Corrections

MICROBIOLOGYCorrection for “Hybrid DNA virus in Chinese patients with se-ronegative hepatitis discovered by deep sequencing,” by BaoyanXu, Ning Zhi, Gangqing Hu, Zhihong Wan, Xiaobin Zheng,Xiaohong Liu, Susan Wong, Sachiko Kajigaya, Keji Zhao, QingMao, and Neal S. Young, which appeared in issue 25, June 18,2013, of Proc Natl Acad Sci USA (110:10264–10269; first pub-lished May 28, 2013; 10.1073/pnas.1303744110).The editors note that on September 10, 2013, PNAS received

a letter to the editor from Naccache et al. (1), which raisedconcerns about a possible contaminant affecting the results ofthis paper. The authors were asked to provide a response tothese concerns, but while we were awaiting their comments, theOffice of Research Integrity (ORI) published a case summary (2)for Baoyan Xu on December 30, 2013. Based on a limited ad-mission by Xu, ORI found that Baoyan Xu had engaged in re-search misconduct. ORI recommended a correction to Fig. 6 andFig. S4. After evaluation of the authors’ response (3) to Naccacheet al. and the detailed information supplied below from the au-thors, the Editorial Board approved the following correction:The authors wish to note, “Correspondence from Stephen

Korsman, Medical Virologist at Groote Schuur National HealthLaboratory Service, South Africa, suggested that some immuno-blots in the above article contained multiple copied images, inwhich images were reproduced for several different patients. Usinggraphics-editing software (varying contrast and lighting of individuallanes of the blots), we confirmed that the images had been ex-tensively manipulated. This finding led to a formal complaint toand subsequent investigation by the Department of Health andHuman Services ORI. ORI has formally concluded that one au-thor engaged in research misconduct (2). Misconduct appeared tobe restricted to assembly of the final, complex figures.“We were able to retrieve and reconstruct digital images from

the original experiments; only immunoblots from patients withseronegative hepatitis, in Fig. 6B and Fig. S4A, had been ma-nipulated. We further repeated immunoblotting experiments,

using existing samples, and we used these data to replace bothFig. 6 and Fig. S4. This correction does not affect the originalfindings of the serology studies. We have evaluated additionalhealthy controls from different ethnicities, which we add to theSupporting Information as Fig. S4C.“We reproduced the experiments using sera samples from 77

Chinese hepatitis patients by immunoblotting as described in ourpaper. Fifteen patient sera samples that were used in the originalstudy were unavailable. In addition, we tested sera samples from 28healthy controls with origins from the United States, France, Korea,Brazil, China, Vietnam, Japan, and Puerto Rico. By immunoblot,86% (66/77) of seronegative hepatitis patients were positive forNIH-CQV IgG, and 16% (12/77) were positive for IgM (Fig. 6Band Fig. S4A). Of healthy controls from different ethnicities, 78%(13/28) were positive for IgG (Fig. S4C). These data confirm thepresence of an antibody response against recombinant capsidprotein of NIH-CQV in the cohort of Chinese seronegative hep-atitis patients, and also in healthy individuals. Reactivity betweenthe polyhistidine-tag and samples also was evaluated and excluded.However, whether the antibody response against recombinantNIH-CQV viral capsid protein detected by immunoblot wasinduced by infection with NIH-CQV or derives from cross re-activity with an epitope present on another microorganism remainsto be clarified. Although we excluded cross reactivity betweenNIH-CQV and other common human parvoviruses, additionalconfirmatory experiments based on serologic reactivity to mul-tiple, nonoverlapping fragments derived from NIH-CQV capsidprotein are in progress and the results will be reported.”The corrected figure and its corrected legend appear below.

1. Naccache SN, Hackett J, Delwart EL, Chiu CY (2014) Concerns over the origin of NIH-CQV, a novel virus discovered in Chinese patients with seronegative hepatitis. Proc NatlAcad Sci USA 111:E976.

2. http://ori.hhs.gov/content/case-summary-xu-baoyan. Accessed January 30, 2014.3. Zhi N, et al. (2014) Reply to Naccache et al: Novel NIH-CQV virus a contaminant of DNA

extraction method. Proc Natl Acad Sci USA 111:E977.

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Fig. 6. Immunoblot of seronegative hepatitis patient using recombinant capsid protein (rCP) of NIH-CQV. (A) Specificity test for the rCP of NIH-CQV. The celllysates derived from the cells transfected with the plasmids that expressed capsid proteins of AAV2, Parv4, HBoV, and B19V and purified rCP were subjected toSDS/PAGE, and then transferred to a nitrocellulose membrane. After blocking with 5% (wt/vol) nonfat milk for 2 h, the membrane was incubated withrespective antisera at 1:1,000 dilution. (B and C) Detection of specific antibodies against the rCP of NIH-CQV. Samples subjected to SDS/PAGE consisted of25 ng of affinity-purified rCP. These proteins were transferred to a nitrocellulose membrane and incubated with a 1:1,000 dilution of individual serum. Abovethe lanes are the identification numbers of individual patients and healthy controls. Only representative results are shown; see Fig. S4 A and B for completedata. Mouse monoclonal antibody against the polyhistidine tag was used as a positive control. The numbers on the left indicate molecular masses inkilodaltons based on the PageRuler Prestained Protein Ladder (Fermentas).

PNAS | March 18, 2014 | vol. 111 | no. 11 | 4345

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ECTIONS

SYSTEMS BIOLOGYCorrection for “mChIP-KAT-MS, amethod tomap protein interactions and acetylation sites for lysine acetyltransferases,” by LeslieMitchell,SylvainHuard,Michael Cotrut, Roghayeh Pourhanifeh-Lemeri, Anne-Lise Steunou,Akil Hamza, Jean-Philippe Lambert,Hu Zhou, ZhibinNing, Amrita Basu, Jacques Côté, Daniel A. Figeys, and Kristin Baetz, which appeared in issue 17, April 23, 2013, ofProc Natl AcadSci USA (110:E1641–E1650; first published April 9, 2013; 10.1073/pnas.1218515110).The authors note “that a mistake was made generating the Epl1 mutant EPcA-Q shown in Fig. 3 B and C and Fig. S4. Instead of

mutating K39, 345, 376 and 379 to Q (glutamine, Gln) these sites were mutated to E (glutamic acid, Glu). Through gene synthesis, wehave generated the correct mutant and assessed its impact on NuA4. As anticipated, detectable acetylation signal on Epl1 mutantsEPcA-R and EpcA-Q was decreased (corrected Fig. 3B). We next sought to determine the function of Epl1 acetylation within the EPcAdomain. Although the EPcA-Q and EpCA-R mutants do not impact coimmunopurification with Eaf5-TAP, global histone H4acetylation was reproducibly decreased in esa1-L254P and EPcA-R (corrected Fig. 3B, lanes 2 and 3), in contrast EPcA-Q displayedglobal histone H4 acetylation similar to wild type Epl1 strains (corrected Fig. 3B, lanes 1 and 4). In agreement with this finding, thestrain expressing EPcA-Q does not display temperature sensitivity (corrected Fig. S4). In conclusion, while mutating K39, 345,376, and 379 to acidic glutamic acid has negative consequences for NuA4 catalytic activity (original Fig. 3), mutating these sites toglutamine does not. Therefore, as reported for other MYST KATs (1), autoacetylation does contribute to NuA4 catalytic activity.The authors apologize for this error.Please see the table below for new strains.”

As a result of these errors, the authors note that the following section should be added to the Materials and Methods.

“Cloning and Mutagenesis of Enhancer of Polycomb Like 1. Acetylation point mutants mimicking acetylated (K-to-Q) state for each acet-ylated lysine were generated by synthesizing a custom-designed DNA fragment (Genscript) of 857 base pairs carrying the mutationK345Q, K376Q, and K379Q and corresponding to nucleotides 600–1456 of EPL1. Following double digestion with BspE1/Nru1 and gelpurification (Qiagen; 28704), this mutated DNA fragment was ligated into pRS415-EPL1-HA3 (pKB41) that was also digested withBspE1/NruI, subjected to calf intestinal phosphatase treatment (New England Biolabs; M0290) and gel purified. Ligation was carried outovernight at 16 °C with T4 DNA ligase (New England Biolabs; M0202S), and then transformed into competent Escherichia coli DH5αcells. K39Q was then generated by using a site-directed mutagenesis kit (Stratagene; 200528). The successful introduction of allpoint mutations pRS415-epl1-EPcA-Q-HA3 (pKB264) was confirmed by sequencing and expression confirmed by Westernblot analysis.”

