Structure/Function Analysis of the Phosphatidylinositol-3-Kinase … · 2007. 9. 20. · Katherine MacKenzie,* Olivier Jobin-Robitaille,† Julie Guzzo,‡ Jacques Coˆte´,† Brenda

Copyright � 2007 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.074476

Structure/Function Analysis of the Phosphatidylinositol-3-KinaseDomain of Yeast Tra1

A. Irina Mutiu,*,1 Stephen M. T. Hoke,*,1 Julie Genereaux,* Carol Hannam,*,2

Katherine MacKenzie,* Olivier Jobin-Robitaille,† Julie Guzzo,‡ Jacques Cote,†

Brenda Andrews,‡ David B. Haniford* and Christopher J. Brandl*,3

*Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario N6A5C1,Canada, †Laval University Cancer Research Center, Hotel-Dieu de Quebec (CHUQ), Quebec City, Quebec G1R-2J6,

Canada and ‡Department of Medical Genetics and Microbiology and the Banting and Best Department ofMedical Research, Terrence Donnelly Centre for Cellular and Biomolecular Research,

University of Toronto, Toronto, Ontario M5S 3E1, Canada

Manuscript received April 12, 2007Accepted for publication July 10, 2007

ABSTRACT

Tra1 is an essential component of the Saccharomyces cerevisiae SAGA and NuA4 complexes. Using targetedmutagenesis, we identified residues within its C-terminal phosphatidylinositol-3-kinase (PI3K) domain thatare required for function. The phenotypes of tra1-P3408A, S3463A, and SRR3413-3415AAA included temperaturesensitivity and reduced growth in media containing 6% ethanol or calcofluor white or depleted of phos-phate. These alleles resulted in a twofold or greater change in expression of�7% of yeast genes in rich mediaand reduced activation of PHO5 and ADH2 promoters. Tra1-SRR3413 associated with components of both theNuA4 and SAGA complexes and with the Gal4 transcriptional activation domain similar to wild-type protein.Tra1-SRR3413 was recruited to the PHO5 promoter in vivo but gave rise to decreased relative amounts ofacetylated histone H3 and histone H4 at SAGA and NuA4 regulated promoters. Distinct from other com-ponents of these complexes, tra1-SRR3413 resulted in generation-dependent telomere shortening andsynthetic slow growth in combination with deletions of a number of genes with roles in membrane-relatedprocesses. While the tra1 alleles have some phenotypic similarities with deletions of SAGA and NuA4components, their distinct nature may arise from the simultaneous alteration of SAGA and NuA4 functions.

IN eukaryotic cells the post-translational modificationof nucleosomes by multisubunit complexes is a key

aspect of transcriptional regulation (reviewed in Berger

2002). Histone modifications including acetylation, meth-ylation, ubiquitylation, and phosphorylation can di-rectly alter chromatin structure or act as a recruitmentsignal for additional factors (Strahl and Allis 2000).As well as regulating transcriptional initiation, nucle-osome modifications affect transcriptional elongationand other nuclear processes such as DNA replication,DNA repair, and RNA export (Iizuka and Smith 2003).

The Saccharomyces cerevisiae Spt-Ada-Gcn5-Acetyltrans-ferase (SAGA) complex modifies chromatin and pro-vides an interface between DNA-binding transcriptionalregulators and the basal transcriptional machinery (re-viewed in Green 2005). The structural core of SAGA is

composed of a subset of the TBP-associated factors(TAFs) (Grant et al. 1998a; Wu et al. 2004), with Spt7,Ada1, and Spt20 also being required for the integrityof the complex (Horiuchi et al. 1997; Roberts andWinston 1997; Sterner et al. 1999). The histone acetyl-transferase Gcn5/Ada4 activates and represses transcrip-tion by modifying histones H3 and H2B (Brownell

et al. 1996; Grant et al. 1997; Kuo et al. 1998; Wang et al.1998; Ricci et al. 2002). In turn, the Ada proteins, Ada2and Ngg1/Ada3 regulate the activity and substrate pre-ference of Gcn5 (Balasubramanian et al. 2001). Fur-ther regulation is provided by the interaction of Spt3and Spt8 with the TATA-binding protein (Eisenmann

et al. 1992, 1994; Dudley et al. 1999). Recruitment ofSAGA to promoters is mediated by Tra1, an essential437-kDa protein (Grant et al. 1998b; Saleh et al. 1998)that interacts directly with transcriptional activators(Brown et al. 2001; Bhaumik et al. 2004; Fishburn

et al. 2005; Reeves and Hahn 2005). The mammalianortholog of Tra1, TRRAP is required for transcriptionalregulation by myc, p53, E2F, and E1A (Mcmahon et al.1998; Bouchard et al. 2001; Deleu et al. 2001; Ard et al.2002; Kulesza et al. 2002). Its deletion results in defectsin cell cycle progression and early embryonic lethality(Herceg et al. 2001).

Microarray data from this article have been submitted to the GEOdatabase at NCBI under accession no. GSE6847.

1These authors contributed equally to this work.2Present address: Department of Molecular and Cellular Biology,

University of Guelph, Guelph, ON N1G 2W1, Canada.3Corresponding author: Department of Biochemistry, University of

Western Ontario, London, ON N6A5C1, Canada.E-mail: [email protected]

Genetics 177: 151–166 (September 2007)

Tra1 is also a component of the multisubunit NuA4complex (Allard et al. 1999) that preferentially acety-lates histones H4 and H2A, the catalytic subunit beingthe essential protein Esa1 (Smith et al. 1998; Clarke

et al. 1999). NuA4 associates with acidic activation do-mains, probably through Tra1-mediated interactions,and activates transcription in an acetylation-dependentmanner (Vignali et al. 2000; Nourani et al. 2004). Acet-ylation by NuA4 is also critical for nonhomologous endjoining of DNA double-stand breaks and for replication-coupled repair (Bird et al. 2002; Choy and Kron 2002;Downs et al. 2004). The role of NuA4 in repair washighlighted by the finding that it acetylates the histonevariant Htz1, which is intimately involved with theseprocesses (Keogh et al. 2006).

Aside from its length, a distinguishing feature of Tra1is its C-terminal domain of �300 amino acids that isrelated to the phosphatidylinositol-3-kinase (PI3K) do-main found in several key cellular regulators includingATM, DNA-PK, and FRAP (Keith and Schreiber 1999).The group also shares less well-defined sequencesflanking the PI3K domain called the FRAP-ATM-TRRAP(FAT) and C-FAT domains (Bosotti et al. 2000). Unlikeother members of the family, Tra1 and TRRAP lack thesignature motifs of kinases and kinase activity (Bosotti

et al. 2000). The exact role of the PI3K domain is thusunclear; although in human cells, the PI3K domain ofTRRAP is required for cellular transformation by mycand E1A (Park et al. 2001).

We have used a mutagenesis approach to determinethe structure/function relationships of Tra1, focusing

in particular on the PI3K domain. We have identifiedeight mutations in the PI3K domain that result in cel-lular inviability and three that result in temperature-sensitive growth and reduced growth on media containing6% ethanol. Characterization of these temperature-sensitive alleles at the permissive temperature confirmsa role for the PI3K domain in transcriptional regulation.These mutations confer altered expression of�7% of theyeast genome and were distinct from those affecting in-dividual components of either SAGA or NuA4 complexes.

MATERIALS AND METHODS

Yeast strains and growth: Yeast strains are listed in Table 1.TRA1 alleles contained on TRP1 centromeric plasmids weretransformed into CY1021 and the wild-type copy was displacedby plasmid shuffling (Sikorski and Boeke 1991). CY1896 is aderivative of Y5565 that has been gene replaced with tra1-SRR3413 and selected for through placement of Tn10LUK atthe downstream BstBI site. CY2437 and CY2438 are derivativesof KY320 and similarly contain integrations of wild-type TRA1and tra1-SRR3413, respectively, in a background lacking BamHIand SalI restriction sites within TRA1.

Growth comparisons were performed on selective platesafter 3–5 days at 30� unless stated otherwise. Assays were per-formed in duplicate on independently constructed strains.Scoring for PI3K domain mutations was done in relation toCY1524, which contains TRA1SB, the background allele used toconstruct mutations within the PI3K domain. TRA1SB is Flag-tagged and contains a BamHI site that converts N3580A and aSalI site of nucleotides A9714G and T9717G. Scoring for theviability of non-PI3K domain mutations was relative to CY1020(Saleh et al. 1998).

TABLE 1

Strains used in this study

Strain Genotype TRA1 plasmid Reference

KY320 MATa ura3-52 ade2-101 trp1-D1 lys2-801his3-D200 leu2TPET56

Chen and Struhl (1988)

CY1021 Isogenic to KY320 but tra1TTn10LUK myc-TRA1 Saleh et al. (1998)CY1962 Isogenic to CY1021 TAP- TRA1SB This workCY1963 Isogenic to CY1021 TAP-tra1-SRR3413 This workCY1524 Isogenic to CY1021 Flag3- TRA1SB This workCY1507 Isogenic to CY1021 Flag3-tra1-P3408A This workCY1531 Isogenic to CY1021 Flag3-tra1-SRR3413 This workCY1514 Isogenic to CY1021 Flag3-tra1-S3463A This workFY630 MATa his4-917 lys2-173R2 leu2D1 ura3-52 trp1D63 Gansheroff et al. (1995)FY1093 Isogenic to FY630 but spt7TLEU2CY1381 Isogenic to FY1093 but leu2THIS3 This workFY86 MATa his3D200 leu2D1 ura3-52 Roberts and Winston (1997)FY1370 Isogenic to FY86 but gcn5THIS3Y5565 MATa can1DTMFA1pr-HIS3 mfa1DTMFa1pr-LEU2

lyp1D ura3D his3D met15D

Tong and Boone (2006)

BY4741 MATa hisD0 leu2D0 met1D0 ura3D0 Winzeler and Davis (1997)BY3281 Isogenic to BY4741 except spt7TKanr

BY4196 Isogenic to BY4741 except vid21TKanr

BY4282 Isogenic to BY4741 except ada2TKanr

QY204 MATa his3D200 trp1D 63 ura3-52 leu2D1 lys2-128d Nourani et al. (2001)QY202 Isogenic to QY204 except yng2TKanr

152 A. I. Mutiu et al.

Construction of DNA molecules: lacZ reporter constructswere cloned as his3-lacZ fusions into the LEU2 centromericplasmid YCp87 (Brandl et al. 1993). ADH2-lacZ contains pro-moter sequences (�440 to �150; Saleh et al. 1998). PHO5(�452 to 147), INO1 (�517 to 140), and HIS4 (�545 to 129)were engineered by PCR as BamHI–HindIII fragments andcloned into YCp87 (see supplemental Table 1 at http://www.genetics.org/supplemental/ for a listing of oligonucleotides).

