Functional Organization of the Yeast SAGA Complex: Distinct …-sterner... · 2020-02-14 · structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1,

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

Jan. 1999, p. 86–98 Vol. 19, No. 1

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

Functional Organization of the Yeast SAGA Complex: DistinctComponents Involved in Structural Integrity, Nucleosome

Acetylation, and TATA-Binding Protein InteractionDAVID E. STERNER,1 PATRICK A. GRANT,2 SHANNON M. ROBERTS,3 LAURA J. DUGGAN,1

RIMMA BELOTSERKOVSKAYA,1 LISA A. PACELLA,3 FRED WINSTON,3

JERRY L. WORKMAN,2 AND SHELLEY L. BERGER1*

The Wistar Institute, Philadelphia, Pennsylvania 191041; Department of Biochemistry and Molecular Biology andCenter for Gene Regulation, The Pennsylvania State University, University Park, Pennsylvania 168022;

and Department of Genetics, Harvard Medical School, Boston, Massachusetts 021153

Received 22 June 1998/Returned for modification 5 August 1998/Accepted 18 September 1998

SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription invivo and possesses histone acetylation function. Here we report both biochemical and genetic analyses ofmembers of three classes of transcription regulatory factors contained within the SAGA complex. We demon-strate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity. Specif-ically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtlestructural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complexand cause severe phenotypes. Interestingly, double mutants (gcn5D spt3D and gcn5D spt8D) causing loss of amember of each of the moderate classes have severe phenotypes, similar to spt7D, spt20D, or ada1D mutants.In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and findthat first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromo-domain. Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo.Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro.Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, includingdirect TBP interaction and nucleosomal histone acetylation. Loss of either of these causes slight impairmentin vivo, but loss of both is highly detrimental to growth and transcription.

The process of transcriptional activation hinges on the abil-ity of various factors to facilitate the function of the transcrip-tion complex. In eukaryotes, chromatin structure is a majorobstacle to transcription by RNA polymerase II: DNA is woundaround histone proteins to form a repeating array of nucleo-somes (81), making it inaccessible to the transcriptional ma-chinery (53, 55). Nucleosomes must therefore be perturbed atpromoter regions prior to or during activation, and such re-modeling has been observed in a number of studies (29, 69,71).

In recent years, a variety of factors relevant to transcriptionhave been identified as involved in nucleosome remodeling ormodification. ATP-dependent remodeling of nucleosomes hasbeen demonstrated by a number of large protein complexesisolated from several eukaryotes (43). The Swi-Snf complex,identified in yeast and human cells, was shown to remodelchromatin in vivo and in vitro and stimulate activator and basalfactor binding to nucleosomal DNA (15, 35, 38, 45, 52). TheDrosophila Nurf (73), CHRAC (76), and ACF (39) complexesand the yeast RSC complex (10) are believed to carry outsimilar functions, also through the hydrolysis of ATP.

Histone acetylation is another mechanism by which nucleo-somes are modified. The core histones (H2A, H2B, H3, andH4) can be acetylated on the lysine side chains of their amino-terminal tail regions (7), reducing their positive charge andpresumably reducing their affinity for negatively charged DNAor other chromatin proteins. Substantial evidence suggests that

acetylated nucleosomes are more permissive for transcription.For example, in vivo, histones associated with active chromo-somal loci were shown to be hyperacetylated (34), while thoseat inactive or heterochromatin regions were shown to be hy-poacetylated (8, 42, 50, 74). In vitro, histone acetylation resultsin increased binding of transcriptional activators to their sitesin nucleosomal DNA (77).

A more definitive link between histone acetylation and tran-scriptional activation was realized with the discovery that theyeast Saccharomyces cerevisiae transcriptional adaptor Gcn5 isa histone acetyltransferase (HAT) (9). Gcn5 is one of a groupof adaptors (also known as mediators or coactivators) thatwere hypothesized to provide a physical bridge between up-stream DNA-bound activators and the transcriptional machin-ery at a promoter (28). Since the discovery of the HAT activityof Gcn5, additional transcriptional cofactors in yeast and high-er eukaryotes, including the TATA-binding protein (TBP)-associated factor TAFII250 (the human homologue of yeastTafII145/130 [49]), p300/CBP (2, 51), and P/CAF (p300/CBP-associated factor [82]), have been identified as HATs, suggest-ing that acetylation may be important in activation. In yeast,genes encoding adaptor proteins were originally identified bymutations that suppress the toxicity caused by a high level ofthe acidic activator, Gal4-VP16 (5). Besides Gcn5 (48), theseproteins include Ada1 (37), Ada2 (5), Ada3 (57), and Ada5(47), and they were subsequently demonstrated to interactphysically and functionally in vivo and in vitro (11, 36, 48). Theability of the adaptors to associate with activation domains (3,14, 68, 75) and TBP (3), a component of TFIID, further indi-cated their function as part of a complex involved in activatedtranscription.

