MYST Family Lysine Acetyltransferase Facilitates AtaxiaTelangiectasia Mutated (ATM) Kinase-mediated DNADamage Response in Toxoplasma gondii*□S

Received for publication, September 14, 2009, and in revised form, January 14, 2010 Published, JBC Papers in Press, February 16, 2010, DOI 10.1074/jbc.M109.066134

Nathalie Vonlaufen‡1, Arunasalam Naguleswaran‡1, Isabelle Coppens§, and William J. Sullivan, Jr.‡2

From the ‡Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202 andthe §Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health,Baltimore, Maryland 21205

The MYST family of lysine acetyltransferases (KATs) func-tion in a wide variety of cellular operations, including gene reg-ulation and theDNAdamage response.Herewe report the char-acterization of the second MYST family KAT in the protozoanparasite Toxoplasma gondii (TgMYST-B). Toxoplasma causesbirth defects and is an opportunistic pathogen in the immuno-compromised, the latter due to its ability to convert into a latentcyst (bradyzoite). We demonstrate that TgMYST-B can gainaccess to the parasite nucleus and acetylate histones. Overex-pression of recombinant, tagged TgMYST-B reduces growthrate in vitro and confers protection from a DNA-alkylatingagent. Expression of mutant TgMYST-B produced no growthdefect and failed to protect against DNA damage. We demon-strate that cells overexpressing TgMYST-B have increased lev-els of ataxia telangiectasia mutated (ATM) kinase and phosphor-ylated H2AX and that TgMYST-B localizes to the ATM kinasegene. Pharmacological inhibitors of ATM kinase or KATsreverse the slow growth phenotype seen in parasites overex-pressing TgMYST-B. These studies are the first to show that aMYSTKATcontributes toATMkinase gene expression, furtherilluminating themechanismof howATMkinase is up-regulatedto respond to DNA damage.

The function of many proteins is regulated by a variety ofpost-translational modifications. Lysine acetylation is rapidlyemerging as a major post-translational modification in eukary-otic cells for a multitude of proteins beyond histones, in whichthis post-translational modification was first described (1).Lysine acetyltransferases (KATs)3 of the MYST family (named

for founding members MOZ, Ybf2/Sas3, Sas2, and TIP60) arebroadly conserved among all eukaryotes, distinguished by ahallmark acetyltransferase domain that serves as the enzymaticcomponent of several diverse multiprotein complexes. MYSTfamily members were initially characterized as histone acetyl-transferases (HATs) involved in epigenetically mediated tran-scription control but have also been implicated in awide varietyof critical cellular functions, including gene regulation, cellcycle progression, and DNA replication (2).MYSTKATs also play a significant role in the cellular response

to DNA damage and apoptosis. TIP60, in particular, has beenshown to activate ataxia telangiectasia mutated (ATM) kinasethrough acetylation of lysine 3016 (3). HeLa cells expressing a“KAT-dead” dominant negative form of TIP60 lacking acetyl-transferase activity display defective DNA repair and fail toundergo apoptosis (4). If ATM kinase is not activated by acety-lation, the cell fails to activate cell cycle checkpoints throughphosphorylation of DNA damage response proteins (5). It isalso possible that MYST KATs participate in the activation ofATM kinase gene expression by virtue of their HAT activity,but this has not been tested.Insight into the evolution of this vital KAT family is lacking

because very little information is available regarding MYSTproteins in eukaryotic cells of distal origin. Previously, we havefound that the protozoan parasite Toxoplasma gondii (phylumApicomplexa) possesses two MYST KATs, which we havenamed TgMYST-A and TgMYST-B, that have high similarityto plant MYST KATs (7). Both TgMYST KATs contain anN-terminal chromodomain (CHD) and C2H2 type zinc fingerwithin the MOZ-SAS KAT domain (PF01853). Histone modi-fications have recently gained much attention in protozoanparasites because they have been linked to modulating criticalfacets of pathogenesis and have been validated as novel drugtargets (6). For example, in Toxoplasma, histone modificationshave been associated with gene-regulatory events relevant tothe clinically important process of parasite differentiation, theprocess by which rapidly growing tachyzoites convert intolatent encysted forms known as bradyzoites (7).We have previously characterized the TgMYST-A KAT (8).

In this report, we present evidence that links theKATactivity ofTgMYST-B to transcription control, parasite proliferation, andtheDNAdamage response.We find thatToxoplasma possessesan ATM kinase orthologue that is up-regulated by TgMYST-B;this up-regulation is dependent on the KAT activity of

* This work was supported, in whole or in part, by National Institutes ofHealth, NIAID, Award R21AI083732 (to W. J. S.). This work was also sup-ported by American Heart Association Grant 0725725Z (to A. N.) and SwissNational Foundation Grant PBBSA-115870 (to N. V.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table S1 and Figs. S1–S4.

