functionally linked in vitro and in vivo to Gcn5-mediated acetylation atlysine 14. Mol. Cell 5:917–926.

25. MacKerell, A. D. J., D. Bashford, M. Bellott, R. L. Dunbrack, Jr., J. D.Evanseck, M. J. Field, S. Fischer, J. Gao, H. Guo, S. Ha, D. Joseph-Mc-Carthy, L. Kuchnir, K. Kuczera, F. T. K. Lau, C. Mattos, S. Michnick, T.Ngo, D. T. Nguyen, B. Prodhom, W. E. Reiher III, B. Roux, M. Schlenkrich,J. C. Smith, R. Stote, J. Straub, M. Watanabe, J. Wiorkiewicz-Kuczera, D.Yin, and M. Karplus. 1998. All-atom empirical potential for molecularmodeling and dynamics studies of proteins. J. Phys. Chem. B 102:3586–3616.

26. Natoli, G., and S. Chiocca. 2008. Nuclear ubiquitin ligases, NF-kappaBdegradation, and the control of inflammation. Sci. Signal. 1:pe1.

27. Nishioka, K., S. Chuikov, K. Sarma, H. Erdjument-Bromage, C. D. Allis, P.Tempst, and D. Reinberg. 2002. Set9, a novel histone H3 methyltransferasethat facilitates transcription by precluding histone tail modifications requiredfor heterochromatin formation. Genes Dev. 16:479–489.

28. Perkins, N. D. 2007. Integrating cell-signalling pathways with NF-kappaBand IKK function. Nat. Rev. Mol. Cell Biol. 8:49–62.

29. Perkins, N. D. 2006. Post-translational modifications regulating the activityand function of the nuclear factor kappa B pathway. Oncogene 25:6717–6730.

30. Phillips, J. C., R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C.Chipot, R. D. Skeel, L. Kale, and K. Schulten. 2005. Scalable moleculardynamics with NAMD. J. Comput. Chem. 26:1781–1802.

31. Pruitt, K., R. L. Zinn, J. E. Ohm, K. M. McGarvey, S. H. Kang, D. N.Watkins, J. G. Herman, and S. B. Baylin. 2006. Inhibition of SIRT1 reacti-vates silenced cancer genes without loss of promoter DNA hypermethyl-ation. PLoS Genet. 2:e40.

32. Sakurai, H., H. Chiba, H. Miyoshi, T. Sugita, and W. Toriumi. 1999. I�Bkinases phosphorylate NF-�B p65 subunit on serine 536 in the transactiva-tion domain. J. Biol. Chem. 274:30353–30356.

33. Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the rangefrom 1 to 100 kDa. Anal. Biochem. 166:368–379.

34. Shi, Y., and J. R. Whetstine. 2007. Dynamic regulation of histone lysinemethylation by demethylases. Mol. Cell 25:1–14.

35. Sims, R. J., III, and D. Reinberg. 2008. Is there a code embedded in proteinsthat is based on post-translational modifications? Nat. Rev. Mol. Cell Biol.9:815–820.

36. Subramanian, K., D. Jia, P. Kapoor-Vazirani, D. R. Powell, R. E. Collins, D.Sharma, J. Peng, X. Cheng, and P. M. Vertino. 2008. Regulation of estrogenreceptor alpha by the SET7 lysine methyltransferase. Mol. Cell 30:336–347.

37. Suganuma, T., and J. L. Workman. 2008. Crosstalk among histone modifi-cations. Cell 135:604–607.

38. Wang, D., S. D. Westerheide, J. L. Hanson, and A. S. Baldwin, Jr. 2000.Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 onSer529 is controlled by casein kinase II. J. Biol. Chem. 275:32592–32597.

39. Yang, S. R., J. Wright, M. Bauter, K. Seweryniak, A. Kode, and I. Rahman.2007. Sirtuin regulates cigarette smoke-induced proinflammatory mediatorrelease via RelA/p65 NF-kappaB in macrophages in vitro and in rat lungs invivo: implications for chronic inflammation and aging. Am. J. Physiol. LungCell. Mol. Physiol. 292:L567–L576.

40. Yang, X. D., B. Huang, M. Li, A. Lamb, N. L. Kelleher, and L. F. Chen. 2009.Negative regulation of NF-kappaB action by Set9-mediated lysine methyl-ation of the RelA subunit. EMBO J. 28:1055–1066.

41. Yang, X. D., A. Lamb, and L. F. Chen. 2009. Methylation, a new epigeneticmark for protein stability. Epigenetics 4:429–433.

42. Yang, X. J., and E. Seto. 2008. Lysine acetylation: codified crosstalk withother posttranslational modifications. Mol. Cell 31:449–461.

43. Yeung, F., J. E. Hoberg, C. S. Ramsey, M. D. Keller, D. R. Jones, R. A. Frye,and M. W. Mayo. 2004. Modulation of NF-kappaB-dependent transcriptionand cell survival by the SIRT1 deacetylase. EMBO J. 23:2369–2380.

44. Zhang, K., and S. Y. Dent. 2005. Histone modifying enzymes and cancer:going beyond histones. J. Cell. Biochem. 96:1137–1148.

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