The ADA Complex Is a Distinct Histone Acetyltransferase Complex in Saccharomyces cerevisiae

1999, 19(10):6621. Mol. Cell. Biol.

WorkmanHassan, John R. Yates III, Shelley L. Berger and Jerry L. Anton Eberharter, David E. Sterner, David Schieltz, Ahmed

Saccharomyces cerevisiaeAcetyltransferase Complex in The ADA Complex Is a Distinct Histone

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MOLECULAR AND CELLULAR BIOLOGY,0270-7306/99/$04.0010

Oct. 1999, p. 6621–6631 Vol. 19, No. 10

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

The ADA Complex Is a Distinct Histone AcetyltransferaseComplex in Saccharomyces cerevisiae

ANTON EBERHARTER,1† DAVID E. STERNER,2 DAVID SCHIELTZ,3 AHMED HASSAN,1

JOHN R. YATES III,3 SHELLEY L. BERGER,2 AND JERRY L. WORKMAN1*

Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, Pennsylvania State University,University Park, Pennsylvania 16802-45001; The Wistar Institute, Philadelphia, Pennsylvania 191042; and

Department of Molecular Biotechnology, University of Washington Health Science Center,Seattle, Washington 98195-77303

Received 21 April 1999/Returned for modification 21 May 1999/Accepted 29 June 1999

We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomycescerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltrans-ferase) complex contains several subunits which also function as part of other protein complexes, including asubset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise thequestion of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HATcomplex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADAcomplex contained subunits that are not present in the SAGA complex. In this study, we report the purificationof the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis andimmunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral com-ponents of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit ofthe ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023Chave not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused byparticular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A(J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999–2009, 1995). Deletion of AHC1 disrupted the integrity of theADA complex but did not affect SAGA or give rise to classic Ada2 phenotypes. These results indicate that Gcn5,Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between thiscomplex and SAGA.

Posttranslational modifications of nucleosomal histoneshave been correlated with the modulation of the structure andfunction of chromatin (7). One of the most extensively studiedmodifications is the acetylation of the highly conserved amino-terminal histone tails. The steady-state level of acetylation ofhistone proteins is accomplished by the action of histoneacetyltransferases (HATs) and histone deacetylases (HDACs)(37). Acetylation affects higher-order folding of chromatin fi-bers (16) and the interaction of nonhistone proteins with his-tones (14). It also plays an important role in histone depositionand nucleosome assembly during S phase (48) and can increasethe affinity of transcription factors for nucleosomal DNA (35,61). Correlations between transcription and histone acetyla-tion are strengthened by reports showing that active chromo-somal domains are hyperacetylated (6, 14, 32), while hetero-chromatic domains are hypoacetylated (10, 31).

A large number of recent studies have provided a directmolecular link between histone acetylation and transcriptionalactivation (24, 63). In these reports, it has been shown thatseveral previously identified coactivators-adapters of transcrip-tion possess intrinsic HAT activity. Among these coactivatorsare yeast Gcn5 (11), human Gcn5 (65, 69), p300/Creb-bindingprotein (CBP)-associated factor (P/CAF) (71), TATA box

binding protein (TBP)-associated factor 250 (TAFII250) (41),p300/CBP (2, 43), ACTR (12), and steroid receptor coactivator1 (SRC-1( (55). Conversely, several transcriptional repressorsand/or corepressors have been shown to be associated withHDACs, including Rpd3 (59), Sin3 (27, 33, 34, 73), and N-CoR/SMRT (1, 28). Moreover, human and Xenopus complexescontaining both HDAC activity and ATP-dependent nucleo-some remodeling activity have been isolated (62, 70, 74).

Many of these chromatin-modifying activities have beenfound within large multisubunit protein complexes that alsocontain several components with homology or identity toknown transcriptional regulators (25, 58). Indeed, the coacti-vator-adapter protein Gcn5 is part of large multisubunit com-plexes in Saccharomyces cerevisiae, which enhances its ability toacetylate nucleosomal histones (20, 30, 46, 50, 51). In yeast,Gcn5 is involved in the regulation of a variety of genes (9, 18,39, 51, 66). The largest of the Gcn5-dependent HAT com-plexes is the 1.8-MDa SAGA complex. SAGA comprises atleast four distinct classes of gene products (22, 23). First, thereare the Ada proteins Ada1, Ada2, Ada3, Gcn5 (Ada4), andAda5 (Spt20), which have been isolated as proteins interactingfunctionally with the yeast activator Gcn4 and the herpes sim-plex virus activation domain VP16 (3, 4). The second groupcomprises Spt3, Spt7, Spt8, and Spt 20 (Ada5). These proteinsare all members of the TBP-related set of Spt proteins, initiallyidentified as suppressors of transcription initiation defectscaused by promoter insertions of the transposable element Ty(68). The third group of proteins found to be part of SAGA area subset of TAFIIs, including TAFII20/17, TAFII25/23,TAFII60, TAFII68/61, and TAFII90 (22). Finally, the productof the essential gene TRA1 has been shown to be a component

* Corresponding author. Mailing address: Howard Hughes MedicalInstitute, Department of Biochemistry and Molecular Biology, ThePennsylvania State University, University Park, Pennsylvania 16802-4500. Phone: (814) 863-8256. Fax: (814) 863-0099. E-mail: [email protected].