The authors also note that Fig. 3 and its corresponding legend appeared incorrectly. The corrected figure and its corrected legendappear below.

www.pnas.org/cgi/doi/10.1073/pnas.1401790111

Strain list

Strain Genotype Source

YKB3402 MATα his3 leu2 lys2 ura3 trp1 epl1Δ::kanMX EAF5-TAP::HIS3 [epl1-K39Q-K345Q-K376Q-K379Q-3HA::LEU2] Present studyYKB 3579 MATa ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1 epl1ΔKAN [epl1-K39Q-K345Q-K376Q-K379Q-3HA::LEU2] Present study

Fig. 3. NuA4 subunits are acetylated in vivo and in vitro.(A) Radioactive KAT assay to assess NuA4 auto-acetylationin vitro. [3H]-acetyl CoA was added directly to bead matrixof a stringently immunopurified NuA4 preparation [Esa1-TAP(YKB440)] versus an untagged control (YPH499) immuno-purification. Histone proteins were added to the reaction toserve as a positive control for acetylation. The reactions wereseparated on a gradient gel, Coomassie-stained to visualizeproteins (Right), treated for fluorography, and finally ex-posed to film for 2 wk (Left). Proteins are identified on theright side and protein size is indicated on the left (kDa).(B) Epl1 acetylation is dependent on Esa1 in vivo. NuA4was purified from cells grown at 25 °C through Eaf5-TAP.Epl1 was expressed as a C-terminal HA fusion proteinin epl1Δ background strain in the presence (lane 2,YKB2876) or absence (lane 1, YKB2862) of the acetyl-transferase-deficient esa1-L254P allele, or as a lysine-to-arginine or -glutamine multipoint mutant [EPcA-R;EPcA-Q (K39,345,376,379R or Q) YKB2781 and YKB3402,lanes 3 and 4, respectively]. Immunopurified (IP) productsand whole cell extract (WCE) were separated by SDS/PAGE(7.5%) and subjected to Western blot using the indicatedantibodies: antiacetyl lysine (α-AcK), antihistone H4 acetyllysine (α-AcK H4), antiglyceraldehyde 6-phosphate de-hydrogenase (α-G6PDH). Image is a representative ofthree independent replicates. (C ) Epl1-EPcA-R reducesNuA4 in vitro KAT activity. NuA4 was purified through Eaf5-TAP (lanes 2–4) relative to an untagged control sample (lane 1, YPH499). Epl1 was expressed fromits endogenous locus (lane 2, YKB1042) or as a C-terminal HA fusion protein of wild type EPL1 (lane 3, YKB2862) or epl1-EPcA-R (lane 4, YKB2781). KAT assayswere performed using NuA4 preparations equalized for Esa1 and nucleosome purified from HeLa cells. Error bars represent SD from duplicate reactions.NuA4 complexes used in assay were separated by SDS/PAGE and subjected to Western blot using the indicated antibody anti-Esa1 (α-Esa1) (Lower).

1. Yuan H, et al. (2012) MYST protein acetyltransferase activity requires active site lysine autoacetylation. EMBO J 31(1):58–70.

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mChIP-KAT-MS, a method to map protein interactionsand acetylation sites for lysine acetyltransferasesLeslie Mitchella, Sylvain Huarda, Michael Cotruta, Roghayeh Pourhanifeh-Lemeria, Anne-Lise Steunoub, Akil Hamzaa,Jean-Philippe Lamberta, Hu Zhoua, Zhibin Ninga, Amrita Basuc, Jacques Côtéb, Daniel A. Figeysa, and Kristin Baetza,1

aOttawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, ON, Canada K1H 8M5;bLaval University Cancer Research Center, Hôtel-Dieu de Québec, Quebec City, QC, Canada G1R 2J6; and cDepartment of Chemical Biology, Broad Instituteof Harvard and MIT, Cambridge, MA 02142

Edited by Jasper Rine, University of California, Berkeley, CA, and approved March 15, 2013 (received for review October 24, 2012)

Recent global proteomic and genomic studies have determined thatlysine acetylation is a highly abundant posttranslational modifica-tion. The next challenge is connecting lysine acetyltransferases(KATs) to their cellular targets. We hypothesize that proteins thatphysically interact with KATs may not only predict the cellular func-tion of the KATs butmay be acetylation targets.We have developeda mass spectrometry-based method that generates a KAT proteininteraction network from which we simultaneously identify both invivo acetylation sites and in vitro acetylation sites. This modifiedchromatin-immunopurification coupled to an in vitro KAT assaywithmass spectrometry (mChIP-KAT-MS)was applied to the Saccha-romyces cerevisiae KAT nucleosome acetyltransferase of histone H4(NuA4). Using mChIP-KAT-MS, we define the NuA4 interactome andin vitro-enriched acetylome, identifying over 70 previously unde-scribed physical interaction partners for the complex and over 150acetyl lysine residues, of which 108 are NuA4-specific in vitro sites.Through this method we determine NuA4 acetylation of its ownsubunit Epl1 is a means of self-regulation and identify a unique linkbetween NuA4 and the spindle pole body. Our work demonstratesthat this methodology may serve as a valuable tool in connectingKATs with their cellular targets.

acetylation map | yeast

Lysine acetyltransferase (KAT) enzymes catalyze the transfer ofan acetyl group from acetyl CoA onto the e-amino group of

lysine residues. Acetylation then regulates protein function ina number of ways, including altering the localization, activity,stability, and physical interactions of the target protein (1, 2).Through the acetylation of histone proteins, KATs have tradi-tionally been associated with a variety of chromatin-based cellularprocesses, such as transcription, silencing, and DNA repair. Morerecently, systematic screens aimed at identifying acetylated lysinepeptides in both prokaryotic and eukaryotic systems have estab-lished acetylation as a ubiquitous and conserved posttranslationalmodification occurring on thousands of proteins (3–10). Further-more, these screens revealed fundamental properties associatedwith lysine acetylation, such as the abundance of acetylation sitesfound on metabolic enzymes and mitochondrial proteins, thetendency of multisubunit protein complexes to be abundantlyacetylated, and that most acetylated proteins do not have obviousroles in chromatin-directed processes. However, in the vast ma-jority of cases the biological consequences of lysine acetylation andthe KAT responsible for catalysis have yet to be determined. Tofully elucidate the cellular functions of KATs in vivo will requirea detailed understanding of the direct pathways in which a KATfunctions, and a focused analysis of the role of acetylation withinthose pathways. (Abbreviations for genes and proteins are pro-vided in Table S1.)A recent proteome-wide survey of acetyl lysine residues in

Saccharomyces cerevisiae identified more than 4,000 lysine acety-lation sites; however, theKATs responsible for thesemodificationswere not discerned (4). Global genetic screens that identify pro-teins whose overexpression cause fitness defects in either KAT

mutants (11) or lysine deacetyltransferase mutants (12) have suc-cessfully predicted proteins whose function is regulated by re-versible acetylation. Together, synthetic dosage lethal geneticscreens have led to the identification of 96 acetylated proteins(11, 12). In another systematic analysis, protein microarray tech-nology, encompassing more than 90% of the proteins encoded bythe S. cerevisiae genome, was used to identify in vitro acetylationsubstrates of the KAT complex nucleosome acetyltransferaseof histone H4 (NuA4) (13). This work identified 91 NuA4 targetsin vitro and ultimately succeeded in confirming 13 substratesin vivo (13). Despite these successes, the genetic and proteomicapproaches currently in use each have unique technologicalchallenges and further share the drawback that the specificlysine residues acetylated by a particular KAT are not imme-diately identified. Thus, complementary techniques need to bedeveloped to connect KAT enzymes to their substrates andacetyl lysine residues in vivo to fully elucidate the pathwaysgoverned by acetylation.To this end, we have developed a unique proteomic method to

generate a KAT-associated protein interaction network in whichthe level of acetylated lysine residues is enriched in vitro. Herewe describe the methodology we have named mChIP-KAT-MS(modified chromatin-immunopurification coupled to an in vitroKAT assay withmass spectrometry) designed to linkKAT enzymesto new substrates and cellular pathways. We showcase this methodusing the essential S. cerevisiae KAT NuA4, a highly conserved,multifunctional enzyme complex (14). UsingmChIP-KAT-MS, wedefine the NuA4 interactome and in vitro-enriched acetylome,identifying over 70 unique physical interaction partners for thecomplex and over 150 acetyl lysine residues, of which 108 are

Significance

Recent proteomic studies have revealed that lysine acetylationis a global and ubiquitous posttranslational modification.However, in the vast majority of cases the lysine acetyl-transferases (KATs) responsible for individual modificationsremain unknown. Here we present a unique methodology thatconnects KATs to their substrates. To validate the methodol-ogy, we use the yeast KAT nucleosome acetyltransferase ofhistone H4 (NuA4) and identify both protein interactions andacetylation targets. Importantly, this methodology can be ap-plied to any KAT and should aid in the linking of KATs to theircellular targets.