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Indiana University School

of Medicine, 635 Barnhill Dr., MS A-525, Indianapolis, IN 46202. Tel.: 317-274-1573; Fax: 317-274-7714; E-mail: wjsulliv@iupui.edu.

3 The abbreviations used are: KAT, lysine acetyltransferase; HAT, histoneacetyltransferase; MOPS, morpholinepropanesulfonic acid; MES, morpho-lineethanesulfonic acid; ATM, ataxia telangiectasia mutated; CHD, chro-modomain; MMS, methyl methanesulfonate; RACE, rapid amplification ofcDNA ends; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)pro-pane-1,3-diol; DAPI, 4�,6-diamidino-2-phenylindole; ChIP, chromatinimmunoprecipitation; UTR, untranslated region; nt, nucleotide(s).

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 15, pp. 11154 –11161, April 9, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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TgMYST-B. Moreover, TgMYST-B is recruited to the ATMkinase gene and protects the parasites from DNA damage. Wealso present evidence that links the up-regulation of ATMkinase to a slowed growth phenotype observed in parasitesoverexpressing TgMYST-B. Our results emphasize the impor-tance of TgMYST-B in parasite physiology and heighten itsattractiveness as a potential drug target.

EXPERIMENTAL PROCEDURES

Parasite Culture and Reagents—Toxoplasma tachyzoitesused in this study were RH strain and maintained in humanforeskin fibroblasts using Dulbecco’s modified Eagle’s mediumsupplemented with 1.0% fetal bovine serum (Invitrogen). Para-sites were grown in a humidified CO2 (5%) incubator at 37 °C.Cultures were routinely monitored for Mycoplasma contami-nation byMycoAlertTM assay (Cambrex Bio Science). Parasiteswere harvested immediately following lysis of host cell mono-layers and purified by virtue of filtration through a 3.0-�m filter(9). In some cultures, theATMkinase inhibitorKU-55933 (Cal-biochemcatalog number 118500), anacardic acids (Calbiochemcatalog number), methyl methanesulfonate (MMS; Sigma), orvehicle control (DMSO) was added to the media.Parasite Growth Assays—Doubling times were determined

as described previously (9). Briefly, T-25 cm2 tissue cultureflasks containing confluent monolayers of host cells wereinoculated with 105 freshly lysed tachyzoites and incubated.The number of parasites in 50 randomly chosen vacuoles wascounted every 8 h. Toxoplasma growth assays based on detec-tion of the parasite-specific B1 gene (10) were carried out asdescribed previously (11). Briefly, 24-well plates were infectedwith 1,000 parasites/well; each day, genomic DNA from in-fected wells was harvested using the DNeasy kit (Qiagen) andused in SYBR� Green-based quantitative PCR with the 7500real-timePCR system (AppliedBiosystems). The parasite countfor a given samplewas calculated by interpolation from a stand-ard curve (11).Antibodies and Western Blot Analysis—Polyclonal antibody

was generated in rabbit to a polypeptide sequence correspond-ing to the C-terminal 100 amino acid residues of TgMYST-B(amino acids 423–523) at Quality Controlled Biochemicals(Hopkinton, MA). Raw antiserum was affinity-purified on thepeptide immobilized on Affi-Gel-15 (Bio-Rad). Specificity ofthe affinity-purified antibody was characterized by immuno-stainingWestern blots containing 20�g of parasite lysate. Anti-body to Toxoplasma tubulin (used at 1:1,000) was provided byDavid Sibley (WashingtonUniversity, St. Louis,MO) to serve asa protein loading control.Mousemonoclonal antibody to phos-phorylated H2AX was from Millipore (05-636) (12). Mousemonoclonal antibody to ATM kinase (2C1 (1A1); used at1:2,000) was purchased from Abcam (ab78). Appropriate anti-mouse or anti-rabbit horseradish peroxidase-conjugated sec-ondary antibodies were employed alongwith the ECL detectionsystem to visualize results (GE Healthcare). Western blottingfor all applications was performed using NuPAGE 4–12% or10% SDS-polyacrylamide gels using MOPS or MES runningbuffer (Invitrogen). For ATM kinase Western blots, proteinswere separated on 4–7% Tris acetate gels (Invitrogen).