† Present address: Department of Molecular Biology, Adolf-Bute-nardt-Institute, 80336 Munich, Germany.

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of SAGA (23, 52). Apparent counterparts of the SAGA com-plex have been isolated from mammalian cells (40, 42, 67).

The second Gcn5-dependent HAT complex is the 0.8-MDaADA complex, which differs from SAGA in many aspects.While the ADA complex is also dependent on and cofraction-ates with Ada2, it is not dependent on Ada1, Ada5 (Spt20), orthe other Spt proteins found in SAGA (20, 57). Both the ADAand SAGA complexes can stimulate in vitro transcription fromnucleosome templates in an acetyl coenzyme A-dependent re-action (56). However, the SAGA complex has been shown tophysically interact with the acidic activators Gcn4 and VP16,whereas ADA failed to do so (60). In addition, we recentlydemonstrated that the ADA and SAGA HAT complexes gen-erate overlapping, yet distinct, patterns of lysine acetylation onhistone H3. While ADA can acetylate lysine residues 14 and 18in histone H3, SAGA acetylates to some extent all four lysinesin H3 (21).

Despite these differences between the two Gcn5-dependentHAT complexes, it remained unclear whether the smallerADA is a subcomplex of the larger SAGA or functions as adistinct HAT complex in yeast. Fourteen subunits contained inthe SAGA complex have been identified so far (22, 23). On theother hand, the proteins contained in ADA, other than thethree adapter proteins (i.e., Ada2, Ada3, and Gcn5), wereunknown. The best way to address whether ADA is distinctfrom SAGA is through the identification of ADA complex-

specific components. We therefore purified the native ADAHAT complex from yeast. Mass spectrometry and immuno-blotting analysis of the purified complex demonstrated that theyeast adapter proteins Ada2, Ada3, and Gcn5 are indeed com-ponents of the ADA complex. Importantly, we demonstrate by

FIG. 1. Purification of ADA. (A) Schematic representation of the chromatographic steps applied for purification of the ADA complex. ADA was followed by HATassay and immunoblotting. (B) Silver staining of purified ADA. Aliquots of ADA peak fractions which were eluted from the last three chromatographic steps (Superose6, Mini Q, and Superose 6 PC 3.2/30) were separated by SDS-polyacrylamide gel electrophoresis and stained with silver. For the final Superose 6 column, side fractionsare also shown. Arrows and asterisks indicate proteins which were coeluted with the purified ADA fraction in amounts apparently stoichiometric with one another.

TABLE 1. Purification of the ADA HAT complex

Purificationstepa

Total protein(mg)

Total units(mU)b

Sp Act(mU/mg)

Purification(fold)

NiAg eluate 580,000 3,000 5.2 120 ml Mono Q 19,500 1,620 83 161 ml Mono Q 4,000 4,800 1,200 214Mono 186 4,350 23,387 4,498Superose 6 52.5 4,125 78,572 14,030DNA cellulose 16.8 3,090 184,000 34,783Histone agarose 7.9 1,680 212,658 40,580Superose 6 2.52 1,305 517,875 98,550Mini Q 0.82 620 755,854 147,826Superose 6 PC

3.2/300.34 525 1.54 3 106 298,550

a Purification was done with whole-cell extract prepared from 90 liters of yeastcell culture.

b One microunit is defined as the activity required to transfer 1 fmol of acetylresidues to histones under standard histone acetyltransferase assay conditions(see Materials and Methods).

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several criteria that the gene product of the open readingframe YOR023C is a novel component of ADA and is notpresent in SAGA. YOR023C in high copy numbers suppressesthe cold sensitivity caused by particular mutations in HTA1(45a), which encodes histone H2A (29). We named this proteinAhc1 for ADA HAT complex component 1. The presence ofAhc1 in the ADA complex indicates that it is a unique HAT

complex in yeast that shares a subset of Ada proteins (Ada2,Ada3, and Gcn5) with the SAGA complex.

MATERIALS AND METHODSYeast strains. ADA was purified from yeast strain CY396 (swi2D::HIS3, HO-

lacZ, SWI2-HA-6HIS::URA3) and was described previously (44). Constructionof a complete disruption of the YOR023C gene was carried out applying theone-step gene disruption (49) method with LEU2 as the disrupting marker.

FIG. 2. Mass spectrometry analysis. Mass spectrometry identified the four ADA subunits as Ada2, Ada3, Gcn5, and Ahc1. (A) Peptide sequences obtained by massspectrometry for Ada2, Ada3, Gcn5, and Ahc1. Numbers at the left and the right of each sequence indicate the first and the last amino acids identified, respectively.For all four proteins, numerous hits were obtained. (B) Complete amino acid sequence for Ahc1. The underlined 16 N-terminal amino acid residues were used togenerate the Ahc1 antiserum. Residues shown in boldface represent amino acids identified by mass spectrometry.