Author contributions: L.M., J.C., D.A.F., and K.B. designed research; L.M., S.H., M.C., R.P.-L.,A.-L.S., A.H., J.-P.L., H.Z., and Z.N. performed research; L.M., S.H., M.C., R.P.-L., J.-P.L., H.Z.,Z.N., A.B., D.A.F., and K.B. analyzed data; and L.M. and K.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. E-mail: kbaetz@uottawa.ca.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218515110/-/DCSupplemental.

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NuA4-specific in vitro sites. To validate our method, we performa series of directed follow-up experiments to decipher the impactof NuA4 acetylation of one of its own subunits, Epl1, and link thecatalytic function of NuA4 to the spindle pole body (SPB) andspindle dynamics. Taking these data together, this work demon-strates the utility and the flexibility of the mChIP-KAT-MS ap-proach as a unique methodology to study KATs in vivo.

ResultsmChIP-KAT-MS as a Unique Method to Study KAT Function. To gaininsight into the mechanisms of action and specific pathways inwhich a KAT functions, we developed a method that can identifythe network of proteins that physically interact with a KAT as wellas simultaneously identify acetyl lysine residues arising from eitherpreexisting in vivo or KAT-dependent in vitro catalysis within thatnetwork. The methodology, called mChIP-KAT-MS, consists ofthree steps: (i) isolating the KAT and its associated protein net-work from cells; (ii) enriching the level of acetylated lysine resi-dues within the network using an in vitro KAT reaction; and(iii) identifying interacting proteins and acetylation sites by LC-MS/MS (Fig. 1). To isolate NuA4 and interacting proteins, wepurify NuA4 through Esa1-TAP (tandem-affinity purification), its

catalytic subunit (15), using a variant of the traditional immuno-purification strategy, termed modified chromatin immunopur-ification (mChIP) (16). The mChIP method, originally developedto study chromatin-associated proteins, has now been used suc-cessfully to increase the depth of coverage of protein interactionsof wide range of bait proteins in vivo (17, 18). Briefly, yeast whole-cell lysates are subjected to mild sonication followed by gentlecentrifugation, thereby promoting retention of poorly solublecellular components in solution. Immunopurification is performedin a single step using magnetic beads coated with IgG antibodiesthat specifically recognize the protein A component of the TAPtag. Next, to boost the level of acetyl lysine residues on proteinswithin the network, an in vitro KAT assay is performed in whichexogenous NuA4, stringently purified from yeast, and isotopicallylabeled acetyl CoA (13C2-acetyl CoA; herein referred to as heavyacetyl CoA) are incubated with the immunopurified bead matrix.Finally, the acetyl lysine enriched network is analyzed by LC-MS/MS. Because of the shift in mass-to-charge ratio, heavy acetylgroups resulting from NuA4 in vitro KAT activity can be distin-guished from unlabeled, preexisting acetyl moieties (herein re-ferred to as light acetyl CoA). Therefore, this methodologyenables: the (i) identification of the network of proteins associatedwith the KAT of interest under normal growth conditions; (ii)generation of a list of light or in vivo acetylation sites, therebyincreasing our general knowledge of yeast acetylation; and (iii)definition of a set of KAT-specific in vitro acetylation sites onproteins that physically copurify with the KAT of interest.

NuA4-Associated Protein Interaction Network. To generate a high-confidence NuA4-associated protein network (Fig. 2A), the Esa1-TAP mChIP assay was repeated six times. Interacting proteinsidentified in at least two experimental replicates were deemedreproducible and included in the final dataset. Proteins identifiedin only one mChIP experiment but found in complexes with re-producible interactors were also included. Finally, all proteinsmodified by lysine acetylation are also presented in Fig. 2A. Ex-cluded proteins were those previously identified as mChIP con-taminants or common mChIP preys (17) (Dataset S1), as well asany protein functioning in a complex with a filtered protein(Dataset S1). The protein network includes the 13 core membersof the NuA4 KAT complex, as well as an additional 84 proteins(Fig. 2A and Dataset S1). As expected, this network containsproteins previously shown to interact with one or more NuA4subunits, such as histone proteins H4 (Hhf1), H2A (Hta1 andHta2), H2B (Htb2), and H3 (Hht1) (19); stress-responsive tran-scription factors (Msn2, Msn4, and Yap1) (20); subunits of theChaperonin Containing Tcp1 (CCT) complex (Tcp1, Cct8, Cct4)(21); and the 14-3-3 protein Bmh1 (22). The unique NuA4 inter-acting proteins dramatically expand our knowledge of pathways inwhich NuA4 may function. New interactions include multiplemembers of protein complexes such as the SPB [8 of 17 corecomponents (23)], the COPI [4 of 7 (24)] and COPII [3 of 13 (25)],transport complexes, and all four members of a multifunctionalmRNA binding stress granule complex [4 of 4 (26)] (Fig. 2A).Furthermore, we identified the copurification of the 26S protea-some: 8 of 33 coremembers of both the 19S regulatory particle and20S catalytic core particle (27), supporting the recent report of thisinteraction (28). In total, more than half of the proteins in thenetwork are nonnuclear (Fig. 2B), suggesting a broad localizationpattern for NuA4 within the yeast cell. This work represents an indepth analysis of proteins that copurify with NuA4 and sub-stantiates the hypothesis that NuA4 participates directly in a diversearray of cellular processes beyond chromatin-related functions.

Lysine Acetylation Identified Within the NuA4-Associated ProteinNetwork. The mChIP-KAT-MS methodology enables the identi-fication of acetylation sites resulting from either in vivo acetylation(light acetyl groups) or in vitro catalysis (heavy acetyl groups).

Fig. 1. mChIP-KAT-MS Methodology. Step 1: NuA4 and its associated pro-tein interaction network are purified from yeast using mChIP technology.Briefly, yeast whole-cell lysate is mildly sonicated and gently clarified beforeNuA4 immunopurification through endogenously TAP-tagged Esa1 withmagnetic beads coupled to IgG antibodies. Step 2: NuA4 and copurifyingproteins are subjected to an in vitro KAT assay. Highly purified exogenousNuA4 and isotopically labeled acetyl CoA (13C2-Acetyl CoA) are added to theNuA4-bead matrix under conditions promoting NuA4 KAT activity. Step 3:Proteins and acetylation sites are identified by LC-MS/MS. Acetyl lysinesidentified on sequenced peptides may derive from preexisting, in vivo ca-talysis (green “Ac” tag; light) or in vitro catalysis, which will yield isotopicallylabeled acetyl lysine residues (red “Ac” tag; heavy).

E1642 | www.pnas.org/cgi/doi/10.1073/pnas.1218515110 Mitchell et al.

Although the mChIP-MS was performed six times, only two rep-licates included the NuA4 KAT assay with isotopically labeledacetyl CoA (Fig. S1). In total, we identified 66 acetyl lysine resi-dues on 23 proteins (Fig. 2A, Table 1, and Dataset S1). Twenty-eight lysines were modified only by a light acetyl group, includingmost previously identified acetylation sites on histone proteins (29,30), the known autoacetylation site of Esa1 (K262) (31), plus anadditional 12 sites on six nonhistone proteins. Five of these 12acetylation sites had also been identified in a recent yeast acety-lome study (4). Because these proteins copurify with NuA4 itsuggests, but does not confirm, that their acetylation could dependonNuA4. Thirty-three lysines were modified by only a heavy acetylgroup; as we detected no other KAT enzymes in the NuA4 proteininteraction network (Fig. 2 and Dataset S1), heavy acetylationimplicates NuA4-dependent catalysis in the acetylation of thesesites in vitro. The set of heavy acetylation sites includes: 13 lysineson seven NuA4 interacting proteins, of which two were previouslyidentified in vivo (4) (Table 1); two heavy sites on histone H2A(Hta1 K124, K127), a protein acetylated by NuA4 in vivo at K5(32) and an in vivo modification reported on the conserved K127residue of human histone H2A (30); and 18 heavy sites on fiveNuA4 subunits themselves. The remaining six acetyl lysine resi-dues, identified on five proteins, were represented by peptidesmodified by both light and heavy acetyl groups. These sites rep-resent strong candidates for NuA4-dependent acetylation targetsin yeast cells, as their in vivo occurrence suggests biological rele-vance, and their heavy acetylation in vitro indicates NuA4 cancatalyze the reaction. All but one of these proteins (Pab1, K7)belongs to the NuA4 complex. Importantly, this set includes theYng2 K170 site, a previously described acetylation target of Esa1in vivo (33). Taken together these data serve to expand ourknowledge of lysine acetylation in yeast by identifying in vivoacetylated proteins and putative targets of NuA4.A unique feature of the acetylation dataset is that it directly links

specific acetylation sites to the catalytic activity of NuA4. Toidentify preferences that may contribute to NuA4 acetylation siteselection, we performed an enrichment analysis on the amino acidsequence surrounding heavy acetyl lysine residues. Previous en-richment analyses have focused on high-throughput acetyl lysinedatasets where the KAT responsible for each identified modifi-cation was unknown (3, 7, 30). Using all heavy acetylation sitespresented in this work, which includes the acetylation sites out-

lined above as well as those identified using the inverse KAT-mChIP described below (Tables 1 and 2 and Tables S2 and S3), wecompared the local amino acid sequences surrounding heavyacetyl lysine residues (six residues to the left and right) for biases ateach position.We identified significant enrichments (Table S2) forlysine residues and small amino acids (serine and alanine) imme-diately surrounding heavy acetyl lysines (Fig. 2C). In the absence ofa clearly defined acetylation motif, one interpretation of this resultis that NuA4 may recognize some other cognate feature of itssubstrate rather than a specific motif surrounding the targetedlysine. Indeed, noncatalytic NuA4 subunits have been implicatedin targeting the complex to specific genomic loci (34), supportingmultiple modes of recognition of acetylation targets.