* Corresponding author. Mailing address: The Wistar Institute, 3601Spruce St., Room 358, Philadelphia, PA 19104. Phone: (215) 898-3922.Fax: (215) 898-0663. E-mail: [email protected].

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The crucial role of the HAT activity for Gcn5 function wasrecently demonstrated through the creation and analysis ofHAT substitution mutants. Specific alanine substitutions (44,78) in the previously identified HAT domain of Gcn5 (13)lower HAT activity, and loss of activity strongly correlates withdefects in growth and transcription in vivo. Moreover, acety-lation of histones at the promoter of the HIS3 gene correlateswith gene activity, and the acetylation at this promoter is re-duced in the presence of substitution mutations in Gcn5 thatimpair its HAT activity (44). In addition, mutations in theHAT domain of Gcn5 correlate with perturbation of the nor-mal chromatin structure at the PHO5 promoter (27).

Gcn5 alone acetylates only free histones; however, as a com-ponent of native yeast complexes, Gcn5 acetylates histones innucleosomes (24, 63). In one study, Gcn5 was shown to be acomponent of two of four distinct nucleosome-acetylating ac-tivities (24). These two Gcn5-containing complexes, whichacetylate histones H3 and H2B in nucleosomes, also containedthe adaptors Ada2 and Ada3. One of these Gcn5-dependentHAT complexes had a molecular size of 0.8 MDa and wastermed the Ada complex.

The other Gcn5-dependent HAT complex, with a molecularsize of about 1.8 MDa, possessed the adaptor components aswell as four Spt proteins. This complex was therefore namedSAGA (Spt-Ada-Gcn5-acetyltransferase). The Spt proteinsknown to be present in the SAGA complex include Spt3, Spt7,Spt8, and Spt20. Spt7 (20) and Spt20 (60) are apparently vitalto the structure of SAGA, since null mutations in either genecauses complete disruption of the SAGA complex and severegrowth defects (24). Interestingly, spt20 null mutants also havean Ada2 phenotype: the gene was independently identified asADA5 in a genetic screen for ada mutants (47).

The SPT genes were originally identified as suppressors ofTy or d insertions in the promoter regions of the yeast HIS4and LYS2 genes. The insertion mutations abolish or otherwisealter transcription of the adjacent gene, resulting in a His2 orLys2 phenotype, and strains with spt mutations restore func-tional transcription of the adjacent gene (reviewed in reference79). A subset of five SPT genes have been grouped togetherbased on common mutant phenotypes. This group includesSPT15, which encodes TBP (19, 30). The other four membersof this group encode the Spt proteins contained in SAGA. Spt3and Spt8 in particular seem to have the most direct relation-ship to TBP, since specific spt3 and spt15 mutations suppresseach other in an allele-specific fashion (17), implying physicalinteraction. Genetic evidence also has suggested that Spt8 isrequired for the functional interaction between Spt3 and TBP(18). Furthermore, TBP, as well as other SAGA components,were pulled down via expressed glutathione S-transferase(GST)–Spt20 from yeast extract (59). Interestingly, TBP alsocoimmunoprecipitated from yeast extract with Ada3, anotherSAGA subunit (64). A further potential relationship betweenSAGA and TBP was identified most recently with the discoverythat a subset of five TafIIs are also present within SAGA (25).Interestingly, the TafII possessing HAT activity, TafII145, is notassociated with SAGA. The precise mechanism of SAGA in-teraction with TBP and its role in vivo remain to be deter-mined.

Mutations in different ADA and SPT SAGA genes causedistinct classes of genetic and biochemical phenotypes (26).Two classes of SAGA mutants have moderate sets of pheno-types. Gcn5, Ada2, and Ada3 form one distinct class withinSAGA because (i) they are also present in the Ada complex,(ii) they are required for HAT activity within both complexes,and (iii) genetic disruptions of them exhibit Ada2 but not Spt2

phenotypes. A second potential subgroup within SAGA is

Spt3/Spt8, mutations of which exhibit a significantly less severerange of phenotypes than disruptions of either Spt20 or Spt7,and in particular exhibit an Spt2, but not Ada2, phenotype.The third subgroup is Spt20/Spt7 mutations, which have severephenotypes and exhibit both Spt2 and Ada2 phenotypes (47,60). Loss of Spt20 or Spt7 completely disrupts the SAGAcomplex.