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Transformation into yeast strain YJW 100 (MATa ade2-1 his3-11 leu2-3,112trp1-1 ura3-1 can1-100) created YJW 103 (MATa ade2-1 his3-11 ahc1D::LEU2trp1-1 ura3-1 can1-100). We verified the correct integration by PCR with twodifferent primer pairs. For the complementation experiment (see Fig. 6) wetransformed YJW 103 with plasmid pJW 100 (pRS314-AHC1:HA3-TRP1/CEN),thereby creating yeast strain YJW 104 (MATa ade2-1 his3-11 ahc1D::LEU2 trp1-1ura3-1 can1-100; pAHC1:HA3 TRP/CEN). For the Ada2 phenotype and in vivotranscription experiments, the strains used were BY4741 (MATa his3D1 leu2Dmet15D ura3D; wild type) and two mutants thereof, which had the mutationsahc1D and ada2D. The former mutant, with a deletion of open reading frameYOR023C, was purchased from Research Genetics (Huntsville, Ala.). The latterstrain, SB608, was prepared by transforming BY4741 with an ada2D::hisG-URA3fragment from plasmid pyADA2-KO (4); Ura1 transformants were then platedon fluoro-orotic acid media to select for loss of the URA3 marker (5). Yeast cellswere transformed with DNA by the lithium acetate method (19). Yeast strainswere grown at 30°C in YPD broth (1% yeast extract, 2% peptone, 2% glucose)or in minimal medium (0.67% yeast nitrogen base without amino acids, 2%glucose) and supplemented as required.

Cloning and epitope tagging of AHC1. The AHC1 coding sequence, includingan upstream sequence of 1 kb spanning the endogenous promoter, was amplifiedby PCR from yeast genomic DNA. The fragment was subsequently digested withApaI-SalI and cloned with these sites into pRS314 (54) bearing the triple hem-agglutinin (HA) and a CYC1 termination sequence at the C-terminal end.Further details about the cloning process will be provided upon request.

Purification of the ADA complex. For purification of the ADA complex, webegan with 90 liters of the yeast strain CY396 grown to mid-log phase. Elutionof ADA from each column was monitored by a combination of immunoblottingand HAT assays. Whole-cell extract was prepared according to a previouslypublished procedure (13, 20). Extracts were incubated on a rotating wheel with90 ml of Ni21 nitrilotriacetic acid (NTA) agarose (Qiagen) overnight at 4°C. Theresin was then sequentially washed in a column with extraction buffer (40 mMHEPES [pH 7.5], 350 mM NaCl, 10% glycerol, 0.1% Tween 20, 2 mg of leupeptinper ml, 2 mg of pepstatin A per ml, 5 mg of aprotinin per ml, 1 mM phenylmeth-ylsulfonyl fluoride [PMSF]) and 20 mM imidazole buffer (100 mM NaCl, 10%glycerol, 0.1% Tween 20, 2 mg of leupeptin per ml, 2 mg of pepstatin A per ml,5 mg of aprotinin per ml, 1 mM PMSF [pH 7.5]), followed by elution of proteinswith 300 mM imidazole buffer. The eluted material from the Ni21 NTA agarosewas directly loaded onto a 20-ml Mono Q HR16/10 (Pharmacia) column equil-ibrated with 100 mM NaCl in buffer A (50 mM Tris-HCl [pH 8.0], 10% glycerol,0.1% Tween 20, 2 mg of leupeptin per ml, 2 mg of pepstatin A per ml, 5 mg ofaprotinin per ml, 1 mM PMSF). Bound proteins were eluted by applying a 500-mllinear salt gradient from 100 to 500 mM NaCl in buffer A. Peak ADA fractionswere pooled, diluted to 100 mM NaCl, and loaded onto a Mono Q HR5/5column (Pharmacia); the elution from this column was done with a 25-ml lineargradient from 100 to 500 mM NaCl in buffer A. ADA fractions from the 1-mlMono Q column (typically fractions 18 to 20) were brought to 100 mM NaCl inbuffer B (50 mM HEPES [pH 7.8], 10% glycerol, 0.1% Tween 20, 2 mg ofleupeptin per ml, 2 mg of pepstatin A per ml, 5 mg of aprotinin per ml, 1 mMPMSF) and applied to a Mono S HR5/5 column (Pharmacia). Proteins were stepeluted with 200 mM NaCl and 500 mM NaCl in buffer B. ADA, which was elutedin the 500 mM NaCl wash, was then concentrated to 0.5 ml in Biomax-30(Millipore) and subsequently loaded onto a Superose 6 HR10/30 size exclusioncolumn (Pharmacia) equilibrated with 250 mM NaCl in buffer B. Peak fractionsof ADA from the Superose 6 column were pooled, diluted to 100 mM NaCl, andloaded onto a 1-ml native DNA cellulose column (Pharmacia). Bound proteinswere eluted with a 12-ml linear gradient from 100 mM to 1 M NaCl. ADA peakfractions were immediately dialyzed against 100 mM NaCl in buffer B andapplied onto a 1-ml histone agarose column (Sigma). Elution from histoneagarose was accomplished with a 12-ml linear salt gradient from 100 mM to 1 M