Inverse Application of mChIP-KAT-MS Provides an Alternative Strategyto Identify Acetylation Sites. More than half of the acetylation siteswe identified by NuA4 mChIP-KAT-MS analysis occurred onNuA4 subunits (Fig. 2A, Table 1, and Dataset S1), likely resultingfrom their relative abundance due to tight copurification withEsa1-TAP and additional supplementation for the in vitro KATreaction. We hypothesized that acetylation sites on other non-NuA4 physical interactors, generally identified at much lowerabundance (Fig. S2), may have been missed. To address this the-ory, we chose four preys from theNuA4 interactome (Msn4, Gds1,Cnm67, and Spc72), and two additional proteins, Cdc11 and Shs1,subunits of the septin protein complex we recently linked to NuA4function (11), and used an inverse mChIP-KAT-MS strategy. Inthis approachmChIP samples of the six TAP-tagged proteins wereindividually subjected to NuA4 in vitro KAT assays, followed byLC-MS/MS to identify acetylation sites. For Msn4, Gds1, and theseptin proteins, only silver-stained bands corresponding to the preyproteins were subjected to tryptic digest and LC-MS/MS; but in thecase of the SPB proteins, each silver-stained lane was subdividedinto 12 bands and all were processed. From our initial NuA4mChIP-KAT-MS dataset (Fig. 2 and Table 1), only Gds1 harboredacetylation sites, all four of which were heavy. We hypothesizedthat the increased abundance resulting from the inverse mChIP-KAT-MS approach would enable identification of previously un-discovered sites of light or heavy acetylation. Indeed, we identifiedmultiple acetylation sites on the bait proteins, as well as additionalsites on NuA4 subunits and on proteins that copurified with thebait proteins (Tables 2 and 3, and Dataset S2). On the stress re-

Fig. 2. The acetyl lysine-enriched NuA4-associated proteinnetwork. (A) The 13 NuA4 subunits, including the baitprotein Esa1-TAP, are represented by black nodes (TopLeft). Interacting proteins are grouped by cellular process(node color/label) and further organized into complexeswhere appropriate (circles). Eighty-four protein inter-actions are represented in the map, 57 of which copurifiedin a minimum of two of six experimental replicates (largenodes); and 27, although only identified in one of sixreplicates, belong to reproducibly copurifying proteincomplexes (i.e., Spc29 of the SPB complex). Furthermore, allproteins harboring one or more acetyl lysine residues areincluded in the map. Previously identified NuA4 proteininteractions are indicated by a black circle around the node(as published at www.theBiogrid.org v3.1.70). Only physi-cal interactions for Esa1, Yng2, Epl1, and Eaf1 were con-sidered, because these proteins function solely withinNuA4 or the related PicNuA4 complex in vivo. (B) Cellularlocalization of proteins in the network. Localization an-notation is based on a global study (62) (see Dataset S2 forindividual annotations). The 13 NuA4 subunits were ex-cluded in this analysis. (C) Frequency distribution of aminoacids surrounding heavy acetyl lysine residues. Frequencyof amino acids (y axis) spanning positions −6 to +6 (x axis)surrounding heavy acetyl lysine residues identified within the NuA4-associated protein network (red tag). Residues in green: basic; red: hydrophobic; pink:small; blue: S/T; black: all other residues.

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sponsive transcription factor Msn4, the transcriptional activity ofwhich we previously demonstrated to be repressed by NuA4 undernonstress conditions (20), we identified two heavy acetyl lysineresidues (Table 2). On the uncharacterized protein Gds1, weidentified six heavy and one light acetyl lysine residues, importantlyreproducing three of the four heavy sites found in the initial NuA4mChIP-KAT-MS experiment (Table 2). Although it is likely thatthe heavy acetylation sites identified on Gds1 and Msn4 are be-cause of NuA4, as peptides of Sgf73 and Spt20 (subunits of theKAT SAGA/SLIK) were identified (Table 3) it is possible that thein vitro acetylation sites detected on these baits could be de-pendent on SAGA/SLIK. Through Cnm67 and Spc72 we purifiedalmost all subunits of the SPB complex and many associated pro-teins (Dataset S3). Nine unique acetylation sites on seven SPB orSPB-associated proteins, including seven heavy sites, were identi-fied (Table 2). Two of the heavy sites were identified on Cnm67,which was previously implicated as a NuA4 target in the in vitroprotein microarray analysis (13), and more recently shown to beacetylated in vivo (12). Notably, application of the inverse mChIP-KAT-MS approach to the two yeast septin proteins (Cdc11, Shs1)revealed two heavy acetylation sites (Table 2), including one thatoverlaps with a previously identified known in vivo site (Shs1 K488)(11). Furthermore, this analysis enabled the identification of anadditional 18 sites on NuA4 subunits, including one Epl1 lysineresidue (K427) represented by peptides harboring both heavy and

light acetyl moieties (Table 2) and one Eaf3 lysine residue (K156)that was recently identified as an in vivo site (4). In addition to theacetylation sites found on target preys and NuA4, 53 additionallysine acetylation sites were identified on copurifying proteins,including 16 in vivo and 37 in vitro (Table 3 and Dataset S2). Insummary, the identification of 20 acetylation sites on 12 targetproteins, 18 acetylation sites on NuA4 subunits (Table 2 andDataset S2), and 53 acetylation sites on copurifying proteins,validates the inverse approach as an effective variant of mChIP-KAT-MS to identify acetyl lysine residues.

NuA4 Autoacetylation. To date, the biological significance of Esa1-dependent acetylation has been reported on two NuA4 subunits(Yng2 K170 and Esa1 K262) (31, 33). Additionally, a radioactivein vitro KAT assay using piccolo NuA4 (PicNuA4) suggested Epl1may also be an Esa1 target (35). Here we confirm these results andidentify a plethora of previously undescribed acetylation sites onseven other NuA4 subunits. In total, we identified 42 acetyl lysineresidues (30 heavy sites, 6 light sites, and 6 sites modified by bothheavy and light acetyl groups) (Tables 1 and 2, and Tables S2 andS3). This identification includes both previously reported sites onEsa1 and Yng2 (among other acetylation sites on these proteins),as well as a total of 20 acetylation sites on Epl1. Furthermore, weidentified acetylation sites on Eaf1, Eaf3, Eaf5, Eaf7, Swc4, Arp4,and Tra1 (Tables 1 and 2, and Tables S2 and S3). To confirm

Table 1. Acetyl lysine residues in the NuA4-associated protein network

Protein Acetylation sites Description

Light (in vivo) acetylationH2A (Hta2) K5*,†, K9*,† HistoneH2B (Htb2) K7*,†, K8†, K12*,†, K17*,†, K22*,†, K23*,† HistoneH3 (Hht1) K19*†, K24*†, K28† HistoneH4 (Hhf1) K6*,†, K9*,†, K13*,†, K17*,† HistoneRps12 K114, K125 RibosomeSec7 K1237, K1238 TransportSsb1 K466, K538 or K539 Protein foldingSsb2 K428† Protein foldingEsa1 K262*,† NuA4Epl1 K345†, K353†, K376, K379 NuA4Tra1 K552† NuA4

Heavy (in vitro) acetylationGds1 K343 or K345, K348, K351, K354 UnknownHca4 K570 RNA processingNop4 K144 RNA processingRpl31A K86, K102 RibosomeRps12 K95 RibosomeSas10 K10 RNA processingSsb1 K90 or K95, K571 or K573 Protein foldingH2A (Hta1) K124, K127* HistoneEsa1 K82, K96 or K97, K97 NuA4Epl1 K96, K100, K116, K118, K395, K446, K470, K496, K512, K569, K721 NuA4Eaf1 K280 NuA4Arp4 K350 NuA4Eaf3 K45, K54 NuA4

Light and heavy (in vivo and in vitro) acetylationEsa1 K135 NuA4Eaf5 K3 NuA4Epl1 K39 NuA4Yng2 K170*, K208 NuA4Pab1 K7 RNA processing

Boldface K represents acetyl lysine residues identified in S. cerevisiae acetylome study (4); ‘or’ indicates thatunambiguous assignment of the acetyl group to one of two lysine residues within a single peptide was notpossible.*Acetyl lysine residues previously identified in vivo (30, 33, 60, 61).†Cannot be distinguished from trimethylation. Note, position number of lysine corresponds to position listed inSGD (www.yeastgenome.org).