Construction of TRA1 alleles: Mutations of DSP171AAA,EFS184AAA, KEL345AAA, VLL837AAA, VRL867AAA, KKK1686AAA,GLW2677AAA, GYH2939AAA, TLP2972AAA, HFQ3168AAA,SRR3413AAA, and PFR3621AAA were engineered into myc-TRA1-Ycplac111 (Saleh et al. 1998). Each allele was synthesized in twoPCR reactions. Reactions contained a listed oligonucleotide andthe appropriate outside flanking primer with a unique cloningsite (oligonucleotides 842, 2542-3, 2337-1, 2323-2, 2323-1,2542-4, 2337-2, and 2346). Mutagenized codons were replacedwith a NotI site such that 59 and 39 halves could be ligated se-quentially into pUC vectors. Fragments were moved into myc-TRA1-Ycplac111 using the outside restriction sites. Mutationswithin the coding region of the PI3K domain (including addi-tional alleles of EER3416AAA and PFR3621AAA) were constructedin TRA1SB. Flag epitopes were introduced at an N-terminal NotIsite. Mutagenized alleles were constructed by PCR and ligated asSalI–BamHI (for alterations N-terminal to N3580) or BamHI–SacI(for alterations C-terminal to N3580). Flanking primers used inthe PCR reactions were 4293-1 and 4225-3 or 4249-1 and 2346/4479-1 depending on the placement relative to N3580. YNG2 andGCN5 were similarly cloned into this molecule.

Tandem affinity purification (TAP)-tagged molecules werecloned into YCpDed-TAP, a derivative of the URA3-containingcentromeric plasmid YCplac33 (Gietz and Sugino 1988) thatcontains a DED1 promoter driving expression of a TAP epi-tope. The DED1 promoter was synthesized by PCR using oligo-nucleotides 4149-1 and 4149-2 and cloned as a PstI–BamHIfragment into YCplac33. The TAP epitope was subsequentlycloned as a BamHI–SacI fragment into this molecule after PCRusing oligonucleotides 4149-4 and 4168-1 and pFA6a as thetemplate (provided by Kathy Gould). The coding sequence forthe Flag-epitope was cloned into this molecule at the engi-neered NotI site to give YCpDed-TAP-Flag. TRA1 was intro-duced into this vector as a NotI–SacI fragment.

TAP: TAP was carried out as described by Rigaut et al.(1999). One liter of cells grown in YPD to an A600 �1.5 wereground in liquid nitrogen (Saleh et al. 1997). Further stepswere at 4�. Cell extract suspended in 5 ml of IPP150 withprotease inhibitors (1 mm phenylmethylsulfonylfluoride, 0.1mm benzamidine hydrochloride, 2 mg/ml pepstatin A, 2 mg/ml leupeptin, and 0.1 mg/ml trypsin inhibitor) was cleared bycentrifugation at 147,000 3 g and incubated with 600 ml ofIgG-Agarose (Sigma-Aldrich Canada, Oakville, Ontario) for 2hr. After washing with 30 ml of IPP150 and 10 ml of TEVcleavage buffer, beads were suspended in 1 ml of cleavage buf-fer and incubated with 100 units TEV protease for 2 hr. Threemicroliters of 1 m CaCl2 were added to the eluent and thendiluted with 3 ml of calmodulin binding buffer. The proteinwas incubated with 600 ml of calmodulin–sepharose (Stratagene,La Jolla, CA) for 1 hr, washed with 30 ml of calmodulin bindingbuffer, and protein eluted.

b-Galactosidase assays: HIS4-lacZ cells grown in minimalmedia to an A600 of�1.5 were pelleted, washed in LacZ buffer,and concentrated approximately fivefold. b-Galactosidase wasdetermined using o-nitrophenol-b-d-galactosidase as substrate,standardizing to cell density (Ausubel et al. 1998). Analysis ofPHO5-lacZ, INO1-lacZ, and ADH2-lacZ was performed similarlyexcept that overnight cultures were washed three times inwater and then grown to an A600 �1.5 in YPD depleted ofphosphate (Han et al. 1988)or depleted of inositol or in

minimal media containing 3% ethanol (v/v) and 0.1%glucose.

Gal4 affinity chromatography: Amino acid residues 764–882 of Gal4 were expressed as a GST fusion in the vectorpGEX1. One liter of Escherichia coli cells was induced with0.6 mm isopropyl b-d-thiogalactopyranoside for 6 hr at 37�.Harvested cells were resuspended in lysis buffer ½40 mm Tris(pH 8.5), 20 mm NaCl, 10% glycerol, 0.08% NP-40, 0.2 mm

EDTA (pH 8.0), 1 mm DTT� with protease inhibitors, treatedwith 1 mg/ml lysozyme on ice for 1 hr, and broken by soni-cation. Cleared extract was incubated on ice with 1 ml glu-tathione sepharose 4B (GE Healthcare, Waukesha, WI) andwashed three times with 10 ml of 140 mm NaCl, 2.7 mm KCl,10 mm Na2HPO4, 1.8 mm KH2PO4 and once with 10 ml of lysisbuffer. TAP-Tra1, purified on IgG-Agarose from 1.0-litercultures of CY1962 and CY1963 containing HA-tagged Ada2(Saleh et al. 1997), was applied to Gal4-containing glutathi-one sepharose in 3 ml of lysis buffer, rotated at 4� for 60 min,and washed with lysis buffer, and bound protein was elutedwith 10 mm reduced glutathione. Thirty microliters of theeluent were separated on a 10% SDS–PAGE gel, transferred toPVDF membrane, and blotted with anti-HA antibody.

Genetic analyses—identification of an intragenic suppres-sor of tra1-SRR3413: Approximately 2 3 106 cells of yeast strainCY1531 (tra1-SRR3413) were plated onto YPD containing 6%ethanol. Two colonies were isolated after 5 days of growthand Tra1 expressing plasmids were recovered (Hoffman andWinston 1987). The SalI–SacI fragment was recloned intoTRA1SB and found sufficient for suppression. Systematicgenetic-array analysis on yeast strain CY1896 (tra1-SRR3413)was performed as described by Tong and Boone (2006).

RNA purification and microarray analysis: Yeast cells weregrown in YP media containing 2% glucose to an A600nm¼ 2.0 at30�. RNA was purified from 108 cells after glass bead disruptionas described previously (Mutiu and Brandl 2005; Mutiu et al.2007). Microarray analyses were performed using the Agilentyeast oligo array kit (Mogene, LC, St. Louis). Comparisonswere made with wild-type strain CY1524 and performed induplicate with dye reversal. Array data for the confirmed set ofprotein-coding genes were log transformed and averagesobtained. Genes displaying an expression change .1.5-fold,where the direction of the response differed between repli-cates, were discarded. Profiles were normalized by subtractingthe mean response of all genes from the response of each geneand then dividing by the standard deviation of all responseswithin a profile. An initial cluster analysis using a subset of thecompendium data (Hughes et al. 2000) that included deletionstrains with gene ontology terms indicating gene expressionidentified microarray profiles of strains with deletions of cka2,ckb2, gcn4, hat2, isw1, mbp1, rpd3, rtg1, sap30, sin3, ste12, andyap3 as being most similar to tra1-SRR3413. Agglomerativehierarchical clustering based on the average linkage of uncen-tered correlations was performed on the profiles from thesestrains and strains with deletions of SAGA (Ingvarsdottir

et al. 2005) and NuA4 (Krogan et al. 2004) components usingCLUSTER 3.0 software (Eisen et al. 1998). Genes not appearingin at least two of the microarray profiles were excluded. Thedata were visualized using MapleTree (http://rana.lbl.gov/EisenSoftware.htm). Gene ontology terms associated with geneclusters were determined using FunSpec (Robinson et al. 2002).

Chromatin immunoprecipitation assays: Assays were per-formed essentially as described by Kuo and Allis (1999). Forassays comparing acetylated histone H3, 50 ml of culture wasgrown to an A600nm� 2.0. To equalize amounts of input DNA,cell extract was reverse crosslinked and PCR reactions per-formed on serial dilutions. For immunoprecipitations, DNAequal to that contained in 50 mg (protein) of cell extract fromthe wild-type sample was incubated with protein A sepharose

Structure/Function of S. cerevisiae Tra1 153

4 Fast Flow (Pharmacia, Piscataway, NJ), transferred to cleantubes, and rotated overnight with 1 ml of antibody (anti-H3,ab1791, Abcam, Cambridge, MA; anti-AcH3, Lys9, 07-352,Upstate Biotechnology). Precipitate was removed by centrifu-gation and the supernatant incubated with protein A beads.After washing and reversal of crosslinks, products were ana-lyzed by PCR (Ricci et al. 2002) using primers for the PHO5(�550 to �300), ADE1 (�505 to 51), PHO84 (�199 to 74),PGK1 (�249 to 28) promoters or PHO5 gene (1035–1322).PCR conditions were 5 min at 95�, followed by 25 cycles of30 sec at 95�, 52�, and 72�, and then 5 min at 72�. Assays ofacetylated histone H4 were performed with 250-ml culturesgrown to A600nm � 2.0 in YPD, 0.5 mg of chromatin (in a totalvolume 0.5 ml), and protein A/G plus 2 ml of anti-AcH4, Lys8(ab1760, Abcam). For immunoprecipitations using TAP-Flag-Tra1, chromatin was prepared in RIPA buffer with washing andelution as described by Alekseyenko et al. (2006).

Western blotting: Yeast extract prepared by glass bead dis-ruption (Brandl et al. 1993) or grinding in liquid nitrogen(Saleh et al. 1997) was separated by SDS–PAGE and trans-ferred to PVDF membranes (Roche Applied Science, Indian-apolis) using a wet transfer system in 48 mm Tris, 40 mm

glycine, 0.0375% SDS, and 20% methanol for 1 hr at 100 V.Anti-Flag antibody (M2, Sigma-Aldrich Canada ) and anti-HAantibody (ascites fluid from the 12CA5 cell line) were used at aratio of 1:4000. Secondary antibody (anti-mouse IgG HRP;Promega, Madison, WI) used at a ratio of 1:10,000 was detectedusing a 20% solution of Immobilon Western (Millipore).Densitometric scanning of films was performed using Al-phaImager 3400 software (Alpha Innotech, San Leandro, CA).

Telomere length assays: Plasmid linearization assay: Strainswere transformed with pVL106 (Lundblad and Szostak

1989). Leu1 Ura1 colonies were grown to saturation in medialacking uracil and then YPD. Ten microliters were spottedonto leucine-depleted plates containing 5-fluoroorotic acid(5-FOA). Values reported are the percentage of 5-FOA re-sistant, Leu1 colonies in the mutant strain as compared to wildtype, normalized to the number of cells plated.

Terminal restriction fragment determination: TRA1 alleles weretransformed into CY1021 and plasmid shuffling was per-formed (generation zero). Ten-milliliter saturated cultureswere grown from these colonies at 30� or 34� and DNA wasprepared (Ausubel et al. 1998). Each culture was also diluted1:1000 into YPD and grown again to saturation, and then theprocess was repeated. Genomic DNA was digested with XhoIand Southern blotting performed (Teng and Zakian 1999).