NaCl. Fractions containing ADA were pooled, concentrated to 0.5 ml withBiomax-30, and loaded onto a Superose 6 HR10/30 column. Peak Superose 6fractions were diluted to 100 mM NaCl, immediately concentrated to 0.5 ml, anddirectly loaded onto a Mini Q PC 3.2/3 column (Pharmacia). Bound proteinswere eluted with a 2-ml linear salt gradient from 100 to 500 mM NaCl. For theisolation of ADA from yeast YJW 100, YJW 103, and YJW 104, we used 6 litersof cells. The strains were grown to an absorbance at 600 nm of 1.0 at 30°C.Whole-cell extracts were prepared as described and subsequently purified with 5ml of Ni21 NTA agarose, Mono Q HR5/5 fractionation, and Superose 6 chro-matography as described above.

HAT assays, Western blotting, antibodies, and immunoprecipitation. HATassays were performed as previously described (13). After each chromatography,equivalent amounts of fractionated samples were subjected to electrophoresiswith sodium dodecyl sulfate (SDS)–10% polyacrylamide gels, transferred tonitrocellulose membranes, and processed for immunoblotting. Anti-Ahc1 anti-bodies were raised in rabbits against a synthetic peptide spanning the amino-terminal 16 amino acids of Yor023C (MMSPAQDKLQHQHHNP) by ResearchGenetics. A monoclonal anti-HA antibody was purchased from BoehringerMannheim (Indianapolis, Ind.). Immunodetection was performed with an en-hanced chemiluminescence kit from Amersham according to the manufacturer’sprotocol. For immunoprecipitation experiments, equivalent titers of anti-Ada2or anti-Ahc1 antibodies were incubated with 20 ml of preequilibrated proteinA-Sepharose resin (Pharmacia) for 1 h at room temperature. Beads were thenwashed with immunoprecipitation (IP) buffer (50 mM HEPES [pH 7.8], 150 mMNaCl, 10% glycerol, 0.1% Tween 20, 2 mg of leupeptin per ml, 2 mg of pepstatinA per ml, 1 mM PMSF), and purified ADA and SAGA fractions were added andincubated in IP buffer for 4 to 16 h at 4°C on a rotating wheel. After incubationsupernatants were collected, the beads were washed five times with IP buffer.Input material, supernatants, and beads were directly assayed for HAT activitywith free-core histones as a substrate. Protein concentrations were determinedaccording to the method described by Bradford (8).

Mass spectrometry analysis. ADA peak fractions after the final Mini Q col-umn were concentrated in Microconcentrator-30 apparatus (Amicon), loadedonto a SDS–10% polyacrylamide gel, and stained with Coomassie blue. Afterdestaining, the bands were excised and digested in gel with trypsin, according tothe method of Shevchenko et al. (53). Identification of proteins was accom-plished by microcolumn high-performance liquid chromatography coupled toelectrospray ionization tandem mass spectrometry and database searching. A100- by 365-mm fused silica capillary (Polymetrics Inc., Phoenix, Ariz.) (17) waspacked to a length of ;10 cm with 10-mm POROS 10 R2 reverse-phase material(Perseptives Biosystems, Framingham, Mass.). The sample was directly loadedonto the microcolumn by helium pressurization of the sample in a stainless-steelbomb (72). Liquid chromatography was performed with a dual-syringe pump(Applied Biosystems, Foster City, Calif.). The mobile phase consisted of 0.5%acetic acid (solvent A) and 80:20 acetonitrile-water containing 5% acetic acid(solvent B). A precolumn split was used to deliver a flow rate of 250 to 400 nl/minthrough the column. The high-performance liquid chromatography pump wasprogrammed to ramp solvent B from 2 to 60% in 30 min. Electrospray ionizationwas done at a voltage of 1.8 kV. Tandem mass spectra were automaticallyacquired during the entire gradient run (36). Tandem mass spectra weresearched against the Saccharomyces genome database obtained from StanfordUniversity with the SEQUEST program (15). Every sequence with high scoresthat matched a tandem mass spectrum was manually verified. To facilitate theidentification of potential contaminants, sequences for human keratin and bo-vine trypsin were included.

b-Galactosidase assays and overexpression of Gal4-VP16. For plate growthexperiments, wild-type, ahc1D, and ada2D yeast strains were transformed withhigh-copy-number Gal4-VP16 plasmid (4) or empty vector (pDB20L-BglII) bear-ing the LEU2 selective marker and plated directly onto synthetic dextrose min-imal medium. Plates were grown for 3 days at 30°C; Gal4-VP16 plates wereincubated for an additional day at room temperature. For in vivo transcriptionassays, wild-type, ahc1D, and ada2D double transformants, containing pLGSD5reporter plasmid (26) and low-copy-number Gal4-VP16 (4) or Gal4-VP16FAplasmid or empty vector (pRS315) (54), were grown to an optical density of 0.8at 600 nm in selective synthetic complete medium. Extracts, prepared by break-ing cells with glass beads, were tested for b-galactosidase activity and proteinconcentration as described previously (47). Reported values are the averages ofthe results from two to four independent transformants for each strain-plasmidcombination.