Cloning, Expression, and Purification of TgMYST-B—RNAligase-mediated rapid amplification of cDNA ends (RACE) wasperformed using GeneRacer (Invitrogen). Amplified productswere gel-purified, subcloned into TA-TOPO vectors (Invitro-gen), and sequenced. Nucleotide sequencing was performed onboth strands at the Indiana University Biochemistry Biotech-nology Facility (GenBankTM accession numberAAZ79483). Toexpress and purify recombinant epitope-tagged TgMYST-Bfrom Toxoplasma, we used a FLAG tag affinity chromatogra-phy approach as described previously (13, 14). TgMYST-B wascloned into a Toxoplasma expression vector containing thetubulin (TUB) promoter and hypoxanthine-xanthine-guaninephosphoribosyltransferase (HXGPRT) selection cassette (15,16) to make ptub-fTgMYST-B::HX. The TgMYST-B codingsequenceswere amplified fromToxoplasma cDNAusing a longrange PCR kit (Qiagen) and primers containing NdeI and AvrIIrestriction enzyme sites (in italic type). The 5� primer alsoencoded the FLAG epitope tag (underlined): sense, 5�-ATAC-CATCATATGAAAATGGACTACAAGGACGACGACGAC-AAGCCTGGCGACTCCGCGTCTCCAGTCTG; antisense,5�-ATACCATCCTAGGTCAGTCTTCTTCTCCCCGCCTC-GGCGGCG. RH�HXparasites were stably transfected by elec-troporating 25 �g of the expression vector into 3.0 � 107 para-sites, and cloneswere obtained by limited dilution. fTgMYST-Bwas purified by sonicating parasite pellets in 0.5 ml of 50 mM

Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol (v/v), and 1%Triton X-100 supplemented with a protease inhibitor mixture(Sigma P8340). The parasite lysate was cleared by centrifugingat 13,000 rpm for 10 min at 4 °C prior to mixing with 40 �l ofequilibrated anti-FLAGM2-agarose affinity resin (Sigma). Theresin and lysate were mixed overnight at 4 °C and spun at8,200 � g for 30 s. The supernatant was removed, and the resinwas washed three times in 500 �l of 50 mM Tris-HCl (pH 7.4),150 mM NaCl, and 10% glycerol (v/v)) and once with 500 �l of1�HATassay buffer (50mMTris-HCl (pH8.0), 5% glycerol, 0.1mM EDTA, 50mMKCl, 1mM dithiothreitol, 1 mM phenylmeth-ylsulfonyl fluoride, 10 mM sodium butyrate) at 4 °C. fTgMYST-Bbound to the resin was used directly in HAT assays as we havedone previously (8). To generate the mutant version, fTgMYST-B(E403G), site-directed mutagenesis was performed usingthe QuikChange� II XL kit (Stratagene) according to themanufacturer’s instructions. Western blots were performedwith anti-FLAG to ensure equal expression between fTgMYST-B(E403G) and fTgMYST-B in the transgenic clones used (datanot shown).HATAssay—In vitroHATassaywas performed as previously

described (17). Briefly, FLAG-tagged recombinant protein waspurified from Toxoplasma lysates using anti-FLAGM2 affinitygel (Sigma) for use in an enzymatic HAT reaction containingrecombinant H3 peptide as substrate. HAT reactions wereincubated at 30 °C for 60min and stopped by the addition of 10�l of 4�NuPAGE loading dye (Invitrogen) containing 2.0 �l of�-mercaptoethanol. Samples were separated on 4–12% BisTrisgel with MES buffer and transferred to nitrocellulose mem-brane for immunoblotting with anti-acetyl lysine (AssayDesigns/Stressgen catalog number KAP-TF 120H, used at a1:1,000 dilution). Densitometry values were determined usingKodak 1D version 3.6.3 software (Scientific Imaging System).

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Immunofluorescence and Immunoelectron Microscopy—Tachyzoites were allowed to infect human foreskin fibro-blast monolayers grown on glass coverslips. Immunofluores-cence assays (IFAs) were carried out as described by Bhattiand Sullivan (15), using a 1:100 dilution of affinity-purifiedanti-TgMYST-B, a 1:1,000 dilution of anti-FLAG antibody(Sigma F7425), or a 1:4,000 dilution of SAG1 as primary anti-body. Secondary antibody consisted of anti-rabbit Alexa Fluor488 or anti-mouse Alexa Fluor 594 (Invitrogen). Extracellulartachyzoiteswere examined after coating coverslipswith 50�l of0.1 mg/ml poly-L-lysine. Parasites in 3% paraformaldehyde andPBS were inoculated onto the coated coverslips and allowed tosit at room temperature for 15 min before being processed forIFA as outlined above. For IFAs, 0.3 �M 4�,6-diamidino-2-phe-nylindole (DAPI) was applied for 5min at room temperature inthe dark as a co-stain. Preparations ofToxoplasmawere fixed in4% paraformaldehyde (ElectronMicroscopy Sciences) in 0.25 M