Fractionation of Tra1-containing complexes on HiTrap Qsepharose Fast Flow: Eighteen milligrams of whole-cell extractcontaining HA-Ada2p and Tra1SB or Tra1-SRR3413 was pre-pared in 40 mm Tris-HCl (pH 7.7) buffer containing 20 mm

NaCl, 10% glycerol, 0.08% Nonidet P-40, 0.2 mm EDTA, and0.1 mm dithiothreitol. Extracts were treated with protaminesulfate (0.2%), rotated at 4� for 30 min, cleared by centrifu-gation at 147,000 3 g, and applied to a HiTrap Q sepharoseFast Flow column (1.0 ml; flow rate of 1.0 ml/min; AmershamPharmacia Biotech). After washing with 4 ml of extractionbuffer, protein was eluted with a 16-ml gradient of 0–1.0 m

NaCl. Aliquots (20 ml) of 200-ml fractions were separated bySDS–PAGE and analyzed by Western blotting with anti-HAantibody. Twenty-five milligrams of whole-cell extract contain-ing TAP-Flag-Yng2 and Tra1SB or Tra1-SRR3413 were fractionatedand blotted using anti-Flag antibody.

RESULTS

The size of Tra1 presents a significant challenge foranalyses of structure and function using molecular gen-

etic approaches. Because of the difficulty in identifyingand characterizing random mutations, we undertook atargeted mutagenesis approach, initially constructing12 triple-alanine scanning mutations scattered in re-gions that might be relevant for function (Table 2).These alleles were expressed on a TRP1-containing cen-tromeric plasmid and transformed into yeast strainCY1021 that contains a disruption of the genomic copyof TRA1 complemented by wild-type TRA1 expressed ona URA3-containing centromeric plasmid (Saleh et al.1998). Each allele was analyzed for growth propertiesafter shuffling out wild-type TRA1 on media containing5-FOA. Phenotypes examined included mating effi-ciency; temperature sensitivity (37�); growth on mediadepleted for inositol; amino acids and phosphate; fer-mentation of galactose and raffinose; and sensitivity tohydroxyurea, methylmethanesulfonate (MMS), ultravi-olet irradiation, g-irradiation, high osmolarity, cyclo-heximide, and tunicamycin. Two of the alleles tested,DSP171AAA and EFS184AAA altered potential phosphor-ylation sites. (The number indicates the first residue ofthe three. We do not include the AAA for each allele.)VLL837 altered a putative LXXLL motif. Many of theother altered residues are conserved in yeast and/or hu-man Tra1 homologs. Only 2 of the 12 alleles, both withmutations within the PI3K domain, had discernible phe-notypes. tra1-PFR3621 would not support growth on me-dia containing 5-FOA and therefore is a nonfunctional

TABLE 2

Growth of strains containing mutations throughoutthe length of Tra1

Viabilityb

Mutationa Reason for analysis 30� 37�

DSP171 Potential phosphorylation Yes YesEFS184 Potential phosphorylation Yes YesKEL345 Conservedc Yes YesVLL837 LXXLL sequence Yes YesVRL867 Conserved Yes YesKKK1686 Found in chromatin

associated Sth1Yes Yes

GLW2677 Conserved in yeastd Yes YesGYH2939 Conserved in yeast Yes YesTLP2972 Conserved in yeast Yes YesHFQ3168 Conserved in FAT domaine Yes YesEER3416 Conserved Yes ReducedPFR3621 Conserved in PI3K domain No No

a Alleles were constructed by placing a NotI linker encodinga triple-alanine tag in place of the indicated three aminoacids. The number refers to the amino acid position of thefirst of the three amino acids.

b Growth on YPD media at 30� or 37�.c Conserved indicates identity in S. pombe and human Tra1

molecules.d Sequence similarity or identity in yeast homologs.e See Bosotti et al. (2000).


allele. tra1-EER3416 showed reduced growth on all mediatested, as well as slight temperature sensitivity.

Targeted mutation of the PI3K domain: On the basisof the above results, we directed additional mutations tothe PI3K domain. As we were concerned that mutationsmight simply alter the fold of the PI3K domain, wefocused on mutagenizing codons for amino acid resi-dues conserved in the Tra1/TRRAP family of moleculesbut not in the broader PI3K domain family. In theabsence of a structure for the Tra1 PI3K domain, thestructure of PI3K-g (Walker et al. 1999; see supplemen-tal Figure 1 at http://www.genetics.org/supplemental/)allows an initial prediction of the positions of theseresidues. The PI3K domain consists of N-terminal andC-terminal lobes connected by a loop between b7 andb8. For PI3K-g, ATP interactions are found with the b3–b4 loop (P-loop), the end of b5, and the b7–b8 loop.The region between a6 and b9 corresponds to thecatalytic loop. The C-terminal lobe also contains anactivation loop that is required for substrate binding ofthe PI3K molecules (Bondeva et al. 1998) and likelybinds phosphatidylinositols in PI3K-g.

The phenotypes of strains containing the mutagen-ized tra1 alleles are shown in Table 3. Of the 19 alleleswith targeted mutations in the PI3K domain, 8 did notsupport viability. Two of the 8, R3456A and R3567A, weresingle-amino-acid substitutions; the others, WRR3317,DIE3351, RFL3374, FRK3544, PFR3621, and RDE3668 weretriple-alanine scanning mutations. Five of the eight mu-tations were within predicted loop regions (WRR3317,DIE3351, RFL3374, R3567, and PFR3621). F3443, which aligns

with the a3–b6 loop, can be considered in this groupsince strains containing tra1-F3443A were marginallyviable in rich media but inviable under conditions ofstress. If their positioning within loops is correct, it is notlikely that these residues are important for structure butinstead may be involved in protein–protein interactionsor have regulatory roles. In agreement with this, R3567 ofTra1 aligns with R947, an essential residue within thecatalytic loop of PI3K-g (Dhand et al. 1994; Stack andEmr 1994). Likewise it is required for the activity of Tra1,though for Tra1/TRRAP the size of the residue may bethe critical factor since human TRRAP contains a leu-cine at this position. Similarly, PFR3621 aligns with theactivation loop and despite the lack of kinase activity,these residues are required for the activity of Tra1. (Wenote that the phenotype of PFR3621 may in part be dueto reduced expression levels; not shown.) Three muta-tions giving rise to inviability were not in predictedloops. R3456 is in a comparable position to G877 ofPI3K-g, which is found within b7 and conserved in manyof the lipid kinases (Walker et al. 1999). FRK3544 andRDE3668 align with a6 and a9, respectively, and areuniquely conserved in the Tra1/TRRAP molecules.

Eleven alleles supported viability in rich media. Theirgrowth characteristics under a variety of conditions thatreflect ada, spt, or NuA4 phenotypes are shown in Table3. Other conditions and assays tested but without sub-stantial effects were growth on MMS (also see below),tert-butylhydroperoxide, galactose, raffinose, 6-azauracil,1.0 m NaCl, and tunicamycin. In addition, none ofthese alleles significantly changed the rate of loss of a

TABLE 3

Phenotypes of tra1 mutations

Mutationa 30�b 37�c Phosd Inoe EtOHf VP16g Benh

K3571 1111 1111 1111 1111 1111 — 1111

P3655 1111 1111 1111 1111 111 — 1111

P3408 111 1 1 111 1 — 11

SRR3413 11 — 1 11 — — 1

EER3416 1111 11 1111 1111 1111 — 1111

S3463 1111 1 11 111 1 — 111

F3443 1 — — — — — —P3446 1111 1111 1111 1111 111 1/� 1111

DKL3491 1111 1111 1111 1111 1111 1/� 1111

P3612 1111 1111 1111 1111 111 1/� 1111

N3617 1111 1111 1111 1111 111 1/� 1111

a The indicated mutations were engineered into TRA1SB, which contains a BamHI restriction site that resultsin N3580A and a silent SalI restriction site that converts nucleotides A9714G and T9717G. All amino acid changeswere to alanine.

b Alleles were transformed into CY1021 and tested for viability by growth on mimimal plates containing 5-FOA. Growth at 30� was determined on YPD. Comparisons were made to a strain containing TRA1SB.

c Growth at 37� on YPD plates.d Growth on YPD plates deficient for phosphate.e Growth on complete minimal media lacking inositol.f Growth on YPD plates containing 6% ethanol.g Strains were transformed with padhGV16-gal4-VP16 (Berger et al. 1992) and growth was examined on min-

imal media lacking leucine.h Growth on synthetic complete media containing 20 mg/ml benomyl.


URA3-containing centromeric plasmid after growth onnonselective conditions or resulted in a significant de-fect in mating (not shown). K3571A and N3580A had littleeffect on growth under any of the conditions tested.Four of the alleles, P3446A, DKL3491, P3662A, and N3617A,showed a very weak classic ada phenotype (Berger et al.1992), being partially resistant to overexpression ofVP16. (Note that a deletion of gcn5 would score as1111 on this scale.) Some of these alleles were alsoslightly sensitive to 6% ethanol, characteristic of an ada2disruption (Takahashi et al. 2001).

Three alleles, tra1-SRR3413, tra1-P3408A, and tra1-S3463A, resulted in a common and distinct phenotype.(EER3416 had a similar phenotype but to a lesser extent.)Strains containing these alleles were temperature sen-sitive for growth at 37�, had dramatically reducedgrowth in media containing elevated concentrationsof ethanol, and grew poorly in media lacking phos-phate. Plates showing the growth of strains containing

these alleles under some of the conditions are shown inFigure 1A. The two alleles with the greatest temperaturesensitivity (tra1-SRR3413 and tra1-P3408A) resulted insomewhat reduced growth on media depleted of inosi-tol or containing benomyl, a microtubule-destabilizingagent. These latter two phenotypes are shared withdeletions of some Spt components of SAGA (Roberts

and Winston 1997) and with components of the NuA4complex (Lemasson et al. 2003; Bittner et al. 2004;Krogan et al. 2004), respectively. The tra1 alleles dis-played similar sensitivity to ethanol as does deletion ofada2 (not shown), and like ada2 they had reducedgrowth on calcofluor white (Figure 1A), suggesting thatthe effect results from changes in cell-wall properties orthe ability of the strains to respond to changes in cell-wall integrity (Takahashi et al. 2001). We examined thegrowth properties of strains containing these alleles inmore detail (Figure 1B). At 34�, the tra1-SRR3413, tra1-P3408A, and tra1-S3463A alleles resulted in a reduced

Figure 1.—Phenotypesof Tra1-SRR3413, P3408A,and S3463A. (A) Yeast strainscontaining the tra1 mutantalleles and Tra1SB (parentmolecule) expressed fromplasmids or endogenouswild-type Tra1 (KY320) weregrown to saturation and 10-fold serial dilutions platedonto YP media containing2% glucose and grown at30� and 37� or at 30� inYPD containing 3% ethanolor 5 mg/ml calcofluor white(bottom). Note that expres-sion of Tra1SB on a plasmidresults in slightly reducedgrowth as compared to en-dogenous wild-type Tra1.(B) Growth curves of Tra1mutant strains after temper-ature shift to 37�. The indi-cated tra1 mutant strains orthe background wild-typestrain (TRA1SB) were grownto saturation and dilutedinto YPD media at 34� (time0). Cells were grown for4 hr with the absorbance at600 nm determined onhourly intervals. One-halfof the culture volume was re-moved and shifted to 37�andabsorbancedeterminedfor cultures at both temper-atures. Note the scale differ-ences for the graphs. Thearrow indicates 30 min after

the temperature shift, the first time point a reading at both temperatures was made. (C) Analyses of a strain containing an integratedversion of the tra1-SRR3413 that lacks the BamHI site found in the plasmid copy. Yeast strains CY2437 (integrated wild-type TRA1) andCY2438 (integrated tra1-SRR3413) were grown to saturation in YPD. Serial dilutions were spotted onto YPD plates and grown at 30� and37� or at 30� in YPD containing 5 mg/ml calcofluor white.


growth rate in rich media relative to the TRA1SB allele.Upon heat shock the mutant strains showed an imme-diate growth arrest but did recover; however, 4 hr afterthe shift to 37�, growth stopped. To ensure that theeffects of these mutations were not due to expressionon a plasmid or the background N3580A mutation fromthe BamHI site within TRA1SB, tra1-SRR3413 lacking theN3580A mutation was integrated into the genome. Asshown in Figure 1C, the integrated allele showed similarslow growth at elevated temperature and in the pres-ence of calcofluor white as did the plasmid version.

