MYST Family Lysine Acetyltransferase Facilitates Ataxia

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  • MYST Family Lysine Acetyltransferase Facilitates AtaxiaTelangiectasia Mutated (ATM) Kinase-mediated DNADamage Response in Toxoplasma gondii*SReceived 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 Vonlaufen1, Arunasalam Naguleswaran1, 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 aKAT-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. S1S4.

    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.

    11154 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 NUMBER 15 APRIL 9, 2010

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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 ReagentsToxoplasma tachyzoitesused in this study were RH strain and maintained in humanforeskin fibroblasts using Dulbeccos modified Eagles 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 AssaysDoubling 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 AnalysisPolyclonal antibody

    was generated in rabbit to a polypeptide sequence correspond-ing to the C-terminal 100 amino acid residues of TgMYST-B(amino acids 423523) 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 20g 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 412% or10% SDS-polyacrylamide gels using MOPS or MES runningbuffer (Invitrogen). For ATM kinase Western blots, proteinswere separated on 47% Tris acetate gels (Invitrogen).

    Cloning, Expression, and Purification of TgMYST-BRNAligase-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 Ind