HEPES (pH 7.4) for 1 h at room temperature and then in 8%paraformaldehyde in the same buffer overnight at 4 °C. Forimmunolabeling, samples were processed as described (18)using undiluted anti-MYSTB antibodies, subsequently revealedby 10-nm protein A-gold particles before examination with aPhilips CM120 electron microscope.Real-time Reverse Transcription-PCR—Primers were de-

signed using the Primer Express 2.0 software (Applied Biosys-tems) and are listed in Supplemental Table S1. The reversetranscription reactionwas performed using 1.0�g of total RNAisolated from tachyzoites, oligo(dT) primers, and Omniscriptreverse transcriptase (Qiagen) according to the manufacturer’sdirections. 1 �l of a 1:10 dilution of the resulting cDNA wasamplified in a 25-�l total volume containing SYBR� GreenPCRMaster Mix (Applied Biosystems, CA) and 0.5 �M of eachforward and reverse primer. The Toxoplasma �-tubulin gene(GenBankTM accession number M20025) was used to normal-ize the real-time reverse transcription-PCR (18). All reactionswere performed in triplicate, using the 7500 real-time PCRsystem and analyzed SDS software version 1.2.1 (AppliedBiosystems).Chromatin Immunoprecipitation (ChIP)—ChIP was per-

formed on tachyzoites stably expressing fMYST-B using poly-clonal anti-FLAG antibody (Sigma F7425) immobilized toDynabeads Protein A (Invitrogen). Quantitative PCR was per-formed as described above. Immunoprecipitated DNA sampleswere normalized using a standard curve created with seriallydiluted inputDNA. 0.1 ng of total ChIPDNAwas added to eachreaction, and reactions were performed in triplicate. Primersused for this study are listed in supplemental Table S1.

RESULTS

Characterization of a Second MYST Family KAT inToxoplasma—Previously, we reported a partial coding se-quence for a second MYST family KAT in Toxoplasma, termedTgMYST-B (GenBankTM accession number AAZ79483) (8). Wehave used 5�- and 3�-RACE to acquire the full-length sequence(supplemental Fig. S1). A single amplicon from a nested5�-RACE reaction was sequenced to reveal an in-frame ATGand 5�-UTR of 133 nt. Interestingly, the 5�-UTR contains asingle intron of 358 nt. The 3�-RACE product confirmed the

previously reported stop codon and revealed the 533-nt3�-UTR. The TgMYST-B genomic locus is 4.7 kb, containingseven introns and eight exons. Our results indicate that thecoding sequence of TgMYST-B is 1,572 nt, which is in agree-ment with the ToxoDB 3.0 prediction, TgTwinScan_2400, notthe latest 3,264-nt gene prediction at ToxoDB (TGGT1_023320). The discrepancy between theTGGT1_023320 predic-tion and our cloning results resides at the 5�-end of the gene.Consequently, we performed reverse transcription-PCRs usingprimers designed to clarify the sequence of the 5�-end ofTgMYST-B. Although products downstream of the transcrip-tional start site we determined by 5�-RACE could be amplified,no amplicons could be generated upstream of this (data notshown). Additionally, no expressed sequence tags or proteom-ics data to date support the longer TGGT1_023320 prediction.According to our cloning results, the deduced amino acid

sequence of TgMYST-B yields a protein composed of 523amino acids with a predicted molecular mass of �60 kDa. Pro-tein structure and motif analysis shows that TgMYST-B pos-sesses the expected domains, including the MYST catalyticdomain, and an upstream C2H2 zinc finger and CHD (Fig. 1A).BLASTp analysis reveals that the closest matches are MYSTacetyltransferases from fellow apicomplexan parasite Crypto-sporidium spp. and plant species including Ricinus communis(castor bean) and Arabidopsis thaliana (�e�86). In a phyloge-netic analysis, TgMYST-B groups with a MYST KAT in Cryp-tosporidium parvum, whereas TgMYST-A is more similar to aMYST KAT in Plasmodium species (Fig. 1B). Protein sequencealignments between TgMYST KATs and those found in otherselect species are shown in supplemental Fig. S2. The afore-mentioned MYST KAT motifs are well conserved betweenTgMYST-A and -B, but the intervening sequences and N-ter-minal sequence were less so (supplemental Fig. S3). Addition-ally, there are two insertions in TgMYST-B that are not presentin TgMYST-A between the CHD and zinc finger/MYSTdomain; the first is basic-rich and composed of 55 amino acids,and the second is composed of 24 amino acids.Affinity-purified, polyclonal antibody generated against the

C-terminal 100 amino acids (amino acids 423–523) recognizesa single protein of the expected size (�60 kDa) on an immuno-blot containing Toxoplasma tachyzoite lysate; no cross-reac-tivity to either form of TgMYST-A (49–53 kDa) wasdetected (Fig. 1C). The protein size supports our cloningdata for the true TgMYST-B transcript.TgMYST-B Displays HAT Activity but Is Found Predomi-

nantly in the Cytoplasm—We sought to confirm that TgMYST-Bexhibited the expected enzymatic activity using an in vitroHATassay as described previously (17). Native TgMYST-B isexpressed at very low levels, making it difficult to obtain suffi-cient quantities of enzyme for analysis. Therefore, we engi-neered a transgenic clone stably expressing an ectopic form ofTgMYST-B under control of the strong Toxoplasma tubulinpromoter; the recombinant TgMYST-B was tagged with FLAGat the N terminus (fTgMYST-B). fTgMYST-B was purifiedfrom parasite lysate using anti-FLAG resin. fTgMYST-B servedas the enzyme source in an in vitroHAT assay that uses recom-binant H3 as substrate. After incubation, the HAT reaction wasresolved on SDS-PAGE for immunoblotting. Acetylated H3