Since the NuA4 complex has a role in DNA double-strand-break repair (Bird et al. 2002), we examinedstrains containing Tra1-SRR3413, P3408A, and S3463Amore closely for sensitivity to MMS compared to a de-letion of the NuA4 component Vid21 (vacuole importand degradation)/Eaf1. tra1-SRR3413 showed a slightsensitivity to MMS but dramatically less so than deletionof vid21/eaf1 (not shown). In support of only minorchanges in double-strand-break repair, none of thethree alleles resulted in enhanced sensitivity to g-irradiation at 30� (not shown).

To evaluate whether the growth phenotypes mightresult from reduced expression of the altered formsof Tra1, the levels of Flag3x-tagged Tra1-P3408A, Tra1-S3463A, and Tra1-SRR3413 were examined by Westernblotting of crude yeast extracts (Figure 2A). Each ofthese forms was expressed at a level approximately equi-valent to Tra1SB, although we observed slightly elevated

levels of Tra1-S3463A. We focused additional studies ontra1-SRR3413 since it displayed the most dramatic phe-notypes. To determine if Tra1-SRR3413 associated withcomponents of NuA4 and SAGA complexes, a TAP-tagged version of the molecule was purified by tandemaffinity purification and the presence of Spt7 and Flag-tagged Yng2 determined by Western blotting (Figure2B). SRR3413 interacted with Flag-Yng2 and with twoforms of Spt7, suggesting its presence in NuA4, SAGA,and SAGA-like (SLIK) complexes. To further examine ifTra1-SRR3413 is found within SAGA and NuA4 com-plexes, crude extracts containing Tra1-SRR3413 werefractionated on a HiTrap Q sepharose column and theelution profile of HA-Ada2 and Flag-Yng2 was comparedto that found in a strain containing Tra1SB (Figure 2Cand 2D). The elution profiles for Tra1-SRR3413 variedlittle with Tra1SB, suggesting that it associates in theappropriate high molecular mass complexes.

Gene expression in the conditional tra1 strains: Todetermine if the tra1-SRR3413, tra1-P3408A, and tra1-S3463Aalleles resulted in changes in transcriptional regulation,we examined expression of PHO5, HIS4, INO1, andADH2 using lacZ-reporter fusions (Figure 3A). Assayswere performed at 30� under conditions that wouldinduce each specific promoter. Strains deleted for gcn5,spt7, and yng2 were examined for comparison. The tra1mutations resulted in expression of PHO5 at a level from3 to 17% of that seen in the wild-type (TRA1SB), theextent paralleling the severity of the growth defects.

Figure 2.—Expression and interactions of theethanol-sensitive Tra1 mutants. (A) Crude extractswere prepared from yeast strain KY320 (�ve) orstrains expressing TAP-Flag3x-tagged versions ofTra1SB, Tra1-S3463A, Tra1-P4308A, and Tra1-SRR3413. Twenty-five micrograms of extract wereseparated by SDS–PAGE (5%) and Western blot-ted with anti-Flag antibody. (Top) Western blotof the �450-kDa band corresponding to Tra1;(bottom) a portion of an equivalent gel stainedwith Coomassie Brilliant Blue to verify protein con-centrations. (B) tra1-SRR3413 associates with Spt7and Yng2. Top section: Crude extracts were pre-pared from strains KY320 expressing wild-typeTra1 (�ve), TAP-Flag-Tra1SB, or TAP-Flag-Tra1-SRR3413 and with Flag-Yng2. One hundred fiftymicrogramsofcrude extractwere separated byelec-trophoresis on 5% SDS–PAGE and visualized byWestern blotting using anti-Flag antibody. Copur-ification: Extracts were subjected to tandem affin-ity purification. Equal volumes were separated byelectrophoresis on 5 and 8% SDS–PAGE and West-ern blotted using anti-Flag antibody to detect Tra1

(top) or Yng2 (middle). The presenceofSpt7(bottom) wasdetectedusing anti-Spt7 antibody(provided byFred Winston). TheSAGAformandSLIKformofSpt7aremarkedasSAandSL, respectively. (C)Tra1-SRR3413 formsAda2-containingcomplexes thatelute froma HiTrap Q sepharose Fast Flow column with a similar profile to Tra1SB. Whole-cell extract from strains containing Tra1-SRR3413 orTra1SB, each with HA-Ada2, were fractionated on a HiTrap Q sepharose Fast Flow column. Protein was eluted with a gradient of0–1.0 m NaCl. Equal volumes of successive fractions were separated on 8% SDS gels and Western blotted for HA-Ada2, and relativeamounts determined by densitometry. (D) Tra1-SRR3413 forms Yng2-containing complexes that elute from a HiTrap Q sepharoseFast Flow column with a similar profile to Tra1SB. Twenty-five milligrams of whole-cell extract from strains containing Tra1-SRR3413

or Tra1SB, each with Flag-Yng2, were fractionated on a HiTrap Q sepharose Fast Flow column as above. Equal volumes of successivefractions were separated on 8% SDS gels and Western blotted with anti-Flag antibody.


Expression of HIS4 and INO1 was relatively unchangedin the tra1 backgrounds. ADH2 was decreased on aver-age to 60% wild-type levels for the three tra1 mutantswith the extent again paralleling the growth defect. Thepattern of expression seen in the tra1 strains mostclosely resembled that seen upon disruption of gcn5;expression of HIS4 is reduced upon disruption of spt7and yng2, unlike the tra1 strains and INO1 is reducedupon disruption of spt7. Elevated temperature did resultin enhanced effects of tra1-SRR3413 as expression ofINO1-lacZ was reduced to �50% of that seen in TRA1SB

when cells were grown at 35� (Figure 3B).A more detailed analysis of gene expression in the

tra1-SRR3413, tra1-P3408A, and tra1-S3463A strains wasobtained by performing microarray analysis. The Agi-lent yeast oligo array representing all independent yeastORFs was probed with cRNA produced from cells grownlogarithmically at 30� in YP media containing 2%glucose (note that this differs from the data shown inFigure 4, which were performed in inducing condi-tions). Two-dye experiments were performed compar-ing expression in the mutant strains to that found in astrain containing TRA1SB. For each mutant, the exper-iment was performed in duplicate with dye reversal. Thetra1-SRR3413 allele resulted in a change in expression oftwofold or greater of �7% of the protein encodinggenes with �70% having decreased expression. The 25genes showing the greatest increase and decrease inexpression for each mutant are shown in Table 4 (thefull data set has been submitted to the GEO database atNCBI under accession no. GSE6847). The genes af-fected in the three strains were highly similar, consistentwith the mutant alleles having a common defect. Geneontology of those most affected indicates a group ofupregulated genes are involved with ion transport andcell cycle regulation, while a number of the down-regulated genes have roles in nucleotide and amino acidmetabolism.

Hierarchical cluster analysis was performed with thearray data for the tra1-SRR3413 strain, strains with dele-

tions of SAGA and NuA4 components, and a subset ofthe compendium data set (Hughes et al. 2000) in-cluding those genes linked to transcription. tra1-SRR3413

clustered in a leaf with a deletion of ada2, although thedepth of the branch is indicative of only weak similarity(Figure 4). Profiles of strains with deletions of the NuA4components (eaf5, yaf9, eaf1/vid21, and yng2) showedless similarity with tra1-SRR3413, than ada2, spt3, or spt8.Overall, the relatively low level of similarity indicatesthat the transcription pattern of tra1-SRR3413 resultsfrom the cumulative effects of this allele on both SAGAand NuA4 complexes and/or from there being one ormore independent roles for Tra1.

Brown et al. (2000) previously identified a tra1 allelethat resulted in decreased binding to the Gal4 transcrip-tional activation domain. To address whether the tran-scriptional changes observed for tra1-SRR3413 were dueto a defect in its interaction with transcriptional acti-vation domains, we compared the ability of Tra1-SRR3413

and Tra1SB to associate with the Gal4 activation domain(Gal4AD). Tandem- affinity-purified Tra1SB and Tra1-SRR3413, prepared from strains containing HA-taggedAda2, were chromatographed on GST-Gal4AD columns.Association of SAGA with Gal4 was detected by Westernblotting for HA-Ada2 (Figure 5A). SAGA containingTra1-SRR3413 bound Gal4AD to the same extent as SAGA-containing Tra1SB (compare lanes 4 and 6), suggestingthat the mutation does not decrease association withtranscriptional activators. This is consistent with the re-gion required for activator interaction being containedwithin amino acid residues 2233–2836 (Brown et al.2001). Recruitment of Tra1-SRR3413 to the PHO5 promoterin vivo was examined by chromatin immunoprecipita-tion using the TAP-tagged derivatives of Tra1. As shownin Figure 5B, Tra1-SRR3413 was recruited as efficiently tothe PHO5 promoter as Tra1SB (compare lanes 2 and 3,top; input DNAs are shown in the bottom).

To determine if Tra1-SRR3413 alters acetylation ofeither histone H3 or histone H4, chromatin immuno-precipitations were performed. We first compared the

Figure 3.—Expression of b-galactosidase reporter constructs intra1 mutant strains. (A) Promoterfragments from PHO5, INO1,HIS4, and ADH2 were cloned ashis3-LACZ reporter fusions intothe LEU2 centromeric plasmidYCp87 and transformed intoFY1093 (spt7D0), FY1370(gcn5D0), CY1514 (tra1-S3463A),CY1507 (tra1-P3408A), andCY1531 (tra1-SRR3413). b-Galacto-sidaseactivitywasdeterminedaftergrowth in low phosphate media

(PHO5), inositol-depleted media (INO1), minimal media (HIS4), or YP containing 3% ethanol plus 0.01% glucose (ADH2) at30�. Activity was compared to that in the appropriate wild-type strains—FY630 for spt7D, FY86 for gcn5D, and CY1524 for the tra1mutant strains—and is shown as percentages. Measurements were made in triplicate in two independent experiments with the stan-dard error indicated. (B) Expression of INO1-lacZ was analyzed at 30� and 35� for strains containing TRA1SB and tra1-SRR3413.


ratio of acetylated histone H4 (at lysine 8) to total his-tone H3 at the ADE1 and PHO5 promoters for the straincontaining tra1-SRR3413 with strains containing dele-tions of the NuA4 components Vid21/Eaf1 and Yng2.Figure 6A shows the relative level of acetylated histoneH4 as a percentage of that found in the wild-type strain.For both ADE1 and PHO5 promoters, tra1-SRR3413 re-sulted in a decrease in relative acetylation to �60% ofthat found in the TRA1SB strain. This decrease was com-parable to that found upon deletion of yng2 and some-what less than deletion of vid21/eaf1. For each mutantstrain, a smaller effect was observed for sequences at the39 end of the PHO5 gene. Acetylation of histone H3 (atlysine 9) was then analyzed at PHO5, PHO84, and PGK1promoters (Figure 6B), the latter being SAGA-indepen-dent. tra1-SRR3413 resulted in a decrease in the ratio ofacetylated histone H3 to total histone H3 at PHO5 andPHO84 promoters that was �35% of that found in thewild-type tra1SB strain, approaching that seen in strainsdeleted for spt7 and ada2. At the PGK1 promoter, thedecrease in acetylation of histone H3 was less apparent(�70% of wild type for tra1-SRR3413). Although wecannot exclude an indirect role of Tra1 on acetylation,the simplest interpretation of these results is that thePI3K domain of Tra1 is required for optimal histoneacetylation by both the NuA4 and SAGA complexes.