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hyperacetylation of NuA4 subunits by Esa1, we performed an invitro KAT reaction on NuA4, stringently purified from yeast,using radiolabeled acetyl CoA. We detected acetylation on Yng2,Eaf5, Eaf1, and Epl1, and one or both of the comigrating pro-teins Esa1-TAP/Eaf7 and Arp4/Swc4 (Fig. 3A). Using antiacetyllysine antibodies to detect acetylation, we observed similar re-sults (Fig. S3).Epl1 was by far the most abundantly acetylated protein in vitro

in the mChIP-KAT-MS data and we also identified two over-lapping heavy/light acetylation sites (K39 and K427) (Tables 1 and2). To assess the in vivo dependence of Epl1 acetylation on Esa1,we introduced a mutant allele of the essential ESA1 gene, esa1-L254P (19), into a strain expressing EAF5-TAP. esa1-L254Pexhibits reduced KAT activity both in vivo and in vitro at thepermissive temperature of 25 °C and is catalytically inactive at37 °C (19). We purified the NuA4 complex through Eaf5-TAP anddiscovered that acetylation of Epl1 was higher in the ESA1 strainbackground compared with the level observed in the esa1-L254Pmutant, even at the permissive temperature of 25 °C (Fig. 3B, lane4). Because equal amounts of Epl1-HA (hemagglutinin) cop-urified with Eaf5-TAP (Fig. 3B, lanes 3 and 4), this finding suggeststhat Epl1 is a bona fide acetylation target of Esa1 in vivo. Epl1contains the highly conserved enhancer of polycomb A (EPcA)domain, spanning residues 50–380, which serves to bridge thephysical interaction between Esa1 and Yng2 and its expression issufficient for cell survival (36, 37). To assess the contribution ofacetyl lysine residues identified within this domain, we generatedarginine and glutamine point mutants to block and mimic lysineacetylation, respectively. The detectable acetylation signal on theEpl1 mutants EPcA-R (K39, 345, 376, 379R) and EPcA-Q (K39,345, 376, 379Q) was virtually eliminated (Fig. 3B). This resultsuggests that these acetylation sites account for the majority of the

signal (detected by this antiacetyl lysine antibody) in the fraction ofEpl1 that copurifies with Eaf5-TAP under these growth con-ditions. We next sought to determine the function of Epl1 acety-lation within the EPcA domain. Although the EPcA-Q and EPcA-R mutants do not impact coimmunopurification with Eaf5-TAP,global histone H4 acetylation was reproducibly decreased in thesestrains, with the greatest reduction seen in the esa1-L254P andEPcA-Q strains (Fig. 3B, lanes 4 and 6). To confirm that the defectin histone acetylation is direct, we performed an in vitroKATassayusing purified oligonucleosomes from HeLa cells with NuA4complexes purified from yeast through Eaf5-TAP. In agreementwith the decrease in global H4 acetylation, NuA4 containing Epl1-EPcA-Q displayed the greatest reduction in in vitro KAT activity(Fig. 3C). Furthermore, the strain expressing Epl1-EPcA-Q dis-played mild temperature sensitivity (Fig. S4), which is consistentwith a decrease rather than an abolishment of acetylation activity.This finding suggests the acetylation state of one, all, or a subset orthe lysines in the EPcA domain (K39, 345, 376, 379) are contrib-uting to the catalytic activity of NuA4, which may reflect subtledefects in Yng2 association with or misorientation, with Epl1resulting in reduction in catalysis. Furthermore, although autoa-cetylation has be largely associated with activation of MYST KATcatalytic activity (31), our work suggests that some lysine acetyla-tion found on KAT complexes may play subtler roles in regulatingfunction, including negatively regulating activity.

NuA4 Is Functionally Connected to the SPB in Yeast.Copurification ofmultiple SPB and SPB-associated proteins with NuA4 (Fig. 2A),coupled with NuA4-dependent acetylation on several of theseproteins (Table 2), suggests that NuA4may regulate some aspect ofSPB function via direct acetylation. The SPB, equivalent to themammalian centrosome, is the sole microtubule-organizing centerin budding yeast and serves as a platform for nucleation of both

Table 2. Acetyl lysine residues identified using the inverse mChIP-KAT-MS approach on Baitproteins and NuA4

Protein Acetylation sites Sequence coverage* (%) Description

Light (in vivo) acetylationSpc110 K331 66 SPBSpc72 K590 35 SPBGds1 K87 43 UnknownCdc10 K128, K166 56 SeptinCdc12 K251 80 SeptinEpl1 K345 — NuA4

Heavy (in vitro) acetylationCnm67 K128, K433 72 SPBNud1 K35 54 SPBSfi1 K868, K869 28 SPBSpc42 K13 66 SPBBfa1 K328 46 SPBMsn4 K357, K557 33 TranscriptionGds1 K348, K351, K354, K365, K366, K408 43 UnknownShs1 K488† 73 SeptinEaf1 K848 — NuA4Eaf3 K156 — NuA4Eaf7 K343†, K381, K399, K409 — NuA4Epl1 K16, K429, K821 — NuA4Swc4 K345, K346, K350 — NuA4Yng2 K145, K146, K170 — NuA4Esa1 K97 — NuA4

Light and heavy (in vivo and in vitro) acetylationCdc3 K3 76 SeptinEpl1 K427 — NuA4

*Sequence coverage is not given for NuA4 subunits as they were identified in multiple MS runs and coveragevaried. Boldface K represents acetyl lysine residues identified in S. cerevisiae acetylome study (4).†Acetyl lysine residues previously identified in vivo (30, 33, 60, 61).

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nuclear and cytoplasmic microtubules (38), playing a critical role inchromosome segregation (23) (Fig. 4A). A connection betweenNuA4 and chromosome segregation has been established. SomeNuA4 mutant genes are sensitive to the microtubule-destabilizingdrug Benomyl (32, 39–44), have elevated rates of chromosome loss(39), and display synthetic genetic interactions with genes thatimpact microtubule dynamics (BIK1, CIN8, BIM1) and the spindleassembly checkpoint (BUB1,BUB2,BUB3,MAD1,MAD2) (20, 33,45, 46). To confirm the interaction between NuA4 and SPB, weperformed a reciprocal coimmunopurification with cells expressingendogenously tagged SPB components, Cnm67-TAP and Spc72-TAP, as well as a MYC-tagged (c-Myc epitope) NuA4 subunit,Eaf7. IgG-coated magnetic beads were used to immunopurify theTAP-tagged proteins, and Western blot analysis confirmed thecopurification of Eaf7-MYC with both SPB bait proteins (Fig.4B). Taking these data together with the unbiased identificationof multiple SPB components through Esa1-TAP in the NuA4interaction network (Fig. 2A), and a previously reported yeasttwo-hybrid interaction between Yaf9 and the SPB protein Mps2(42), we conclude that NuA4 and the SPB physically interact.As the sole microtubule-organizing center in yeast, disruption

of SPB function by mutation of any subunit can lead to specific

defects in nuclear or astral microtubule dynamics that can bemonitored by fluorescence microscopy in cells expressing a greenfluorescent protein (GFP)-tagged tubulin protein. To assess spe-cific defects associated with loss of NuA4 acetylation, we examinedcells expressingGFP-Tub1 using fluorescencemicroscopy in eithera wild-type strain or a strain harboring the mutant allele esa1-L254P described above (19). In unbudded and small-budded cells,we observed no difference in microtubule morphology betweencells expressing the wild-type or mutant alleles of ESA1. However,in large-budded cells in which the mitotic spindle had begun toextend, we observed multiple defects in esa1-L254P mutant cells(Fig. 4C). Specifically, although 90% of large-budded wild-typecells had straight anaphase mitotic spindles, extending continu-ously to opposing edges of themother and daughter bud, only 40%of large-budded mutant cells displayed this expected morphology.Rather, in about 5% of cells, the mitotic spindle had a “hooked”phenotype, 30% of cells exhibited bent or broken spindles, and inabout 20% of cells the spindle was characterized as “mis-ex-tended,” as it had elongated within the mother cell (Fig. 4C).Similarly, a high-throughput screen to identify mutants withmicrotubule defects uncovered the hooked microtubule pheno-type in multiple nonessential NuA4 mutants (47). In agreement