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was detectedwith antibody raised against acetylated lysine. Theresults verify that TgMYST-B is capable of acetylating histonesubstrate in vitro (Fig. 2A). The point-mutated version ofTgMYST-B (E403G) is described in detail below.To determine the subcellular localization of TgMYST-B, we

used our affinity-purified polyclonal antibody in IFAs of intra-cellular Toxoplasma. Results indicate that TgMYST-B appearsto be predominantly cytoplasmic with virtually no proteindetectable in the nucleus or apicoplast in either intracellular orextracellular tachyzoites (Fig. 2B). Predominantly cytosolic dis-tribution was also seen in transgenic parasites stably expressingectopic fTgMYST-B (Fig. 2C). For better resolution, we usedthe anti-MYST-B antibody in immune electron microscopy ofintracellular wild type tachyzoites. Density (gold particles/�m2) of labeled structures was determined from 22–26 cellularcryosections. Percentage of cytosolic and intranuclear densitywas determined from the sum of gold density normalized forthe variation in expression of TgMYST-B. Results confirm thecytosolic location for the majority of TgMYST-B (Fig. 2D andsupplemental Fig. S4) but also revealed 20% of gold particles tobe in the nuclei of the parasites, consistent with the well char-acterized HAT activity for a member of the MYST family.

TgMYST-B Protects against DNA Damage Induced byAlkylation—The mammalian MYST HAT TIP60 has beenlinked with the DNA damage response (19). We found thatparasites containing extra TgMYST-B are significantly moreresistant to the alkylating DNA-damaging agent MMS (Fig.3A). To test if the protection against MMS is dependent on theKATactivity of TgMYST-B,we stably expressed a pointmutant(E403G) version deficient in catalytic activity (20) (Fig. 2A).Parasites expressing the mutant form of TgMYST-B are nolonger protected from MMS (Fig. 3A). At the higher level ofMMS (500 �M), the parasites harboring mutant TgMYST-Bare even more susceptible to DNA damage than wild type,suggesting that the mutant is exerting a dominant negativeeffect as previously reported for TIP60 (4) and further sup-porting a link between TgMYST-B and the DNA damageresponse.The DNA damage response in eukaryotic cells is strongly

correlated with an increased level of histone H2AX phosphor-ylation (21). H2AX is rapidly phosphorylated in response todouble-stranded breaks (termed �H2AX), which are inducedby MMS. We have established that Toxoplasma containsH2AX that becomes phosphorylated in response to double-

FIGURE 1. TgMYST-B, the second MYST KAT family member in Toxoplasma. A, schematic diagram and amino acid sequence of TgMYST-B with relevantdomains highlighted: CHD (blue), C2H2 zinc finger (ZnF) (red), and MYST KAT domain (green). The Glu residue targeted for point mutation is underlined in theKAT domain. The polypeptide used to generate anti-TgMYST-B antiserum is in italic type. B, phylogenetic analysis of TgMYST-B compared with other MYST KATsfrom other species; tree drawn by Phylodendron (available on the World Wide Web). Species used are as follows. At, A. thaliana (Q9LXD7); Bb, Babesia bovis(A7ASC0); Cp, C. parvum (Q5CX42); Cr, Chlamydomonas reinhardtii (A8J386); Dc, Daucus carota (Carrot) (O80378); Eh, Entamoeba histolytica (gi�54306302); Gi1,Giardia intestinalis (gi�52857636); Gi2, G. intestinalis (gi�52857638); Hs, Homo sapiens (O95251); Mm, Mus musculus (Q5SVQ0); Os, Oryza sativa subsp. japonica(rice) (Q8LI34); Pf, Plasmodium falciparum (Q8III2); Py, Plasmodium yoelii yoelii (Q7RR28); Sc, Saccharomyces cerevisiae Esa1 (YOR244W); Tb1, Trypanosoma bruceiHAT1 (gi�25992510); Tb2, Trypanosoma brucei HAT2 (gi�25992512); TgA, T. gondii MYST-A (AAT81527); TgB, T. gondii MYST-B (AAZ79483); Tp, Theileria parva(Q4N7S1); Vitis vinifera (grape) (A7NV61); Zm, Zea mays (maize) (Q8W513). C, Western blot containing the polypeptide antigen (lane 1) or 20 �g of tachyzoitelysate (lane 2) probed with anti-TgMYST-B used at 1:10,000. Arrowhead, TgMYST-B.