Tra1-SRR3413 results in shortened telomere length:The similarity of Tra1 to many of the PI3K-domain-containing proteins involved in telomere biology (for

example, Tel1 and Mec1) led us to test if the tra1-SRR3413, tra1-P3408A, and tra1-S3463A alleles affectedtelomere length. As an initial test we assayed the effectsof these alleles on the ability of strains to generate andstably maintain linearized plasmids after transformationwith circular plasmids containing inverted repeats oftelomeric sequences (Lundblad and Szostak 1989).Strains containing tra1-SRR3413, tra1-P3408A, and tra1-S3463A were transformed with the LEU2-containingcentromeric plasmid pVL106 (kindly supplied by V.Lundblad), which contains URA3 flanked by invertedtelomeric sequences. Cells were grown in nonselectivemedia and plated onto media containing 5-FOA andlacking leucine. 5-FOA-resistant/Leu1 colonies werecounted and normalized to the number of total cellsplated. As shown in Figure 7A, tra1-SRR3413 resulted in areduced ability to linearize and maintain pVL106. Todirectly examine telomere length, Southern blottingwas used to compare the terminal restriction fragments(TRFs) of chromosomes in the tra1-SRR3413 and TRA1SB

strains. CY1021 containing wild-type TRA1 on a URA3-containing plasmid was transformed with TRA1SB ortra1-SRR3413 on TRP1-centromeric plasmids. The wild-type TRA1 allele was lost after selection on 5-FOA,establishing generation zero. Strains were grownthrough multiple generations, genomic DNA was iso-lated, and TRF length was determined after digestion ofthe genomic DNA with XhoI. Cells were grown at both30� and 34�. The latter significantly reduces growth rate

Figure 4.—Hierarchical cluster analysis oftra1-SRR3413 microarray expression data. Micro-array analyses were performed for yeast strainCY1531 containing tra1-SRR3413 as the sole copyof Tra1 grown in YPD at 30� using the Agilentyeast oligo array kit. The experiment was per-formed in duplicate with dye reversal usingTRA1SB (yeast strain CY1524) as the control.Comparisons were made to the expression pro-files of strains with deletions of NuA4 (Krogan

et al. 2004) and SAGA (Ingvarsdottir et al.2005) components. Also included were profilesof strains with deletions of cka2, ckb2, gcn4,hat2, isw1, mbp1, rpd3, rtg1, sap30, sin3, ste12,and yap3, which were identified as being mostsimilar to tra1-SRR3413 in an initial clusteringanalysis using a subset of the compendium data(Hughes et al. 2000). Gene families are indicatedto the right.


TABLE 4

Gene expression profile in tra1-P3408A, -SRR3413, and -S3463A

tra1-P3408A tra1-SRR3413 tra1-S3463A

Genea Foldb Gene Fold Gene Fold

1 FET3 3.2 TKL2 4.5 YGL262W 6.02 PCL1 3.2 YHR033W 3.9 AFT2 3.93 ATF2 3.2 SCW10 3.5 COS12 3.54 RNR1 3.1 YLR327C 3.4 SCW10 3.05 SCW10 3.1 YER053C 3.4 FET3 3.06 HXT4 2.9 PDR12 3.3 RNR1 3.07 SVS1 2.8 YLR312C 3.1 PDR12 2.98 YKL097W-A 2.7 SVS1 3.1 PCL1 2.89 PDR12 2.7 MNN1 3.1 PUS2 2.710 DBP2 2.6 RNR1 3.0 KAP122 2.711 YHR033W 2.6 ATF2 3.0 SVS1 2.712 RMD6 2.5 PCL1 3.0 RRN3 2.713 MNN1 2.5 RRN3 2.9 MNN1 2.714 NCE4 2.5 MND1 2.8 HXT4 2.615 RRN3 2.4 ECM11 2.8 DBP2 2.616 APG10 2.4 KCC4 2.7 AGP10 2.517 YER053C 2.4 TRA1 2.7 YHR033W 2.518 HO 2.4 APG14 2.7 HO 2.519 RPL7B 2.3 PUS2 2.7 TRA1 2.420 PLB3 2.3 INO1 2.6 YPL207W 2.321 RNA14 2.3 FET3 2.6 FDH2 2.322 MIR1 2.3 BDF2 2.6 DHR2 2.323 YLR312C 2.3 HRP1 2.6 YER053C 2.324 MEP3 2.3 SIT1 2.6 MDV1 2.325 PUS2 2.3 ATP7 2.5 TOP3 2.3Total no.c 85 152 97

1 IMD2 �14.7 IMD2 �19.3 IMD2 �18.32 ADE17 �14.2 ADE17 �13.5 SPL2 �10.13 SPL2 �10.9 SPL2 �11.4 YBR285W �7.94 HIS4 �6.9 AAD4 �11.3 ADE17 �7.25 YBR285W �6.9 YLR053C �8.7 AAD4 �6.36 AAD4 �6.8 YBR285W �8.5 HIS4 �5.57 ADE1 �6.5 ADE1 �8.5 YAL061W �5.28 LAP4 �6.3 PHO84 �7.9 TSA2 �5.29 VPS73 �6.1 HIS4 �7.4 YMR090W �5.110 KHS1 �5.8 VPS73 �7.3 ZTA1 �5.111 YAL061W �5.8 GTT2 �7.2 EMI2 �5.012 SNZ1 �5.8 YMR090W �7.2 SNZ1 �4.913 YLR053C �5.5 YAL061W �6.5 YBR047W �4.914 SHM2 �5.2 TSA2 �6.5 YLR460C �4.815 PH05 �5.1 BNA1 �6.1 YLR053C �4.716 ZTA1 �5.1 LYS20 �6.1 YBR147W �4.617 PHO84 �5.0 ADE5,7 �6.1 YPS73 �4.518 TSA2 �5.0 LAP4 �5.8 YJL038C �4.319 EMI2 �4.9 IMD1 �5.8 SNO1 �4.320 IMD1 �4.8 ZTA1 �5.8 YMR118C �4.221 ADE5,7 �4.7 LYS9 �5.7 YKL107W �4.222 YJL038C �4.7 LYS7 �5.5 LAP4 �4.223 YBR147W �4.7 GCV2 �5.4 ECM12 �4.224 YNL194C �4.6 YPS6 �5.3 PHO84 �4.125 GCV2 �4.6 YKL107W �5.3 IMD1 �4.1Total no.c 258 294 261

a Genes underlined are found a minimum of either twofold elevated or fourfold decreased in one of the other strains. Thoseitalicized are threefold decreased in one of the other strains. Dubious ORFs are not listed. HIS4 was downregulated in the genearray data more so than in the b-galactosidase assays, likely reflecting different levels of Tra1 contribution in different media.

b Values are the fold-change in expression relative to that in CY1524. Cells were grown in YPD media to an A600nm of�1.5. Valuesare the average of two experiments with dye reversal.

c Total number of genes affected twofold or greater.


and from the linearization assay appeared to suppressthe effects of SRR3413 on telomere shortening (notshown). As shown in Figure 7B, at 10 generations ofgrowth, the TRFs were of approximately equal lengths

in TRA1SB and tra1-SRR3413 strains. By 50 generations,TRFs were appreciably shorter in the tra1-SRR3413 strain,with a further shortening apparent at 100 generationswhen cells were grown at 30�. The median difference was�120 and 140 bp at 50 and 100 generations, respectively.The effect of SRR3413 was not apparent when cells weregrown at 34�, perhaps the result of increasing the periodavailable for telomere extension.

Genetic analysis: To examine the molecular basis forthe tra1-SRR3413 phenotype, we initiated a suppressoranalysis. tra1-SRR3413 was introduced into yeast strainCY1021 by plasmid shuffling. Approximately 2 3 106

cells were plated onto YPD containing 6% ethanol.Growth of two colonies was observed after 5 days. Todetermine if these were intragenic, the tra1-expressingplasmid was isolated from both strains and the pheno-type reexamined after plasmid shuffling in yeast strainCY1021. In both cases the plasmid enabled growth inYPD containing 6% ethanol. The 39 SalI–SacI fragmentof each allele was sequenced and found to contain theSRR3413 mutation and a common mutation convertingH3530Y. This mutation was shown to be sufficient forsuppression by recloning the SalI–SacI fragment intoTRA1SB and plasmid shuffling. H3530Y suppressed thegrowth defects associated with SRR3413 on both 6%ethanol-containing plates (Figure 8A) and at 37� (notshown). Allele specificity was examined by introducingH3530Y into wild-type and tra1-P3408A-containing back-grounds. H3530Y on its own had no effect on growth butdid suppress the ethanol sensitivity due to P3408A andtherefore is a general suppressor of the ethanol-sensitiveclass of tra1 alleles. As shown in Figure 8B, the effects ofH3530Y are most likely due to suppression of transcrip-tional defects since this mutation suppressed the slowinduction of PHO5 seen in a tra1-SRR3413 strain.

To detect genes whose disruption in parallel with tra1-SRR3413 generates a synthetic slow-growth phenotype,

Figure 5.—Effects of Tra1-SRR3413 on interaction with theGal4 activation domain and recruitment to the PHO5 pro-moter. (A) Interaction of Tra1-SRR3413 with the Gal4 activationdomain. Yeast extract was prepared from CY1021 (lane 1, neg-ative control), CY1962 containing HA-tagged Ada2 (Tra1SB;lane 2), and CY1963 containing HA-tagged Ada2 (Tra1-SRR3413; lane 3). Purified TAP-Tra1SB was applied to the gluta-thione sepharose bound with GST-Gal4AD (lane 4) or controlglutathione sepharose bound with GST (lane 5). SimilarlyTAP-Tra1-SRR3413 was applied to GST-Gal4AD beads (lane 6)or GST beads (lane 7). Bound protein was eluted with reducedglutathione and the eluant separated on a 10% SDS–PAGE gel.Interaction with Gal4AD was monitored by Western blotting forHA-Ada2. (B) Recruitment of TAP-Tra1 to the PHO5 promoterwas determined by chromatin immunoprecipitation usingstrains BY4741 (lane 1) and BY4741 containing TAP-Tra13413

(lane 2) or TAP-Tra1-SRR3413 (lane 3). The presence of PHO5promoter DNA was determined by PCR and separation on5% native polyacrylamide gels stained with ethidium bromide.PCR products from the input DNA used for analysis are shownin the bottom. A serial dilution of the sample for Tra1-SRR3413

is shown in lanes 3–5. Lane 6 is a no-template control.