Table 3. Acetyl lysine residues identified using the inverse mChIP-KAT-MS approach oncopurifying proteins

Protein Acetylation sites Purification Description

Light (in vivo) acetylationBir1 K920 SPB Chromosome segregationCdc1 K451 Septin Cell cycleDbp10 K76 SPB Ribosome biogenesisHac1 K38 SPB Transcription factorHul5 K24, K31 SPB Ubiquitin ligaseItc1 K317, K319 SPB Chromatin remodelingLtv1 K453 Septin Ribosomal processingMss2 K348 SPB Electron transport chainNam2 K200 SPB tRNA synthetaseNup159 K5 SPB Nuclear pore complexRad24 K291 Gds1 cell cycleRps18A K56 SPB RibosomeSrf1 K206, K214 Septin Phospholipase D regulation

Heavy (in vitro) acetylationNop1 K85, K102 SPB/Septin Ribosome biogenesisNup116 K796 SPB Nuclear pore complexNup53 K392, K394, K399 SPB Nuclear pore complexPbp1 K433 SPB RNA processingRpl2A K155 SPB RibosomeRpl35B K5, K14 SPB RibosomeRpl4A K343 SPB RibosomeRpl8A K15 SPB RibosomeRpl8B K23 SPB RibosomeRps1A K45 SPB RibosomeRps25A K21 SPB RibosomeRtt106 K315 SPB Histone modificationSfi1 K868, K869 SPB SPBSgf73 K180, K199, K211, K212 Msn4 SAGA complexSkt5 K634 SPB Chitin biosynthesisSpt20 K570, K573, K574 Msn4/Gds1 SAGA complexSpt5 K317, K318, K803 SPB/Septin DSIF complexStm1 K74 SPB Stress responseTef2 K454, K457 SPB eEF1ATif4631 K153 SPB eIF4GVid31 K141 SPB Transcription regulationXrn1 K1272 SPB RNA processingYer138C K394, K396 SPB/Septin Retrotransposon

Boldface K represents acetyl lysine residues identified in S. cerevisiae acetylome study (4).

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with a role for reversible acetylation playing a relevant role inmicrotubule function, deletion of RPD3, a proposed NuA4KDAC antagonist (33), suppresses the Benomyl sensitivity ofeaf1Δ mutant cells and partially suppresses the Benomyl sensi-tivity of esa1 mutant cells, and the single rpd3Δ mutant displaysremarkably robust growth on this microtubule-destabilizing drug(Fig. 4D). Further deletion of RPD3 partially rescues the micro-tubule defects of esa1-L254P mutant cells (Fig. S5). Importantly,our work connects NuA4-dependent acetylation to this pheno-type, and moreover indicates the critical importance of lysineacetylation, as only a moderate reduction in NuA4 acetyl-transferase activity results in dramatic, pleiotropic defects.

DiscussionAcetylome studies aimed at identifying acetylated lysine peptidesin vivo have dramatically expanded our knowledge of acetylationsites (3–10); however, the identity of the KATs responsible forthese acetylation events remain elusive. Even in studies comparingthe acetylome of wild-type versus KAT mutant strains, differen-tially acetylated peptides may not represent direct targets of themutant KATs. Here we describe a unique method called mChIP-KAT-MS that enables the generation of a KAT protein-interactionmap enriched for lysine acetylation. Importantly, we have in-corporated an isotopically labeled acetyl CoA into the in vitroKAT reaction, thus allowing the differentiation of preexisting invivo acetyl lysine residues from those resulting from in vitro ca-talysis. In a nutshell, the mChIP-KAT-MS connects KATs to cel-lular pathways, targets and acetylated lysine residues and shouldbe universally applicable to the study of KATs in all model systems.

Validation of the mChIP-KAT-MS Methodology.We chose to validatethe mChIP-KAT-MS methodology on the S. cerevisiae KATNuA4. Before this work, the known protein interactions of NuA4were minimal. High-throughput affinity purification surveys inyeast (17, 48–50) had identified a limited number of NuA4 in-teraction partners, and thus most published interactions were de-fined in the course of directed experiments (20–22, 51). The workwe present here dramatically increases our knowledge of physical

interaction partners for the NuA4 complex (Fig. 2A), as itencompasses most previously known interactors and identifiesover 70 hitherto unknown ones (Fig. 2A). It is important to notethat although some of the proteins in the network directly interactwith at least one NuA4 subunit [e.g., Msn4 (20)], many others—inparticular those belonging to protein complexes (e.g., the SPB)—could have copurified through an intermediate protein in thenetwork. Moving forward, additional studies will be able to dis-tinguish these possibilities; however, proteins modified by heavyacetylation represent an excellent candidate list of direct proteininteraction with Esa1. Further, we cannot exclude the possibilitythat at least some interactors presented in this network copurifywith Esa1 as part of PicNuA4, theKAT-competent trimer complex(Esa1-Epl1-Yng2) thought to globally acetylate chromatin (36).Finally, it is also possible that some interactions may be mediatedthrough DNA, although the nonnuclear localization pattern ofmore than half of the proteins in the network (Fig. 2B) suggeststhis is not the case for the majority of the interactors.NuA4 has been linked to a wide variety of cellular processes,

but the molecular pathways in which the complex directly func-tions are largely unclear. Importantly, the physical interactionsidentified in this study may help to explain the phenotypes andgenetic interactions associated with the complex and also impli-cate NuA4 in new biological pathways. For example, NuA4 hasbeen implicated in chromosome stability (39, 52) andmicrotubuledynamics (47), but our identification of the physical interactionbetween NuA4 and the SPB strongly suggests that NuA4 is notmediating its pleotropic effects on the spindle solely throughhistone acetylation or transcriptional events. Rather, NuA4 maybe regulating many aspects of chromosome segregation throughmultiple nonhistone targets, for which our interactome/acetylomemay provide valuable clues for deciphering this complex cel-lular event.In addition to the unique NuA4 interactions we confirmed, the

network also suggests additional roles for NuA4. For example, weidentified a large number of proteins involved in RNA process-ing, including four proteins that together bind to and regulatepolyadenylation of mRNA (Pab1, Pbp1, Pbp4, Lsm12) (26), as

Fig. 3. NuA4 subunits are acetylated in vivo and invitro. (A) Radioactive KAT assay to assess NuA4autoacetylation in vitro. [3H]-acetyl CoA was addeddirectly to bead matrix of a stringently immunopuri-fied NuA4 preparation [Esa1-TAP (YKB440)] versus anuntagged control (YPH499) immunopurification. His-tone proteins were added to the reaction to serve asa positive control for acetylation. The reactions wereseparated on a gradient gel, Coomassie-stained tovisualize proteins (Right), treated for fluorography,andfinally exposed tofilm for 2wk (Left). Proteins areidentified on the right side and protein size is in-dicated on the left (kilodaltons). (B) Epl1 acetylationis dependent on Esa1 in vivo. NuA4 was purifiedfrom cells grown at 25 °C through Eaf5-TAP (lanes2–6) relative to an untagged control sample (lane 1,YPH499). Epl1 was expressed from its endogenouslocus (lane 2, YKB1042), as a C-terminal HA fusionprotein in the presence (lane 4, YKB2876) or absence(lane 3, YKB2862) of the acetyltransferase-deficientesa1-L254Pallele, or as a lysine-to-arginineor -glutaminemultipoint mutant [EPcA-R; EPcA-Q (K39,345,376,379Ror Q) YKB2781 and YKB2782]. Immunopurified (IP)products and whole cell extract (WCE) were separateby SDS/PAGE (7.5%) and subjected to Western blotusing the indicated antibodies: antiacetyl lysine(α-AcK), antihistone H4 acetyl lysine (α-AcK H4), anti-glyceraldehyde 6-phosphate dehydrogenase (α-G6PDH).(C) Epl1-EPcA-Q reduces NuA4 in vitro KAT activity. NuA4 was purified through Eaf5-TAP (strains described in B). KAT assays were performed using NuA4preparations equalized for Esa1 and nucleosome purified from HeLa cells. Error bars represent SD from duplicate reactions. NuA4 complexes used in assay wereseparated by SDS/PAGE and subjected to Western blot using the anti-Esa1 antibody (α-Esa1) (Lower).