RESULTS

Purification of the ADA HAT complex. To gain a betterinsight into the protein composition of the ADA complex andto investigate its relationship to SAGA, we purified the ADAcomplex from S. cerevisiae whole-cell extract prepared from 90liters of yeast cell culture. The purification strategy is outlinedin Fig. 1A. After each chromatographic separation, we moni-tored for the ADA complex by two criteria. Column fractionswere tested for their HAT activity, including monitoring the

FIG. 3. Western blot analysis of Mono Q chromatography. Ten microliters ofindicated fractions was separated after Mono Q chromatography by SDS–10%polyacrylamide gel electrophoresis and transferred onto nitrocellulose mem-branes. Membranes were incubated with antibodies (a) against Ahc1, Ada2,Ada3, and Spt8.



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substrate specificity, since the ADA complex preferentiallyacetylates nucleosomal histones H3 and H2B (20). We alsoexamined column fractions by Western blot analysis with an-tibodies to Ada2 and Gcn5. Only the peak fractions containingADA complex activity from each column were used for subse-quent chromatographic steps. The results of the purificationare presented in Table 1. Aliquots of fractions from the 10thcolumn, a Superose 6 PC 3.2/30 size exclusion column, wererun on an SDS–7.5% polyacrylamide gel and analyzed by silverstaining (Fig. 1B). Eight proteins with approximate molecularmasses of 50, 55, 65, 90, 97, 110, 180, and 250 kDa coeluted

with the ADA HAT activity (Fig. 1B, lanes 17 and 18). Theseprotein bands were excised and analyzed by mass spectrome-try.

The proteins migrating with molecular masses of 50, 55, and97 kDa were identified as the adapter proteins Ada2, Gcn5,and Ada3, respectively (Fig. 2). Sixteen peptides from p97identified this protein as Ada3. Ada2 and Gcn5 were identifiedby nine and five peptides, respectively. These findings con-firmed previous genetic and biochemical studies with less-pu-rified material (20). Mass spectrometry also tentatively identi-fied the remaining subunits of the ADA complex. However, to

FIG. 4. Ahc1 is coeluted with purified ADA. (A) Fractions from the seventh column, Superose 6 size exclusion chromatography, were tested in a nucleosomal HATassay and Western blotting with the indicated antibodies. The upper panel shows the fluorogram from the HAT assay depicting the specificity of ADA. Histones H3and H2B were acetylated by ADA. (B) Fluorogram after nucleosomal HAT assay and Western blotting with the indicated antibodies (a) of purified fractions from theMini Q column. (C) Immunoprecipitation with purified ADA and SAGA complex. ADA and SAGA were incubated with preimmune serum, anti-Ada2 antiserum, oranti-Ahc1 antiserum immobilized on protein A-Sepharose beads. The fluorogram of HAT reaction products obtained with free histones is shown.



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confirm that each putative subunit is not a contaminant in thefinal fraction, it is necessary to generate antibodies againstpeptides from the protein. This allows an examination of thecopurification and immunoprecipitation of the putative sub-unit with the ADA complex. Moreover, by generating a yeaststrain where the corresponding open reading frame is deleted,its importance for the activity and integrity of the ADA com-plex can be examined. Using these criteria, we have thus farconfirmed the presence of one novel subunit of the ADAcomplex (see below).

Identification of a novel component of the ADA complex.Mass spectrometry analysis of the p65 band revealed the pres-ence of three peptides from the same open reading frame,YOR023C (Fig. 2). This previously uncharacterized open read-

ing frame has been renamed AHC1 for ADA HAT complexcomponent 1. The amino acid sequence of the entire protein isshown in Fig. 2B. We generated antibodies against the peptidespanning the first 16 N-terminal amino acid residues (Fig. 2B)and used this antiserum to follow this protein during the courseof purification. Fractions from the initial Mono Q column weretested with the Ahc1 antiserum, since this column separatesthe ADA, NuA4, NuA3, and SAGA complexes (13). TheWestern blot results in Fig. 3 demonstrate that Ada2 and Ada3cofractionated, as expected, with the ADA and SAGA com-plexes. Spt8 cofractionated only with SAGA and was not de-tected in fractions containing ADA. By contrast, Ahc1 cofrac-tionated only with the ADA complex and was not found in thefractions containing the SAGA complex. Thus, while Ada2,