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stranded DNA breaks (12). In TgMYST-B-overexpressing par-asites, however, we found high levels of basal�H2AXand foundthat the increase in �H2AX is dependent on the KAT activity ofTgMYST-B (Fig. 3B). The higher basal level of �H2AX is likelyto be a contributing factor explaining why the TgMYST-B-overexpressing parasites are more resistant to MMS.KAT Activity of TgMYST-B Increases Levels of ATM Kinase—

We hypothesized that the increase in �H2AX may be due toinappropriately activated ATM kinase. In human cells, ATMkinase responds to genotoxic stresses that lead to double-stranded DNA breaks following activation by TIP60 (22). Tox-oplasma possesses a predicted ATM kinase (50.m03243/TGGT1_024330), and we examined ATM kinase protein levelsby immunoblotting Toxoplasma lysate with a cross-reactiveantibody. ATM kinase was only detectable in the fTgMYST-B-overexpressing parasites; we could not detect ATM kinasein wild type or parasites expressing mutant fTgMYST-B(Fig. 3C).

To test if the increase in ATKkinasemay be due to increased tran-script levels, we analyzed ATMkinase mRNA levels in wild typeversus parasites overexpressingfTgMYST-B or the mutant versionfTgMYST-B(E403G). We detectedincreased levels of ATM kinasemRNA in fTgMYST-B-expressingparasites compared with wild typeand transgenics expressing mutantfTgMYST-B (Fig. 4A). Actin wasassayed as a control to show thatgene expression is not globally up-regulated in the TgMYST-B-over-expressing clone.We then determined if fTgMYST-

B was being recruited to the ATMkinase promoter, which would beconsistent with the idea thatTgMYST-B activates ATM kinasegene expression. Four regions span-ning the predicted start codon of theATM kinase locus were selected foranalysis by ChIP. The results dis-play the expected enrichment offTgMYST-B in regions most proxi-mal to the predicted start site forATM kinase (Fig. 4B). IdenticalChIP assays were performed onparasites expressing the mutatedfTgMYST-B(E403G). fTgMYST-B(E403G) shows a similar localiza-tion pattern at the ATM kinasegene, indicating that KAT activitydoes not interfere with TgMYST-Brecruitment to target genes. Toensure that fTgMYST-B is not beingrecruited to promoters randomly,we performed ChIP at the Toxo-

plasma actin promoter because actin mRNA is not increasedin the fTgMYST-B-overexpressing parasites (Fig. 4A). In con-trast to theATMkinase gene, the presence of fTgMYST-B doesnot significantly increase at genes like actin that are not up-reg-ulated (Fig. 4B). Collectively, these data strongly suggest thatthe KAT activity of TgMYST-B protects cells from DNA dam-age by contributing to the activation of the ATM kinase gene.TgMYST-B KAT Activity Is Associated with Slowed Re-

plication—It was observed that the parasites expressing addi-tional TgMYST-B were taking considerably longer to lyse theirhost cell monolayer than wild type parasites. Toxoplasmagrowth assays comparing wild type and fTgMYST-B-express-ing parasites confirmed a significant decrease in parasite pro-liferation (Fig. 5A). Additional clones isolated from an indepen-dent transfection with fTgMYST-B also displayed the sameretardation in growth (data not shown). The doubling time ofwild type parasites is �8 h, but it increases to �16–18 h in thefTgMYST-B overexpressor (Fig. 5B). Toxoplasma expressing

FIGURE 2. HAT activity and localization of TgMYST-B protein. A, in vitro HAT assays. The designated recom-binant FLAG-tagged protein was purified from parasite lysate over anti-FLAG resin and used as the enzymesource in a HAT assay with recombinant H3 peptide substrate as described previously (17). Acetylation wasdetected using an acetyl-lysine antibody on immunoblots of the HAT assay. Lane 1, fTgGCN5B (used as apositive control); lane 2, fMYST-B; lane 3, wild type lysate; lane 4, fMYST-B(E403G). The -fold induction of H3acetylation (listed below the gel) was determined by densitometry of the autoradiogram. B, localization ofnative TgMYST-B by IFA in intracellular (top) and extracellular (bottom) tachyzoites, using anti-TgMYST-B at1:100 (green). For reference, DNA is stained with DAPI (blue), and for intracellular parasites, the surface antigenSAG1 is stained with anti-SAG at 1:4,000 (red). C, IFA of intracellular tachyzoites engineered to stably expressrecombinant TgMYST-B tagged with FLAG epitope at the N terminus (shown in green, contrasted with DAPI(blue) and SAG1 (red) for reference). D, immunoelectron microscopy using anti-TgMYST-B performed on intra-cellular tachyzoites showing gold particles both in the cytoplasm and nucleoplasm (circled particles). n, nucle-us; ne, nuclear envelope (dotted line); dg, dense granule; m, microneme.