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well as proteins involved in both mRNA decay (Lsm1) and rRNAmaturation (Sik1, Hca4, Pwp1, Rcl1) (Fig. 2A). Intriguingly, Esa1acetylates Pab1 in vitro at K7, which is a modification we also detectin vivo. These physical interactions are the first indication that NuA4may participate directly in one ormore aspects ofmRNAprocessing.It is of interest to note that both Esa1 and Eaf3 contain chromodo-mains, best known for their methylated histone-binding capability,but are also able to bind RNA (53). Another pair of protein com-plexes identified in the NuA4-associated network are the COPI andCOPII transport complexes, which promote vesicle formation andcargo transport at the Golgi and endoplasmic reticulummembranes,respectively (54). These interactions may provide functional insightinto previously reported synthetic lethal genetic interactions thatsuggest a role for NuA4 in vesicle-mediated transport, and pheno-typic analysis indicating that acetyltransferase-deficient NuA4mutants have defects in vacuole morphology (20).

mChIP-KAT-MS to Identify Putative Acetylation Targets.OurmChIP-KAT-MS methodology addresses two hurdles associated withdissecting pathways mediated by lysine acetylation: the reducedlikelihood of detecting low-abundance acetyl lysine residues bymass spectrometry and the need to identify the KAT responsiblefor catalysis. Specifically, the heavy in vitro KAT reaction, makinguse of 13C2-acetyl CoA, addresses these issues by: (i) enriching thepool of acetyl-lysine residues within the copurified proteins; and(ii) covalently attaching an isotopically labeled acetyl group, whichunequivocally distinguishes in vitro catalysis from preexistingmodifications that originated in vivo. Therefore, the mChIP-KAT-MS technique not only builds on the success of the KAT in vitroprotein acetylation microarray (13) in predicting novel substrates,

but also introduces significant advantages. Specifically, our meth-odology defines both a list of substrates and the specific lysineresidues modified by acetylation, because mass spectrometry isused to detect the modification. Moreover, because enzymaticactivity demands a physical interaction between the substrate andenzyme in vivo, acetylated proteins identified using mChIP-KAT-MS inherently meet this important constraint, thereby providinga highly relevant functional connection in vivo between NuA4 andthe acetylated proteins. As proof for the relevance of the KAT-mChIP-MS in finding relevant in vivo substrates, we show that thein vivo acetylation status of Epl1 (Fig. 3) is dependent on NuA4acetyltransferase activity.We postulate the low abundance, and hence minimal MS pro-

tein sequence coverage of many NuA4 copurifying proteins, mightresult in undetected acetyl lysine residues. To this end, we used aninverse application of the mChIP-KAT-MS to identify both in vivoacetylation sites and NuA4-dependent in vitro acetylation siteson SPB and septin subunits, plus Msn4 and Gds1 (Table 2). Al-though themajority of the in vitro sites we identified have, as of yet,no known biological relevance in vivo, similar approaches usingkinases have yielded biologically relevant targets (55). Indeed ofthe 158 lysine acetylation sites identified in this study (DatasetS4), more than 20% of sites were identified as in vivo sites in aS. cerevisiae acetylome study (4). As one would predict, the overlapbetween the sites identified in the acetylome and ours was greaterfor sites that we identified as being both heavy and light peptides(50%). The overlap decreased to 34% for in vivo or light sites and14% for NuA4-dependent in vitro sites. As it has been predictedthat most posttranslational modifications likely have no biologicalrole (56), increasingly it will be important to focus on the acety-

Fig. 4. NuA4acetylates spindlepolebodyproteins invitro and regulates spindle dynamics in vivo. (A) Car-toon of the yeast SPB. The relative positions of all coreSPB components are shown with respect to the nu-clear and cytoplasmic faces of the nuclear lipid bi-layer. Astral and nuclear microtubules (MT) emanateinto the cytoplasm and nucleus, respectively. SPBproteins identified in the Cnm67 and/or Spc72 SPBmChIP-KAT-MS experiments are indicated by bluetext, otherwiseprotein names are in black.Acetylatedproteins are noted (as described in the legend). (B)NuA4 interacts with the SPB in vivo. Protein extractsexpressing the indicated tagged proteins [Bbp1-TAP(YKB1996), Spc72-TAP (YKB1999), Eaf7-MYC (YKB518),Bbp1-TAP Eaf7-MYC (YKB1296), Spc72-TAP Eaf7-MYC(YKB1306)] or an untagged control (no TAP tag)(YPH499) were immunoprecipitated with magneticbeads coated with IgG antibodies that recognizethe protein A component of the TAP tag. Totalprotein extracts (WCE) and immunoprecipitates(α-TAP IP) were resolved by 7.5% SDS/PAGE and sub-jected to Western blot analysis with anti-MYC andanti-TAP (α-MYC, α-TAP, respectively), as indicated atthe right side of the panels. (C) esa1-L254P mutantshave microtubule morphology defects. Fixed cellsexpressing GFP-Tub1 encoding either a wild-type(YKB1233) or mutant allele of ESA1 (YKB1250) (wild-type or esa1-L254P) were examined by fluorescencemicroscopy. Cells were grown to midlog phase instandardYPDmedium supplementedwith adenineat25 °C or 30 °C, as indicated. The average of three ex-perimental replicates is shown and at least 50 large-budded cellswith extendedmicrotubuleswere scoredfor each replicate. (D) Acetylation regulates Benomylsensitivity. Wild-type (YPH499), eaf1Δ (YKB44), esa1(esa-L254P, YKB859), rpd3Δ (YKB1130), rpd3Δeaf1Δ(YKB1154), and rpd3Δ esa1 (YKB2158) cultures werediluted to an OD600 of 0.1 and 10-fold serial dilutionswere plated on YPD plates containing vehicle control(DMSO) or Benomyl, as indicated.

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lation sites identified by multiple approaches. Our ability toidentify previously known acetyl lysine residues, including thosefrom both directed studies and unbiased global proteomic ap-proaches, indicates that in vitro acetylation sites identified bymCHIP-KAT-MS should uncover relevant target sites. Directedstudies assessing the biological consequence of additional acety-lation sites will provide greater insight into this.We postulate that themChIP-KAT-MSmethodology presented

here can serve as a valuable complementary analysis tool in theelucidation of pathways governed by lysine acetylation because ofits ability to connect KATs to their substrates and modified lysineresidues. Pairing this information with phenotypes previously as-sociated with KATs through genetic analyses provides a powerfulsystem toward unraveling the biological consequences of lysineacetylation. Overall, our data suggest that subjecting purificationsof novel interactors to KAT-MS represents a promising tool toidentify novel KAT-specific acetylation sites and to ultimatelydefine novel enzyme–substrate relationships.

Materials and MethodsYeast Strains. Yeast strains used in this study are listed in Table S3. Genomicdeletions or epitope tag integrations made for this study were designedwith PCR-amplified cassettes, as previously described (57).

NuA4 mChIP. NuA4 and its associated protein network were isolated fromexponentially growing yeast cultures through mChIP (16) of endogenouslyTAP-tagged ESA1. Six replicates of the experiment were performed anda flowchart is presented in Fig. S1 with specific details of each replicate.Briefly, cells from 400- to 700-mL midlog phase cultures grown in YPD at30 °C were collected by centrifugation, washed in 25 mL of ice-cold water,transferred to 1.5-mL Eppendorf tubes, and frozen on dry ice. Cell pelletswere resuspended in 300 μL of lysis buffer [100 mM Hepes pH 8.0, 20 mMmagnesium acetate, 10% glycerol (vol/vol), 10 mM EGTA, 0.1 mM ETDA, 300mM sodium acetate, and fresh protease inhibitor mixture (Sigma; P8215)]plus an equal volume of glass beads, and cells were lysed through vortexing(six 1-min blasts with incubation on ice between vortexing). Lysates weresubjected to sonication (3 × 20 s; 1-min incubation on ice between eachpulse) using a Misonix Sonicator 3000 at setting four. Before centrifugation(10 min, 800 × g, 4 °C), Nonidet P-40 was added to a final concentration of1% (vol/vol). Next, 40–150 mg of whole-cell extract was incubated with 100–600 μL magnetic beads (Invitrogen; 143.02D) cross-linked to rabbit Ig (IgG)(Chemicon; PP64) as per the manufacturer’s instructions. Following 2 h of end-over-end rotation at 4 °C, the beads were collected on a magnet and washedthree times with 1 mL of ice cold wash buffer [100 mM Hepes pH 8.0, 20 mMmagnesium acetate, 10% glycerol (vol/vol), 10 mM EGTA, 0.1 mM EDTA,300 mM sodium acetate, 0.5% Nonidet P-40]. At this point, immunopurifiedproteins were either eluted from the magnetic beads in 1× loading dye[50 mM Tris, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% (vol/vol) glycerol]with gentle heating (65 °C for 10 min) (three replicates); eluted from themagnetic beads by incubating in 1 mL of elution buffer (0.5 M NH4OH, 0.5 MEDTA) at room temperature for 20 min (one replicate); or washed once in 1 mL1× KAT buffer (50 mM Tris pH 8.0, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT) andsubjected to a KAT reaction using isotopically labeled acetyl CoA (see below;two replicates). The loading dye eluate samples were transferred to new tubesand boiled for 10 min at 95 °C following the addition of β-2-mercaptoethanolto 100 mM. Proteins were separated by SDS/PAGE (NuPAGE Novex 4–12%Bis·Tris Gel; Invitrogen, NP0321), visualized by silver stain, and bands wereexcised and processed for mass spectrometry (see below). The sample in elu-tion buffer was transferred to a new tube and evaporated to dryness usinga speed vac and processed on the proteomic reactor (see below).