Figure 6.—Histone acetylationin Tra1-SRR3413-containing strains.(A) Acetylation of lysine 8 of his-tone H4. Yeast strains CY1531(tra1-SRR3413), CY1524 (TRA1SB),QY202 (yng2D0), QY204 (YNG2),BY4916 (vid21D0), and BY4741(VID21) were grown to A600 ¼ 1.5in YPD and ChIP analysis was per-formed using antibody directedagainst acetylated K8 of histoneH4 and against histone H3. Levelsof immunoprecipitated ADE1 pro-moter, PHO5 promoter, and the39-coding region of PHO5 were de-

termined after PCR and separation by electrophoresis and staining with ethidium bromide. Serial dilutions were examined to ensuredetection in a linear range. The histogram shows the ratio of acetylated histone H4 to total histone H3 for each of the mutant strains asa percentage of that found in the relevant wild-type strain for experiments performed in duplicate. (B) Histone H3 acetylation. Yeaststrains CY1531 (tra1-SRR3413), CY1524 (TRA1SB), BY3281 (spt7D0), BY4282 (ada2D0), and BY4741 (wild type) were grown in phos-phate-depleted media (PHO5) or YPD (PHO84 and PGK1) to an A600¼ 1.5. Equal amounts of chromatin were immunoprecipitatedwith anti-acetylated (K9) histone H3 antibody or anti-histone H3 antibody and levels of promoter determined after PCR. The ratio ofacetylated histone H3 to total histone H3 for each mutant is shown as a percentage of that found in the relevant wild-type strain.


we performed a systematic genetic array (SGA) analysisin which a strain containing tra1-SRR3413 was crossedinto the array representing the collection of �4700viable haploid deletion strains. Candidate genetic inter-actions, scored as slow-growing colonies through twoindependent screenings, were examined individuallyafter generation of double-mutant strains by tetrad

dissection. Growth for these strains was examined onsynthetic complete media at 34�. The 23 genes identi-fied and their functions are listed in supplemental Table2 at http://www.genetics.org/supplemental/. Consis-tent with the finding that tra1-SRR3413 resulted in sen-sitivity to ethanol and calcofluor white, 10 of theseappear involved in cell-membrane and cell-wall pro-cesses. This number is greater if indirect roles, forexample a contribution to phosphatidylserine synthesisby SER1 and SER2, are considered. The other prevalentgroups represented were genes involved with transportand mitochondria function. A hierarchal cluster analy-sis was performed with the tra1-SRR3413 interactinggenes and the data sets of Tong et al. (2004) and Pan

et al. (2006). tra1-SRR3413 did not cluster closely with anyof the strains used in these analyses. In fact, no commonsynthetic growth defects were found among those listedin the Saccharomyces Genome Database for NuA4 andSAGA components and those for tra1-SRR3413.

DISCUSSION

Tra1 is one of the largest yeast proteins and is essentialfor viability. As a component of both NuA4 and SAGAcomplexes, Tra1 interacts directly with DNA-bindingtranscriptional regulators recruiting these HAT com-plexes to promoters. On the basis of the similarity of thisdomain with that found in other cellular regulators it islikely that the PI3K domain of the molecule has a sig-nificant function. Indicative of a key role for the PI3Kdomain, eight targeted mutations within this regionresulted in loss of viability. Although we lack detailedstructural information for the Tra1/TRRAP subfamily,the alignment of these residues in comparison to thestructure of porcine PI3K-g is interesting. ResiduesR3567 and PFR3621 are not unique to Tra1/TRRAP andare found in the catalytic loop and activation loop,respectively. As is the case for the kinase members of thePI3K family (Dhand et al. 1994; Stack and Emr 1994),R3567 is required for function of Tra1. Similarly, PFR3621

is generally conserved within the activation loop of thePI3K family. The activation loop plays a critical role insubstrate binding for these molecules (Bondeva et al.1998), suggesting a similar essential role for this regionin Tra1. Substrates for Tra1 are unknown. Potentiallythese could include phospholipids; however, we havedetected only very weak binding of recombinant Tra1-PI3K domain to PIP strips (Echelon Biosciences, SaltLake City; not shown).

Eight alleles with mutations in the PI3K domainsupported viability and displayed phenotypes, whichfell into two groups. The first group, tra1-P3446, -DKL3491,-P3612, and -N3617, was slightly resistant to overexpressionof VP16, although to a much lesser extent than the adagenes, and was partially sensitive to hydroxyurea,characteristic of defects in DNA replication. This latterphenotype has not been previously ascribed to SAGA or

Figure 7.—TRA1-SRR3413 results in a generation-depen-dent reduction in telomere length. (A) Plasmid linearizationassay. Yeast strains CY1524 (TRA1SB), CY1531 (tra1-SRR3413),CY1507 (tra1-P3408A), and CY1514 (tra1-S3463A) were trans-formed with pVL106 (supplied by V. Lundblad). Leu1 Ura1

transformants were grown sequentially in media lacking uraciland then in YPD, washed, and spotted onto plates containing5-FOA and lacking leucine. The number of 5-FOA-resistantLeu1 colonies was counted after 5 days of growth at 30�and normalized to the total number of cells plated. The graphindicates the percentage of 5-FOA-resistant Leu1 colonies ineach of the tra1 mutant strains as compared to that observedfor TRA1SB for five experiments each performed in duplicate.(B) Southern blot of genomic DNA from TRA1SB (wt) andtra1-SRR3413 (SRR) containing strains cut with XhoI and hy-bridized to a 32P-labeled TG1-3/C1-3A DNA probe. The termi-nal restriction fragment (TRF) is derived from a subset ofyeast telomeres that have a Y9 telomere element. The Y9 ele-ment has a conserved XhoI site that is located proximal tothe terminal telomeric repeat tract. The larger DNA frag-ments are derived from either telomeres lacking the Y9 ele-ment or subtelomeric DNA sequences containing TG1-3

tracks. Cells were serially passaged for the indicated numberof generations. After 10 generations the average TRF lengthfor the two strains (�1110 bp) did not differ significantly(left) at either growth temperature. After 50 and 100 gener-ations at 30� (right) the average TRF length for the tra1-SRR3413 strain was reduced by �125 and 140 bp, respectively.


NuA4 components and may reflect a novel function ofone or both of these complexes or perhaps defects inexpression of genes involved in this process. The secondgroup of alleles containing mutations of P3408, SRR3413,S3463, and to a lesser extent EER3416, was more marked.These alleles resulted in temperature-sensitive growthand reduced growth in low-phosphate media and inmedia containing 6% ethanol. The latter result has alsobeen noted for deletions of ada2 and on the basis oftheir reduced growth in the presence of calcofluorwhite, this phenotype likely reflects changes in cell-wallproperties (Takahashi et al. 2001) or the response tocell-wall stress. Unlike deletions of Ada components, thetra1-P3408A, -SRR3413, and -S3463A alleles did not resultin resistance to overexpression of VP16, nor is theirtemperature-sensitive growth indicative of deletions ofada genes in the KY320 strain background. tra1-P3408

and tra1-SRR3413, the alleles with the most pronouncedphenotypes, were partially sensitive to the microtubule-destabilizing agent benomyl. This is characteristic ofdeletions of NuA4 components, but again for the tra1alleles the effect was not as severe. The tra1-SRR3413

allele was the only allele showing sensitivity to MMS butdramatically less so than for deletions of components ofthe NuA4 complex. Growth of tra1-P3408, SRR3413, andS3463 strains was modestly reduced on media depletedof inositol, a characteristic of deletions of spt genes;however, unlike deletion of spt7, the inositol auxotrophyof the tra1 alleles was not the result of reduced expres-sion of INO1. Cumulatively, these phenotypes suggestthat Tra1 possesses functions overlapping yet distinctfrom SAGA or NuA4 components.

The transcriptional effects of the tra1-P3408A, SRR3413,and S3463A alleles were compared to deletions of spt7,gcn5, and yng2 using lacZ fusion reporters. The tran-scriptional change for the tra1 alleles was less than thatseen upon deletion of the NuA4 or SAGA componentsbut most closely resembled that seen upon deletion ofgcn5. Microarray analyses confirmed the transcriptiondefect arising from these alleles and that they were act-ing by altering a common function. Cluster analysis vs.

the expression profiles seen for deletions of SAGAand NuA4 components showed the greatest similarity,though not robust, to ada2. Comparison of the growthphenotypes of strains containing deletion of strainscarrying single and double mutations of ada2D0 andtra1-P3408A confirmed that the tra1 mutations were notfunctioning exclusively through effects on the ADAgenes (data not shown). This lack of similarity suggeststhat the transcriptional effects of the tra1 alleles resultfrom the integrative effect of disturbing histone acety-lation by both SAGA and NuA4 complexes and/or thepossibility that the PI3K domain of Tra1 has one or moreroles distinct from other members of these complexes.The mechanism by which the tra1 mutations affecthistone acetylation is unclear; interestingly, in the three-dimensional structure of the SAGA complex revealed byelectron microscopy, Tra1 and Gcn5 are positioned onopposite sides of a nucleosome-sized cleft (Wu et al.2004), suggesting that Tra1 may play a role in orientingnucleosomes for acetylation.

The finding of a generation-dependent telomere short-ening in the tra1-SRR3413 strain points to a role of Tra1 intelomere elongation or maintenance. The shortenedtelomeres may be the indirect result of Tra1 regulatingexpression of genes required for these processes. In thisregard 11 genes whose deletion results in shortened telo-meres (Askree et al. 2004) have decreased expressionof $1.5-fold in the tra1-SRR3413 strain. Most notable ofthese is EST3 with decreased expression of 2.4-fold. Al-ternatively, Tra1 may play a direct role in telomere elon-gation or maintenance. We do not believe that the Tra1effect is mediated solely through one of SAGA or NuA4-dependent histone acetylation, as component genes werenot found in the genomewide screen of Askree et al.(2004) or in our independent analyses of Spt7, Gcn5,and Eaf7 (data not shown). Additional studies will be re-quired to determine if Tra1 is recruited to telomeres andthe role of the PI3K domain in telomere elongation.