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fTgMYST-B(E403G) grow at the same rate as wild type. Theslowed growth is not a result of conversion to the bradyzoitestage as determined byDolichos lectin staining and PCR for the

bradyzoite-specific marker gene BAG1 (data not shown). Weconclude that the acetyltransferase activity of TgMYST-B isimportant in properly regulating growth rate in Toxoplasma

tachyzoites.Inhibition of ATM Kinase or KAT

Activity Reverses the Slow GrowthDefect in TgMYST-B-overexpressingParasites—To explore the possibil-ity that inappropriately activatedATM kinase contributes to theslowed replication of the fTgMYST-B-overexpressing parasites, we addedKU-55933, a well characterizedATMkinase inhibitor with an IC50 of 13.0nM (23), to the cultures. As shownin Fig. 6A, inclusion of 10 nM KU-55933 restores the growth rateof fTgMYST-B-expressing para-sites to wild type levels. Becausewe have established that excessKAT activity is responsible for theslowed growth of fTgMYST-B-overexpressing parasites (Fig. 5), weexamined the effect of pharmaco-logical inhibition of in vivo KATactivities. The addition of anacardicacids (24) also restored the growthrate of fTgMYST-B-overexpressingparasites to levels similar to wildtype (Fig. 6B). These data suggestthat the mechanism of the reducedreplication observed inTgMYST-B-

FIGURE 3. TgMYST-B confers protection against DNA damage induced by MMS. A, wild type (WT), fMYST-B, orfMYST-B(E403G) parasites were grown in the presence of 100 or 500 �M MMS; growth rate was monitored using theB1 assay. B, higher basal levels of phosphorylated H2AX (�H2AX) exist in fMYST-B tachyzoites. Equal amounts ofparasite protein were loaded for immunoblotting with anti-�H2AX (1:5,000); probing with anti-tubulin serves as aloading control. C, immunoblot as described for B, but probed with antibody to ATM kinase at 1:2,000.

FIGURE 4. A, relative gene expression of TgMYST-B, ATM kinase, and actin in WT, fMYST-B, or fMYST-B(E403G) tachyzoites as assayed by real-time PCR. B, ChIP followedby PCR analysis of four regions proximal to the ATM kinase start site. ChIP was performed on both fMYST-B and fMYST-B(E403G) expressing tachyzoites using anti-FLAGor a nonspecific antibody (antibody to an eIF2 kinase, TgIF2K-A (18)). Four primer pairs that amplify a �93-bp region were used, and their approximate position isdepicted in the diagram. C, ChIP was performed to show that MYST-B is not enriched on the actin promoter. Results are represented as a ratio of ChIP/input DNA.

Functions of TgMYST-B in Toxoplasma

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overexpressing parasites involves inappropriate activation ofToxoplasma ATM kinase by TgMYST-B KAT.

DISCUSSION

Here we report the full-length cloning and characterizationof the secondMYST familyKAT in the parasiteT. gondii, build-ing from a partial clone first described by Smith et al. (8).5�-RACE for TgMYST-B revealed an unusual 5�-UTR thatharbors an intron, which has been reported for other genesin Apicomplexa (e.g. Plasmodium HGPRT (25)). Similar toTgMYST-A, TgMYST-B can be classified as a “MYST �CHD”KAT,which includes yeast Esa1, humanTIP60, and human andDrosophilaMOF (2). BothTgMYST-A and -B are similar in size(between 50 and 60 kDa) and subcellular localization and bearthe strongest homology to MYST family proteins found inplants.We present several lines of data suggesting that a subset of

total TgMYST-B protein must gain access to the parasitenucleus. Although difficult to detect by IFA, immune electronmicroscopy shows that 20% of TgMYST-B localized to thenuclear compartment (Fig. 2D). TgMYST-B contains a basic-rich stretch of amino acids beginning at residue 125, which ischaracteristic of a nuclear localization signal (15). Most con-vincingly, we are able to purify TgMYST-B associated withgenomic DNA via ChIP (Fig. 4B). It is intriguing, however, thatthe bulk of protein is in the cytosol; similar results wereobserved for TgMYST-A (8). Moreover, we did not detect byIFA any shift in the localization pattern following alkaline pHstress or MMS treatment (data not shown). In other species,MYSTKATs are largely nuclear, presumably operating primar-ily as histone acetyltransferases. However, human TIP60 hasbeen shown to be translocate into the cytoplasm to form“speckles” that facilitate the stabilization of Nmi (N-Myc andSTAT interactor) protein (26). In another example, an alterna-tively spliced form of TIP60 called TIP60� is present in bothnuclear and cytosolic compartments (27). Toxoplasma is thefirst cell reported to have such high proportions of MYST KATprotein in the cytosol relative to the nucleus. We speculate twopossible reasons for the unusual distribution of TgMYSTKATs. First, the distribution may simply be for regulatory rea-sons to control the amount of TgMYST protein in the nucleus;however, a more tantalizing possibility is that the TgMYSTKATs regulate non-histone proteins via acetylation. Severalproteomic studies have recently revealed that acetylation is awidespread post-translational modification in prokaryotes andeukaryotes (28, 29). Yeast proteome microarray studies haveshown that the nucleosome acetyltransferase of H4 (NuA4)complex, which containsMYSTKAT Esa1, acetylates cytosolicsubstrates, such as Pck1 (phosphoenolpyruvate carboxykinase)(30).Previously, when we attempted to overexpress TgMYST-A

in Toxoplasma, we could not do so unless the catalytic domainwas mutated (8). Here we report that expression of additionalTgMYST-B, but not a mutant form, dramatically slows thegrowth of transgenic tachyzoites. This result suggests that theKAT activity of TgMYST-Bmay be important in the regulationof cell cycle, which could be connected to the established role ofMYST KATs in the DNA damage response (19). TgMYST-B