FIG. 5. An AHC1 mutant specifically affects the ADA HAT complex. Whole-cell extracts from a wild-type strain and a yeast strain bearing a mutation in AHC1were partially purified with Ni21 NTA agarose and Mono Q chromatography. (A) In the top panel, a typical fluorogram from a nucleosomal HAT assay with fractionsfrom a Mono Q column prepared from a wild-type strain is presented. The four HAT complexes are indicated at the top. The lower panels show results from Westernblotting with antibodies (a) raised against Ada2 and Gcn5. (B) Fluorogram from a nucleosomal HAT assay of Mono Q fractions prepared from the ahc1D yeast strain.NuA4, NuA3, and SAGA were eluted in the same fractions as in the wild type. ADA was absent in the AHC1 mutant. Western blots (lower panels) demonstrate thatanti-Ada2 and anti-Gcn5 antibodies showed immunoreactivity only for SAGA (fractions 38 to 40).



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Ada3, and Gcn5 are contained in both complexes, each alsoappears to have unique subunits not found in the other, i.e.,Spt8 in SAGA and Ahc1 in ADA.

To confirm the copurification of Ahc1 with the ADA com-plex, we performed Western blotting on highly purified ADAfractions from further chromatographic steps. Figure 4 showsWestern blots with antibodies against ADA subunits and Ahc1on fractions from columns, that were used very late in thepurification process, Superose 6 and Mini Q. As shown in Fig.4A, Gcn5, Ada2, and Ahc1 coeluted with ADA HAT activityon the Superose 6 column that represented the eighth chro-matographic step. Similarly, Ada2, Ada3, Gcn5, and Ahc1 coe-luted with ADA complex HAT activity on the Mini Q column,the ninth column. Thus, Ahc1 copurifies with the other sub-units and the HAT activity of the ADA complex through mul-tiple chromatographic steps, suggesting that it is a bona fidesubunit of the complex.

To confirm the physical association of Ahc1 with the ADAcomplex, we tested whether the anti-Ahc1 antisera were ableto immunoprecipitate the HAT activity of the complex. Asshown in Fig. 4C, anti-Ahc1 antibodies immunoprecipitatedthe ADA complex as efficiently as antibodies against Ada2.Both antibodies were able to deplete ADA HAT activity fromthe supernatant, in contrast to the preimmune serum. In ad-dition, the ADA HAT activity in each case was detected on the

beads (Fig. 4C). By contrast, anti-Ahc1 antisera failed to im-munoprecipitate the SAGA complex, confirming that Ahc1 isa component of ADA but not SAGA.

The ADA complex is dependent on the AHC1 gene. From thebiochemical data presented above, we concluded that the pro-tein Ahc1 is a distinct component of the ADA HAT complex.To examine the importance of Ahc1, we generated an AHC1deletion strain and investigated the effects of this deletion onthe ADA complex. To do this, we prepared whole-cell extractsfrom a wild-type strain and the mutant strain bearing thecomplete deletion of AHC1. Extracts from both strains werepartially purified with Ni21 NTA agarose and Mono Q anionexchange chromatography. Fractions eluting from the Mono Qcolumn were then subjected to HAT assays with both nucleo-somes and core histones as substrates. As demonstrated in Fig.5, partially purified ADA prepared from the wild-type strainwas eluted in fractions 18 to 22 (identified by H3-H2B HATactivity and Ada2 and Gcn5 Western blotting). In addition, theother previously identified HATs, NuA4, NuA3, and SAGA,were eluted as predicted (13). Figure 5B shows the fraction-ation of complexes from extract prepared from the strain bear-ing a disruption in AHC1. In this instance, we found thatextracts from ahc1D cells specifically lacked the ADA complex(fractions 18 to 22), while the SAGA, NuA4, and NuA3 com-plexes were unaffected. There was neither detectable nucleo-

FIG. 6. The ADA HAT complex can be rescued by plasmid expression of AHC1. (A and B) Mono Q fractionation of partially purified whole-cell extracts preparedfrom YJW 103 (ahc1D) and YJW 104 (ahc1D-pAHC1:HA3). HAT assay fluorograms and Western blots of fractions containing ADA and NuA4 (fractions 14 to 24)are shown. Immunodetection of Ahc1 was accomplished with an anti-HA antibody. The ADA HAT complex was specifically restored in YJW 104 (B) and was absentin the AHC1 deletion (A). HAT assays (C) and Western blots (D) from Superose 6 size exclusion chromatography of Mono Q fractions (A) are shown. Fractions 14to 20 from a Mono Q column were pooled, concentrated, and fractionated on a Superose 6 column. Ahc1 was immunodetected by monoclonal anti-HA antibody. ADAis present only in YJW 104 bearing pAHC1-HA3 and was eluted at a molecular mass of ;800 kDa.



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somal ADA HAT activity (Fig. 5) nor free-core histone HATactivity for ADA (results not shown). Furthermore, immuno-blotting analysis with antibodies against Ada2 and Gcn5 indi-cated that, in addition to the loss of activity, the ADA complexitself was lost in the ahc1D preparation. Importantly, SAGAwas unaffected (fractions 36 to 38). Therefore, Ahc1 behavedlike Ada2 and Ada3 (20) in that it was required for the overallstructural integrity of the ADA complex (19a).