High-Stringency Purification of NuA4 from Yeast for in Vitro KAT Assays. NuA4immunopurified from yeast was carried out using Esa1-TAP as previouslydescribed (20), except the complex was eluted from magnetic beads by en-zymatic cleavage in TC Buffer [50 mM Tris, pH 8.0, 1 mM DTT, 0.1% NonidetP-40, 150 mM NaCl, 10% (vol/vol) glycerol] using tobacco etch virus (TEV)protease, which was prepared and generously provided by the laboratory ofJean-François Couture (Ottawa Institute of Systems Biology, University ofOttawa, Ottawa, Canada). Briefly, 1 L of exponentially growing yeast cells (inYPD, at 30 °C) expressing endogenously TAP-tagged ESA1 were lysed andNuA4 was purified in a single step using 600 μL of magnetic beads coupledto IgG. After washing, the NuA4-bead matrix was resuspended in TC buffer(100 μL) to which was added 20 μL of tobacco etch virus (TEV). The cleavage

reaction was incubated overnight at 4 °C with end-over-end rotation. Fi-nally, the supernatant was isolated from the beads, aliquoted, and stored at−80 °C. The purity of each NuA4 preparation was assessed by silver stainusing 2 μL of TEV-cleaved NuA4 separated by SDS/PAGE (7.5%). Activity of allhigh-stringency NuA4 complex preparations was confirmed by performinga KAT reaction using 2 μg of chicken core histones (Upstate; 13–107) and2 μg of standard, unlabeled acetyl CoA (Sigma; A2056) in a final volume of15 μL. The acetylation signal was assessed by Western blot using an anti-acetyl lysine antibody (Upstate; 06–933). An untagged control strain was alsotaken through immunopurification procedure to ensure the purity of thepurification (by silver stain) and to confirm that KAT activity did not non-specifically associate with the IgG-coated magnetic beads.

In Vitro Heavy KAT Reactions for NuA4 mChIP. NuA4 in vitro heavy KAT assaysusing isotopically labeled acetyl CoA (herein referred to as “heavy”) werecarried out with immunopurified proteins still bound to the magnetic beadsby adding to the protein-magnetic bead matrix 5× KAT buffer (250 mM TrispH 8.0, 250 mM NaCl, 25 mM MgCl2, 5 mM DTT), 13C2-acetyl CoA (Isotec;658650), and stringently purified, exogenous NuA4 (see above). As the ex-ogenous NuA4 preparation included the TEV enzyme, immunopurifiedproteins still bound to magnetic beads were enzymatically cleaved duringthe KAT reactions. Heavy KAT reactions were performed on two NuA4mChIP replicates (Fig. S1) and were carried out in a total volume of 100 uLusing 6 μg of 13C2-acetyl CoA and 10 μL of exogenous NuA4 at 30 °C for 1 hwith end-over-end rotation. Samples were separated from the beads andprocessed directly on the proteomic reactor, as described below (58).

Inverse mChIP-KAT-MS. Cnm67-TAP, Spc72-TAP, Msn4-Tap, Gds1-TAP, Cdc11-TAP, and Shs1-TAP were isolated from 700 mL of exponentially growing yeastcultures in YPD at 30 °C using strains expressing endogenously tagged genes.The mChIP procedure was carried out identically as described above for NuA4except 100 μL of magnetic beads coupled to IgG and 100 mg of whole-celllysate were used. Following immunopurification and washing, beads wereequilibrated in 1× KAT buffer, and then subjected to a heavy KAT reaction ina final volume of 20 μL including 5 μL highly purified exogenous NuA4, 2 μgof 13C2-acetyl CoA, and 4 μL 5× KAT buffer. Following incubation at 30 °C for1 h with end-over-end rotation, an equal volume of 2× loading dye wasadded directly to the beads and the samples were heated at 65 °C for 10 min.The loading dye eluate samples were transferred to new tubes and boiled for10 min at 95 °C following the addition of β-2-mercaptoethanol to 100 mM.Inverse mChIP-KAT-MS assays were performed once and proteins were sep-arated by SDS/PAGE (NuPAGE Novex 4–12% Bis·Tris Gel; Invitrogen, NP0321),and visualized by silver stain. For the Cnm67-TAP and Spc72-TAP, entire laneswere excised and digested (23 bands in total for the two samples). For Cdc11-TAP and Shs1-TAP, 11 bands were processed, and for Gds1-TAP and Msn4-TAP, only the bait protein band was analyzed. To identify the proteome ofGds1-TAP, the mChIP procedure was repeated without the addition of KATassay, and the entire lane was excised and digested before MS.

MS to Detect Protein Interactions and Acetyl Lysine Residues. For three rep-licates of NuA4, samples were separated by SDS/PAGE (NuPAGE Novex4–12% Bis·Tris Gel; Invitrogen, NP0321), the entire lane was excised into 15–20bands, reduced, alkylated, and digested as previously described (59). Theother three replicates of NuA4 mChIP, including the two subjected to theheavy KAT assay, were prepared using a proteomic sample processing devicetermed the “proteomic reactor” (58). Briefly, the proteomic reactor enablesthe enrichment, clean up, and chemical and enzymatic processing withincapillary tubing packed with either strong anion or strong cation exchange(SAX or SCX, respectively) beads. Processed peptides were eluted using 10-step pH buffers as described previously (58). One of three replicates wassubjected to both SAX and SCX proteomic reactor conditions, and theremaining two subjected to only SAX conditions (Fig. S1). LC-MS/MS wasperformed as previously described (16) using an Agilent 1100 HPLC system(Agilent Technologies) coupled to either an LTQ or an LTQ-Orbitrap XL massspectrometer (Thermo-Electron) as indicated (Dataset S1). Acetylated lysineresidues were identified as previously described (3). MS/MS corresponding toputative lysine acetylation sites were all manually validated.

Lysine Acetyltransferase Assay on Nucleosomes. NuA4 complexes were puri-fied from the indicated Eaf5-TAP strains as described above and eluted fromthe IgG magnetic beads by incubation with TEV protease (an untagged strainwas used for mock purification). Histone acetyltransferase reactions wereperformed in 15-μL final volume of 50 mM KCl, 50 mM Tris (pH 8), 1 mM DTT,5% (vol/vol) glycerol, 10 mM Na-Butyrate, and 0.1 mM EDTA with 500 ng ofpurified H1-depleted oligonucleosomes (from HeLa cells) and 1.25 μL of [3H]

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acetyl-CoA (0.1 μCi/μL, 4.9 Ci/mmol) for 60 min at 30 °C. Amounts of wild-type and mutant complexes used in the reaction were normalized byWestern blot based on the Esa1 signal. Each reaction was spotted onto p81filters, which were then washed three times with 50 mM Na Carbonate (pH9.2). The amount of incorporated [3H] acetyl was determined using a scin-tillation counter. Error bars represent SD from duplicate reactions.

Additional methods are provided in the SI Materials and Methods.

ACKNOWLEDGMENTS. The computation analysis performed by A.B. wascarried out in the laboratory of C. David Allis at Rockefeller University. Thisresearch is funded by Canadian Cancer Society Grant 20309 (to K.B.);

a Natural Sciences and Engineering Research Council of Canada Discoverygrant (to D.A.F.); Canadian Institutes of Health Research (CIHR) OperatingGrant MOP-14308 (to J.C.); a CIHR Canada Graduate Scholarship (to L.M.);CIHR Master’s Awards (to R.P.-L. and A.H.); an Ontario Graduate Scholarshipand a postdoctoral fellowship from the Natural Sciences and EngineeringResearch Council of Canada (to J.-P.L.); and a postdoctoral fellowship fromthe Strategic Training Initiative in Health Research/CIHR Training Grant inNeurodegenerative Lipidomics postdoctoral fellowship TGF-96121 (to Z.N.).K.B. is a Canada Research Chair in Chemical and Functional Genomics, D.A.F.is Canada Research Chair in Proteomics and Systems Biology, and J.C. isa Canada Research Chair in Chromatin Biology and Molecular Epigenetics.

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