FIGURE 5. TgMYST-B KAT activity is associated with slowed replication.A, transgenic parasites expressing fTgMYST-B exhibit decreased growth ratecompared with wild type (WT) or transgenic parasites expressing a mutant ver-sion of the fMYST-B (fMYST-B(E403G)). The growth rate was measured for 5 daysby quantitative real-time PCR using the Toxoplasma B1 gene as described under“Experimental Procedures.” B, doubling assay was performed by counting thenumber of parasites in 50 randomly chosen vacuoles. The y axis displays themean number of parasites/vacuole at 8, 16, and 24 h postinfection. Error bars, S.D.

FIGURE 6. Restoration of fMYST-B growth rate with pharmacologicalinhibitors. Wild type (WT) or fMYST-B-expressing parasites were cultivated inthe presence of KU-55933 ATM kinase inhibitor (A) or anacardic acid histoneacetyltransferase inhibitor (B). DMSO was used as a vehicle control. Thegrowth rate was monitored using a B1 assay at day 5. **, a significant differ-ence (p � 3.23545 � 10�5) as determined by t test for unequal variance.

Functions of TgMYST-B in Toxoplasma

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overexpressors show increased protection against the alkylat-ing agentMMS (Fig. 3A). The presence of ATMkinase explainsthe increased basal levels of �H2AX in the TgMYST-B-overex-pressing parasites. In turn, increased �H2AX causes cell cyclearrest and a decrease in the number of cells inmitosis (31), thusexplaining why parasites containing extra TgMYST-B exhibitretarded growth in culture.We postulated two mechanisms how TgMYST-B may in-

crease the levels of ATM kinase. One mechanism has alreadybeen established in humans; TIP60 activates ATM kinasethrough direct acetylation of Lys3016 (3, 32). Although Toxo-plasma ATM kinase contains the conserved lysine residue(Lys1966), we could not test this possibility because the anti-ATM kinase antibodies do not immunoprecipitate the Toxo-plasma ATM kinase. In this report, we present evidence for anovel second mechanism that involves TgMYST-B increasingATM kinase mRNA levels. Further analysis showed thatTgMYST-B is specifically recruited to the ATM kinase pro-moter and that this recruitment is independent of KAT activity.The recruitment of enzymatically active TgMYST-B to theATM kinase gene correlates with an increase in ATM kinasemRNA levels; conversely, KAT-deficient TgMYST-B, despitebeing recruited to the ATM kinase gene, is unable to up-regu-late mRNA levels. These data strongly suggest that TgMYST-BKAT activity is critical for ATM kinase-mediated DNA repairin Toxoplasma. Our study does not rule out other possibilitiesthat could contribute to increase ATM kinase protein in theTgMYST-B-overexpressing parasites. For example, it is possi-ble that KAT activity of TgMYST-B directly or indirectly leadsto stabilization of ATM kinase protein.Toxoplasma is now the earliest eukaryote demonstrated to

have a link between MYST KATs and the response to DNAinjury. These findings also suggest that TgMYST-B is ortholo-gous to mammalian TIP60, which is known to be critical forDNA damage response (19). Ongoing studies are attempting toelucidate the TgMYST-B complex to determine the identity ofthe associating proteins.Toxoplasma causes congenital birth defects and life-threat-

ening disease in immunocompromised patients.More tolerabledrug therapies are urgently needed because the current treat-ment of pyrimethamine and sulfadiazine has toxic side effects.The data we present here, considered with our previous find-ings reported by Smith et al. (8), strongly suggest that MYSTKATs are involved in diverse critical functions in the parasite,therefore underscoring this protein family as important candi-dates for future drug design.

Acknowledgments—We thank Gustavo Arrizabalaga and membersof the Sullivan laboratory for helpful discussion and for criticallyreading the manuscript.

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Functions of TgMYST-B in Toxoplasma

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Sullivan, Jr.Nathalie Vonlaufen, Arunasalam Naguleswaran, Isabelle Coppens and William J.

Toxoplasma gondii(ATM) Kinase-mediated DNA Damage Response in MYST Family Lysine Acetyltransferase Facilitates Ataxia Telangiectasia Mutated

doi: 10.1074/jbc.M109.066134 originally published online February 16, 20102010, 285:11154-11161.J. Biol. Chem. 

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