To further substantiate the importance of Ahc1 for ADAintegrity, we wished to address the possibility that the complexwould be restored when AHC1 is expressed on a low-copy-number plasmid in ahc1D cells. To this end, we cloned AHC1bearing a triple HA epitope tag at the C terminus into theARS-CEN vector pRS314 (54) and expressed it under its en-dogenous promoter in the AHC1 deletion strain. Whole-cellextracts were prepared from yeast strains YJW 103 (ahc1D)and YJW 104 (ahc1D pAHC1:HA3), and ADA was fraction-ated as described (see Materials and Methods). While ADAwas missing in YJW 103 (Fig. 6A), we found that ADA HATactivity was present in Mono Q fractions 16 to 20 in YJW 104(Fig. 6B). Immunodetection with antibodies against Ada2 anda monoclonal anti-HA antibody to detect epitope-tagged Ahc1confirmed the presence of ADA in this preparation. The ADAcomplex was apparently fully restored in YJW 104 cells asdemonstrated by size exclusion chromatography (Fig. 6C andD). The complex eluted in fractions 22 to 24 from a Superose6 column, giving it a size of about 800 kDa. Again, no ADA

complex was detectable in ahc1D cells; however, the ADAcomplex was present in ahc1-pAHC1-HA as detected by its H3HAT activity (Fig. 6C) and by Western blotting (Fig. 6D). Notethat the slight histone H4 activity in fraction 24 on Superose 6is from a slightly smaller contaminating HAT complex whichpeaks in Mono Q fractions 14 and 15 (Fig. 5) and is unrelatedto ADA (12a).

An AHC1 deletion does not display an Ada phenotype. Mu-tations in ADA2, ADA3, and GCN5 (ADA4) were isolated in aselection for mutants that confer resistance to toxicity fromoverexpressed Gal4-VP16. In addition, mutations in any ofthese genes reduced transcriptional activation by the acidicactivators VP16 and GCN4 (4, 38, 45). Therefore, we wished toask whether a mutation in AHC1 is resistant to overexpressionof Gal4-VP16 and shows reduced Gal-VP16-mediated tran-scription levels. Overexpression of Gal4-VP16 and in vivo tran-scription assays were performed as described in Materials andMethods. As demonstrated in Fig. 7, AHC1 deletion did notexert the typical adapter (Ada2) phenotypes as described fordeletions of ADA2, ADA3, or GCN5 (4, 38, 45). First, we foundthat the ahc1D strain did not have a growth defect on minimalmedium (Fig. 7A, upper panels). The wild type and the AHC1deletion strain showed similar growth on minimal media whilethe adapter ada2D mutant grew more poorly and resulted insmaller colonies. Second, the AHC1 deletion was unable torelieve the toxicity of overexpressed Gal4-VP16 (Fig. 7A, lowerpanels). No significant growth was seen for either the wild type

FIG. 7. An AHC1 deletion does not display a classic Ada phenotype. (A) Transformants of wild-type, ahc1D, and ada2D (adapter control) cells containinghigh-copy-number empty vector were plated on minimal medium to assess overall growth phenotype (upper panels). To test for adapter phenotype (Ada2; relief oftoxicity of overexpressed chimeric activator Gal4-VP16), cells were transformed with high-copy-number activator plasmid and plated on minimal medium (lowerpanels). (B) Quantitation of acidic-activator-mediated in vivo transcription is presented. Wild-type, ahc1D, and ada2D cells were transformed with pLGSD5 reporterplasmid and low-copy-number empty vector, Gal4-VP16, or Gal4-VP16FA plasmids, and extracts were assayed for b-galactosidase activity.



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or the ahc1D strain, whereas ada2D cells were able to grow.Third, Gal4-VP16-mediated transcription levels were similarin the wild type and the mutant (Fig. 7B). Thus, results fromthese three assays measuring Ada2 phenotypes indicate that astrain lacking Ahc1 does not exhibit these defects.

DISCUSSION

The discovery that several transcriptional coactivator pro-teins are HATs provided a direct molecular link between his-tone acetylation and activation of transcription. One of thesecoactivators possessing HAT activity, the yeast protein Gcn5,functions as the catalytic subunit in several native high-molec-ular-weight complexes (20, 46, 50, 51). The largest of theseGcn5-dependent native HAT complexes, the 1.8-MDa SAGAcomplex, has recently been purified and characterized (22, 23).SAGA contains Tra1, several Ada proteins, the TBP class ofSpt proteins, and a subset of TAFII proteins. Members fromeach of these classes of proteins have been demonstrated to beessential for structural integrity (20, 57), transcriptional stim-ulation (64), or nucleosomal histone acetylation (22) bySAGA.