The ethanol and calcofluor white sensitivity of tra1-SRR3413 as well as its genetic interactions with genes in-volved in cell-wall and membrane processes is consistent

Figure 8.—H3530Y suppresses the ethanol sen-sitivity and transcription defects due to tra1-SRR3413. (A) Yeast strains containing the TRA1alleles indicated in the legend to the right wereplated onto YPD or YPD containing 6% ethanoland grown at 30�. (B) Delay in the induction ofPHO5 resulting from SRR3413 is suppressed byalteration of H3530Y. Yeast strains containingTra1SB, Tra1-SRR3413, and Tra1-SRR3413-H3530Ywere transformed with PHO5-lacZ, grown to satu-ration in YPD, washed four times in water, and di-luted 1:12 into low-phosphate media. Sampleswere taken at the indicated time points and b-galactosidase activity was determined.


with the involvement of SAGA in the response to stress(Huisinga and Pugh 2004). Tra1 may be required to trig-ger transcriptional changes necessary when membraneprocesses are disturbed during cellular stress. Associa-tions between SAGA components and membrane pro-cesses have previously been suggested by the finding thatGcn5 and Spt20 are required for the unfolded proteinresponse (Welihinda et al. 1997, 2000). Interestingly,deletion of a number of genes involved in vesicular traf-ficking, including vps9, vps15, vps28, vps34, vps36, vps39,bro1, and sur4, results in both telomere shortening andethanol sensitivity similar to that in tra1-SRR3413. Theother two gene deletions with both of these phenotypesare bem2 and sit4, each of which is involved in cytoskel-etal and cell-wall organization. (Expression of these genesis not reduced in the tra1-SRR3413 background.) Otherstudies connect NuA4 components with membrane pro-cesses; most notably, vid21/eaf1 (vacuole import and deg-radation) was identified in a screen for defects in sortingof carboxypeptidase Y to the vacuole (Bonangelino

et al. 2002), is sensitive to ethanol (Fujita et al. 2006),and interacts with Vac8, which is required for severalaspects of vacuolar function (Tang et al. 2006).

Given the importance of PI3K domains in key signal-ing proteins and the conservation of this domain in theTra1/TRRAP family of molecules, it is reasonable toconsider possible activities of the PI3K domain. In thisregard P3408, SRR3413, and S3463 map proximally to whatwould be the cleft between N- and C-terminal lobes ofthe PI3K-g structure. P3408, in fact, aligns with K833,which in PI3K-g interacts with the a-phosphate ofATP and is covalently modified by the kinase inhibitorwortmanin (Wymann et al. 1996). While clearly an ap-proximation of the Tra1 structure, S3463 may be locatedwithin the cleft at a position that putatively could beinvolved with substrate interaction. Though speculative,it is attractive to propose that the putative cleft of Tra1 isa substrate-binding pocket, where the domain catalyzesthe transfer of a modifying unit other than phosphatemoieties, transfers water as a hydrolase, or perhaps has arole in the nuclear signaling by inositol polyphosphates(for example, Steger et al. 2003).

We thank Brian Shilton, Catherine Bateman, David Litchfield, andGreg Gloor for their comments on the manuscript; Victoria Lundblad,Fred Winston, Kathy Gould, Joan Curcio, V. Zakian, and RaymundWellinger for reagents; Shayna Oldegard, Dana Abrassart, MonicaPiasecki, Courtney Coschi, and Kim Grant for assistance in prepara-tion of DNA constructs; and Joe Martens and Melissa Bradford forassistance with the chromatin immunoprecipitations. This work wassupported by Canadian Institutes of Health Research grant to C.J.B.S.M.T.H. is a holder of a Natural Sciences and Engineering ResearchCouncil studentship. C.H. was supported by an Ontario GraduateScholarship in Science and Technology scholarship. A.I.M. is a holderof a Western Graduate Research Scholarship.

LITERATURE CITED

Alekseyenko, A. A., E. Larschan, W. R. Lai, P. J. Park and M. I. Kuroda,2006 High-resolution ChIP-chip analysis reveals that the Dro-

sophila MSL complex selectively identifies active genes on themale X chromosome. Genes Dev. 20: 848–857.

Allard, S., R. T. Utley, J. Savard, A. Clarke, P. Grant et al.,1999 NuA4, an essential transcription adaptor/histone H4acetyltransferase complex containing Esa1p and the ATM-relatedcofactor Tra1p. EMBO J. 18: 5108–5119.

Ard, P. G., C. Chatterjee, S. Kunjibettu, L. R. Adside, L. E. Gralinski

et al., 2002 Transcriptional regulation of the mdm2 oncogeneby p53 requires TRRAP acetyltransferase complexes. Mol. Cell.Biol. 22: 5650–5661.

Askree, S. H., T. Yehuda, S. Smolikov, R. Gurevich, J. Hawk et al.,2004 A genome-wide screen for Saccharomyces cerevisiae deletionmutants that affect telomere length. Proc. Natl. Acad. Sci. USA101: 9515–9516.

Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman

et al., 1998 Protocols in Molecular Biology. Greene/Wiley-Interscience,New York.

Balasubramanian, R., M. G. Pray-Grant, W. Selleck, P. A. Grant

and S. Tan, 2001 Role of the Ada2 and Ada3 transcriptional co-activators in histone acetylation. J. Biol. Chem. 277: 7989–7995.

Berger, S. L., 2002 Histone modifications in transcriptional regula-tion. Curr. Opin. Genet. Dev. 12: 142–148.

Berger, S. L., B. Pina, N. Silverman, G. A. Marcus, J. Agapite et al.,1992 Genetic isolation of ADA2: a potential transcriptionaladaptor required for function of certain acidic activation do-mains. Cell 70: 251–265.

Bhaumik, S. R., T. Raha, D. P. Aiello and M. R. Green, 2004 In vivotarget of a transcriptional activator revealed by fluorescence res-onance energy transfer. Genes Dev. 18: 333–343.

Bird, A. W., D. Y. Yu, M. G. Pray-Grant, Q. Qiu, K. E. Harmon et al.,2002 Acetylation of histone H4 by Esa1 is required for DNAdouble-strand break repair. Nature 419: 411–415.

Bittner, C. B., D. T. Zeisig, B. B. Zeisig and R. K. Slany, 2004 Di-rect physical and functional interaction of the NuA4 complexcomponents Yaf9p and Swc4p. Eukaryot. Cell 3: 976–983.

Bonangelino, C. J., E. M. Chavez and J. S. Bonifacino, 2002 Ge-nomic screen for vacuolar protein sorting genes in Saccharomycescerevisiae. Mol. Biol. Cell 13: 2486–2501.

Bondeva, T., L. Pirola, G. Bulgarelli-Leva, I. Rubio, R. Wetzker

et al., 1998 Bifurcation of lipid and protein signals of PI3Kgam-ma to the protein kinases PKB and MAPK. Science 282: 293–296.

Bosotti, R., A. Isacchi and E. L. Sonnhammer, 2000 FAT: a noveldomain in PIK-related kinases. Trends Biochem. Sci. 25: 225–227.

Bouchard, C., O. Dittrich, A. Kiermaier, K. Dohmann, A. Menkel

et al., 2001 Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and his-tone acetylation at the cyclin D2 promoter. Genes Dev. 15: 2042–2047.

Brandl, C. J., A. M. Furlanetto, J. A. Martens and K. S. Hamilton,1993 Characterization of NGG1, a novel yeast gene required forglucose repression of GAL4p-regulated transcription. EMBO J.12: 5255–5265.

Brown, C. E., L. Howe, K. Sousa, S. C. Alley, M. J. Carrozza et al.,2001 Recruitment of HATcomplexes by direct activator interac-tions with the ATM-related Tra1 subunit. Science 292: 2333–2337.

Brownell, J. E., J. Zhou, T. Ranalli, R. Kobayashi, D. G. Edmondson

et al., 1996 Tetrahymena histone acetyltransferase A: a transcrip-tional co-activator linking gene expression to histone acetylation.Cell 84: 843–851.

Chen, W., and K. Struhl, 1988 Saturation mutagenesis of a yeasthis3 ‘‘TATA element’’: genetic evidence for a specific TATA-bindingprotein. Proc. Natl. Acad. Sci. USA 85: 2691–2695.

Choy, J. S., and S. J. Kron, 2002 NuA4 subunit Yng2 function inintra-S-phase DNA damage response. Mol. Cell. Biol. 22: 8215–8225.

Clarke, A. S., J. E. Lowell, S. J. Jacobson and L. Pillus,1999 Esa1p is an essential histone acetyltransferase requiredfor cell cycle progression. Mol. Cell. Biol. 19: 2515–2526.

Deleu, L., S. Shellard, K. Alevizopoulos, B. Amati and H. Land,2001 Recruitment of TRRAP required for oncogenic transfor-mation by E1A. Oncogene 20: 8270–8275.

Dhand, R., I. Hiles, G. Panayotou, S. Roche, M. J. Fry et al.,1994 PI3-kinase is a dual specificity enzyme: autoregulation


by an intrinsic protein-serine kinase activity. EMBO J. 13: 522–533.

Downs, J. A., S. Allard, O. Jobin-Robitaille, A. Javaheri, A. Auger

et al., 2004 Binding of chromatin-modifying activities to phos-phorylated histone H2A at DNA damage sites. Mol. Cell 16:9979–9990.

Dudley, R. M., C. Rougeulle and F. Winston, 1999 The Spt com-ponents of SAGA facilitate TBP binding to a promoter at a post-activator-binding step in vivo. Genes Dev. 13: 2940–2945.

Eisen, M. B., P. T. Spellman, P. O. Brown and D. Botstein,1998 Cluster analysis and display of genome-wide expressionpatterns. Proc. Natl. Acad. Sci. USA 95: 14863–14868.

Eisenmann, D. M., K. M. Arndt, S. L. Ricupero, J. W. Rodney andF. Winston, 1992 SPT3 interacts with TFIID to allow normaltranscription in Saccharomyces cerevisiae. Genes Dev. 6: 1319–1331.

Eisenmann, D. M., C. Chapon, S. M. Roberts, C. Dollard andF. Winston, 1994 The Saccharomyces cerevisiae SPT8 gene enco-des a very acidic protein that is functionally related to SPT3 andTATA-binding protein. Genetics 137: 647–657.

Fishburn, J., N. Mohibullah and S. Hahn, 2005 Function of a eu-karyotic transcription activator during the transcription cycle.Mol. Cell 18: 369–378.

Fujita, K., A. Matsuyama, Y. Kobayashi and H. Iwahashi,2006 The genome-wide screening of yeast deletion mutantsto identify the genes required for tolerance to ethanol and otheralcohols. FEMS Yeast Res. 6: 744–750.

Gansheroff, L. J., C. Dollard, P. Tan and F. Winston, 1995 TheSaccharomyces cerevisiae SPT7 gene encodes a very acidic proteinimportant for transcription in vivo. Genetics 139: 523–536.

Gietz, R. D., and A. Sugino, 1988 New yeast E. coli shuttle vectorsconstructed with in vitro mutagenized yeast genes lacking six-basepair restriction sites. Gene 74: 255–269.

Grant, P. A., L. Duggan, J. Cote, S. M. Roberts, J. E. Brownell

et al., 1997 Yeast Gcn5 functions in two multisubunit complexesto acetylate nucleosomal histones: characterization of an Ada com-plex and the SAGA (Spt/Ada) complex. Genes Dev. 11: 1640–1650.

Grant, P. A., D. Schieltz, M. G. Pray-Grant, D. J. Steger, J. C.Reese et al., 1998a A subset of TAFIIs are integral componentsof the SAGA complex required for nucleosome acetylation andtranscriptional stimulation. Cell 94: 45–53.

Grant, P. A., D. Schieltz, M. G. Pray-Grant, J. R. Yates, 3rd andJ. L. Workman, 1998b The ATM-related cofactor Tra1 is a com-ponent of the purified SAGA complex. Mol. Cell 2: 863–867.

Green, M. R., 2005 Eukaryotic transcription activation: right on tar-get. Mol. Cell 18: 399–402.