While many subunits of the SAGA complex have been iden-tified, proteins other than Ada2, Ada3, and Gcn5 comprisingthe 0.8-MDa ADA complex were not known. A long-standingquestion to address was whether ADA functions as a distinctcomplex in S. cerevisiae or is a subcomplex of the larger SAGAcomplex. There are several lines of evidence which suggestedthat the ADA complex may be distinct from SAGA. First,ADA is capable of acetylating nucleosomal histones althoughlacking TAFII68. A depletion of TAFII68 from SAGA re-sulted in the loss of both nucleosomal acetylation and tran-scriptional stimulation for SAGA (22). Second, ADA andSAGA show different lysine specificities within histone H3(21). One can speculate that there are distinct proteins withineither complex that are required for this specificity. Third, incontrast to SAGA, ADA fails to interact with the acidic acti-vators Gcn4 and VP16 in vitro (60), even though it containsAda2, which has been shown to physically interact with theseactivation domains (3). Fourth, a recent study has demon-strated that a deletion of the bromodomain within Gcn5 sig-nificantly reduced nucleosomal HAT activity of SAGA, whilethe ability of ADA to acetylate nucleosomes was unchanged(57). All of these observations suggest distinct activities of theADA complex relative to the SAGA complex. However, thesedifferences could arise from the fact that ADA lacks manysubunits found in SAGA and/or that the ADA complex maycontain distinct subunits not found in the SAGA complex.

In this study, we have purified the ADA HAT complex andhave identified a novel subunit of this complex. Indeed, we findthat a protein of previously unknown function, Yor023C,herein named Ahc1, is a unique subunit of the ADA complex.Several lines of evidence demonstrate that Ahc1 is an integralcomponent of the ADA complex. First, Ahc1 was identified bymass spectrometry analysis as a protein in the highly purifiedADA complex (Fig. 2). Second, Western blot experiments us-ing an anti-Ahc1 antiserum confirmed the copurification ofAhc1 with the HAT activity of ADA. Third, anti-Ahc1 anti-bodies immunoprecipitated the ADA complex, demonstratingthat Ahc1 is a stably interacting component of the purifiedcomplex (Fig. 4A and B). Fourth, an AHC1 deletion strainspecifically lacked the ADA complex, while the larger SAGAcomplex was unaffected by this deletion. Thus, the structuralintegrity of the ADA complex was dependent on the presenceof the AHC1 gene product. Fifth, reintroducing Ahc1 on aplasmid restored the ADA complex, as shown in Fig. 6.

The finding that the ADA complex contains unique subunitsnot found in the SAGA complex illustrates that it is a distinctcomplex and not merely a subcomplex of SAGA. While thefunctions of the ADA complex remain under investigation, it isclear that it is not responsible for the classic Ada2 phenotypes(e.g., the lower Gal4-VP16 function in vivo) (4). While thedeletion of AHC1 disrupted the ADA complex, it did not resultin an Ada2 phenotype (Fig. 7). It is therefore likely that ADAis not involved in transcriptional activation mediated by acidicactivators in the same manner as SAGA is. Consistent with thisis the observation that SAGA, but not ADA, interacts with theacidic activators VP16 and Gcn4 (60). Moreover, deletion ofthe SAGA components Ada1, Spt20, and Spt7, which are notin the ADA complex, disrupts the SAGA complex and alsoresults in Ada2 phenotypes (57). Thus, the functions of Ada2,Ada3, and Gcn5 which give rise to the Ada2 phenotypes aremost likely attributable to the functions of the SAGA complex.However, a genetic link between the ADA complex and his-tone function is suggested by the fact that AHC1 in high copynumbers suppresses the cold sensitivity mediated by particularmutations in histone H2A (29, 45a). The presence of theadapter proteins Ada2, Ada3, and Gcn5 in two unique com-plexes indicates that these proteins may perform importantroles in complexes with distinct functions. In this regard, theAda proteins are similar to other proteins that are involved intranscriptional regulation (e.g., several TAFIIs and Tra1 [22,23]).

ACKNOWLEDGMENTS

We thank members of the Workman lab for many helpful discus-sions. A.E. also thanks David Steger, Sam John, and Patrick Grant fortheir continuous encouragement during this work. We also thankLeAnn Howe for her help during the initial cloning steps.

This work was supported by the National Center for Research Re-sources, National Institutes of Health, and by grants from the NationalInstitute of General Medical Sciences awarded to J.L.W., NationalInstitutes of Health grant 11823-02 and NSF Science and TechnologyCenter grant BIR9214821AM awarded to J.R.Y., and National Insti-tutes of Health and NSF grants to S.L.B. A.E. was a recipient of apostdoctoral fellowship from the Austrian Science Foundation (Fondszur Forderung der wissenschaftlichen Forschung, J1571-GEN) andwas a postdoctoral associate of the Howard Hughes Medical Institute.D.E.S. was supported by an NIH postdoctoral fellowship. J.L.W. is anassociate investigator of the Howard Hughes Medical Institute.

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The ADA Complex Is a Distinct Histone Acetyltransferase Complex in Saccharomyces cerevisiae

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