Han, M., U. J. Kim, P. Kayne and M. Grunstein, 1988 Depletion ofhistone H4 and nucleosomes activates the PHO5 gene in Saccha-romyces cerevisiae. EMBO J. 7: 2221–2228.

Herceg, Z., W. Hulla, D. Gell, C. Cuenin, M. Lleonart et al.,2001 Disruption of Trrap causes early embryonic lethalityand defects in cell cycle progression. Nat. Genet. 29: 206–211.

Hoffman, C. S., and F. Winston, 1987 A ten-minute DNA prepara-tion from yeast efficiently releases autonomous plasmids fortransformation of Escherichia coli. Gene 57: 267–272.

Horiuchi, J., N. Silverman, B. Pina, G. A. Marcus and L. Guarente,1997 ADA1, a novel component of the ADA/GCN5 complexhas broader effects than GCN5, ADA2, or ADA3. Mol. Cell. Biol.17: 3220–3228.

Hughes, T. R., M. J. Marton, A. R. Jones, C. J. Roberts, R. Stoughton

et al., 2000 Functional discovery via a compendium of expressionprofiles. Cell 102: 109–126.

Huisinga, K. L., and B. F. Pugh, 2004 A genome-wide housekeepingrole for TFIID and a highly regulated stress-related role for SAGAin Saccharomyces cerevisiae. Mol. Cell 13: 573–585.

Iizuka, M., and M. M. Smith, 2003 Functional consequences of his-tone modifications. Curr. Opin. Gen. Dev. 13: 154–160.

Ingvarsdottir, K., N. J. Krogan, N. C. Emre, A. Wyce, N. J. Thompson

et al., 2005 H2B ubiquitin protease Ubp8 and Sgf11 constitute adiscrete functional module within the Saccharomyces cerevisiae SAGAcomplex. Mol. Cell. Biol. 25: 1162–1172.

Keith, C. T., and S. L. Schreiber, 1999 PIK-related kinases: DNA re-pair, recombination, andcell cycle checkpoints. Science 270: 50–51.

Keogh, M. C., T. A. Mennella, C. Sawa, S. Berthelet, N. J. Krogan

et al., 2006 The Saccharomyces cerevisiae histone H2A variant Htz1is acetylated by NuA4. Genes Dev. 20: 660–665.

Krogan, N. J., K. Baetz, M. C. Keogh, N. Datta, C. Sawa et al.,2004 Regulation of chromosome stability by the histone H2Avariant Htz1, the Swr1 chromatin remodeling complex, andthe histone acetyltransferase NuA4. Proc. Natl. Acad. Sci. USA101: 13513–13518.

Kulesza, C. A., H. A. van Buskirk, M. D. Cole, J. C. Reese, M. M.Smith et al., 2002 Adenovirus E1A requires the yeast SAGA his-tone acetyltransferase complex and associates with SAGA compo-nents Gcn5 and Tra1. Oncogene 21: 1411–1422.

Kuo, M. H., J. Zhou, P. Jambeck, M. E. Churchill and C. D. Allis,1998 Histone acetyltransferase activity of yeast Gcn5p is re-quired for the activation of target genes in vivo. Genes Dev.12: 627–639.

Kuo, M. H., and C. D. Allis, 1999 In vivo cross-linking and immu-noprecipitation for studying dynamic Protein:DNA associationsin a chromatin environment. Methods 19: 425–433.

Le Masson, I., D. Y. Yu, K. Jensen, A. Chevalier, R. Courbeyrette

et al., 2003 Yaf9, a novel NuA4 histone acetyltransferase sub-unit, is required for the cellular response to spindle stress inyeast. Mol. Cell. Biol. 23: 6086–6102.

Lundblad, V., and J. W. Szostak, 1989 A mutant with a defect intelomere elongation leads to senescence in yeast. Cell 57: 633–643.

McMahon, S. B., H. A. Van Buskirk, K. A. Dugan, T. D. Copeland

and M. D. Cole, 1998 The novel ATM-related protein TRRAP isan essential cofactor for the c-Myc and E2F oncoproteins. Cell 94:363–374.

Mutiu, A. I., and C. J. Brandl, 2005 RNA isolation from yeast silicamatrices. J. Biomol. Tech. 16: 316–317.

Mutiu, A. I., S. M. T. Hoke, J. Genereaux, G. Liang and C. J.Brandl, 2007 The role of histone ubiquitylation and deubiqui-tylation in gene expression as determined by the analysis of anHTB1K123R Saccharomyces cerevisiae strain. Mol. Genet. Gen. 277:491–506.

Nourani, A., W. Doyon, R. T. Utley, S. Allard, W. S. Lane et al.,2001 Role of an ING1 growth regulator in transcriptional acti-vation and targeted histone acetylation by the NuA4 complex.Mol. Cell. Biol. 21: 7629–7640.

Nourani, A., R. T. Utley, S. Allard and J. Cote, 2004 Recruitmentof the NuA4 complex poises the PHO5 promoter for chromatinremodeling and activation. EMBO J. 23: 2597–2607.

Pan, X., D. S. Yuan, X. Wang, J. S. Bader and J. D. Boeke, 2006 ADNA integrity network in the yeast Saccharomyces cerevisiae. Cell10: 1069–1081.

Park, J., S. Kunjibettu, S. B. McMahon and M. D. Cole, 2001 TheATM-related domain of TRRAP is required for histone acetyl-transferase recruitment and Myc-dependent oncogenesis. GenesDev. 15: 1619–1624.

Reeves, W. M., and S. Hahn, 2005 Targets of the Gal4 transcriptionactivator in functional transcription complexes. Mol. Cell. Biol.25: 9092–9102.

Ricci, A. R., J. Genereaux and C. J. Brandl, 2002 Components ofthe SAGA histone acetyltransferase complex are required for re-pressed transcription of ARG1 in rich medium. Mol. Cell. Biol.22: 4033–4042.

Rigaut, G., A. Shevchenko, B. Rutz, M. Wilm, M. Mann et al.,1999 A generic protein purification method for protein complexcharacterization and proteome exploration. Nature Biotech. 17:1030–1032.

Roberts, S. M., and F. Winston, 1997 Essential functional interac-tions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, andGcn5 proteins, with the Snf/Swi and Srb/mediator complexes.Genetics 147: 451–465.

Robinson, M. D., J. Grigull, N. Mohammad and T. R. Hughes,2002 FunSpec: a web-based cluster interpreter for yeast. BMCBioinformatics 3: 35.

Saleh, A., V. Lang, R. Cook and C. J. Brandl, 1997 Identificationof native complexes containing the yeast coactivator/repressorproteins NGG1/ADA3 and ADA2. J. Biol. Chem. 272: 5571–5578.

Saleh, A., D. Schieltz, N. Ting, S. B. McMahon, D. W. Litchfield

et al., 1998 Tra1 is a component of the yeast Ada-Spt transcrip-tional regulatory complexes. J. Biol. Chem. 273: 26559–26565.

Sikorski, R. S., and J. D. Boeke, 1991 In vitro mutagenesis andplasmid shuffling: from cloned gene to mutant yeast. MethodsEnzymol. 194: 302–329.


Smith, E. R., A. Eisen, W. Gu, M. Sattah, A. Pannuti et al.,1998 ESA1 is a histone acetyltransferase that is essential forgrowth in yeast. Proc. Natl. Acad. Sci. USA. 95: 3561–3565.

Stack, J. H., and S. D. Emr, 1994 Vps34p required for yeast vacuolarprotein sorting is a multiple specificity kinase that exhibits bothprotein kinase and phosphatidylinositol-specific PI-3- kinase ac-tivities. J. Biol. Chem. 269: 31552–31562.

Steger, D. J., E. S. Haswell, A. L. Miller, S. R. Wente and E. K.O’Shea, 2003 Regulation of chromatin remodelling by inositolpolyphosphates. Science 299: 114–116.

Sterner, D. E., P. A. Grant, S. M. Roberts, L. J. Duggan, R.Belotesrkovskaya et al., 1999 Functional organization ofthe yeast SAGA complex: distinct components involved in struc-tural integrity, nucleosome acetylation, and TATA-Binding pro-tein interaction. Mol. Cell. Biol. 19: 86–98.

Strahl, B. D., and C. D. Allis, 2000 The language of covalent his-tone modifications. Nature 403: 41–45.

Takahashi, T., H. Shimoi and K. Ito, 2001 Identification of genes re-quiredforgrowthunderethanolstressusingtransposonmutagenesisin Saccharomyces cerevisiae. Mol. Genet. Genomics 2656: 1112–1119.

Tang, F., Y. Peng, J. J. Nau, E. J. Kauffman and L. S. Weisman,2006 Vac8p, an armadillo repeat protein, coordinates vacuole in-heritance with multiple vacuolar processes. Traffic 7: 1368–1377.

Teng, S. C., and V. A. Zakian, 1999 Telomere-telomere recombina-tion is an efficient bypass pathway for telomere maintenance inSaccharomyces cerevisiae. Mol. Cell. Biol. 19: 8083–8093.

Tong, A. H., and C. Boone, 2006 Synthetic genetic array analysis inSaccharomyces cerevisiae. Methods Mol. Biol. 313: 171–192.

Tong, A. H., G. Lesage, G. D. Bader, H. Ding, H. Xu et al.,2004 Global mapping of the yeast genetic interaction network.Science 303: 808–813.

Vignali, M., D. J. Steger, K. E. Neely and J. L. Workman,2000 Distribution of acetylated histones resulting from Gal4–VP16 recruitment of SAGA and NuA4 complexes. EMBO J. 19:2629–2640.

Walker, E. H., O. Perisic, C. Ried, L. Stephens and R. L. Williams,1999 Structural insights into phosphoinositide 3-kinase cataly-sis and signaling. Nature 402: 313–320.

Wang, L., L. Liu and S. L. Berger, 1998 Critical residues for histoneacetylation by Gcn5, functioning in Ada and SAGA complexes,are also required for transcriptional function in vivo. GenesDev. 12: 640–653.

Welihinda, A. A., W. Tirasophon, S. R. Green and R. J. Kaufman,1997 Gene induction in response to unfolded protein in theendoplasmic reticulum is mediated through Ire1p kinase interac-tion with a transcriptional coactivator complex containingAda5p. Proc. Natl. Acad. Sci. USA 94: 4289–4294.

Welihinda, A. A., W. Tirasophon and R. J. Kaufman, 2000 Thetranscriptional co-activator ADA5 is required for HAC1 mRNAprocessing in vivo. J. Biol. Chem. 275: 3377–3381.

Winzeler, E. A., and R. W. Davis, 1997 Functional analysis of theyeast genome. Curr. Opin. Genet. Dev. 7: 771–776.

Wu, P. Y., C. Ruhlmann, F. Winston and P. Schultz, 2004 Molec-ular architecture of the S. cerevisiae SAGA complex. Mol. Cell 15:199–208.

Wymann, M. P., G. Bulgarelli-Leva, M. J. Zvelebil, L. Pirola,B. Vanhaesebroeck et al., 1996 Wortmannin inactivates phos-phoinositide 3-kinase by covalent modification of Lys-802, a res-idue involved in the phosphate transfer reaction. Mol. Cell. Biol.16: 1722–1733.

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