Further genetic support for multiple functions within SAGAcomes from an analysis of double-mutant phenotypes betweenmutations in SAGA genes and mutations in genes that encodeother transcription regulatory factors (59). Specifically, spt20Dand spt7D are synthetically lethal in combination with muta-tions in genes that encode members of the Swi-Snf or Srb-mediator complex. In contrast, null mutations in the SAGAgenes GCN5, SPT3, and SPT8 do not cause inviability in com-bination with mutations in these other transcription factorgenes.

In this study, we sought to establish the basic functionalorganization of SAGA, using genetic and biochemical analysesof certain key components. Our results suggest that the mul-tiple discrete biochemical functions relate in a direct way to thedistinct genetic phenotypes and that mutant SAGA complexes,lacking certain components, contain subfunctional activity.These data suggest a model for overall SAGA function in theprocess of transcriptional activation.

MATERIALS AND METHODS

Yeast strains and media. The yeast strains used in this study are listed in Table1. All FY strains are congenic and were originally derived from the S288Cderivative FY2 (80). To introduce the snf5D2 null mutant allele (46) into S288Cbackground, integrating plasmid pLY17 was used for transplacement (1, 66) inhaploid strain FY37. All other deletion derivatives except ada mutations havebeen described previously (reference 59 and references therein). Null mutantswere constructed by transforming diploids with a restriction fragment designedto delete the gene being studied (62). In each case, the null mutation containeda selectable nutritional marker. Transformants were then sporulated, and tetradswere dissected to generate haploid mutant progeny (1). In the ada1D::HIS3,ada2D::HIS3, and ada3D::HIS3 alleles, the entire open reading frame of eachcorresponding gene was replaced with the HIS3 gene, using a fragment gener-ated by PCR (1). The following PCR primer pairs were used to create and verifycorrect integration of the corresponding ada knockout mutations: ADA1KOLEFT (59-ATAGGGAAAACAAGCCCAGTAGTTTTGATTTCTTCTATCCTGTGCGGTATTTCACACCG-39), ADA1KORITE (59-TATAATTACAACATACCGCATACACACACTTTTTATACAAGATTGTACTGAGAGTGCAC-39), ADA1LCHECK (59-TGTGCCTTGAACATCGAACTTAC-39), ADA1RCHECK (59-GCTCACTTAAGCTCGGTCACA-39), ADA2KOLEFT (59-ATCAGCGTAGTCTGAAAATATATACATTAAGCAAAAAGATGCGGTATTTCACACCG-39), ADA2KORITE (59-ACTAGTGACAATTGTAGTTACTTTTCAATTTTTTTTTTGAGATTGTACTGAGAGTGCAC-39), ADA2LCHECK(59-CCAGACGTTCCAAACAAATAAGTG-39), ADA2RCHECK (59-CAAGGTCCCTTTATGACTTGGCC-39), ADA3KOLEFT (59-TAACAAAGACGGAGCGACGAGAAGTATTGGACAGGACATCTGTGCGGTATTTCACACCG-39), ADA3KORITE (59-GTTATTATGCTACGTATTTTTCCTTAGAGTTCGTATATTAGATTGTACTGAGAGTGCAC-39), ADA3LCHECK (59-GAGCTCCGACGTGCAACGCGA-39), and ADA3RCHECK (59-CCCGCGATCGGAAGTTCATGC-39). For each ADA gene, the corresponding KOLEFTand KORITE primers were used in a standard PCR to amplify the HIS3 genefrom pRS403 (67). The resulting PCR fragments, which contained the HIS3 geneflanked by 78 bp of 59 and 39 noncoding sequence from the respective ADA gene,were used to transform diploid S288C yeast strains. We verified the correctintegration event for each mutation by using PCR with three different primerpairs (data not shown).

ADA1 deletion mutant SB11 was created by PCR amplification of the HIS3gene with chimeric primers that included sequence upstream from or near theend of the ADA1 gene; these oligonucleotides were 59-CGTTGCTCTTTTCATTCGTCTGTTCGTTATCATCATTGGATAGGGCTTACTCTTGGCCTCCTCTAG-39 (upstream) and 59-CATCCAAAGGCGTGAAAGTCGGCTTTTCATTTAAGAAGTCCGGCAAGTCGTCGCCTCGTTCAGAATGACACG-39(downstream). The resulting PCR product was transformed into a trp1D deriv-ative of strain PSY316 (12), and ada1D clones were selected on His2 selectiveplates.

To investigate the relationship between the Gcn5 and Spt7 bromodomains,GCN5 deletion strain SB326 was produced from spt7 DBrD strain FY1009 (20)by transformation with the BglII/XhoI fragment of gcn5D::URA3 hisG plasmidpyGCN5.KO (12) followed by selection on 5-fluoroorotic acid (5-FOA) plates to

VOL. 19, 1999 FUNCTIONAL ANALYSIS OF SAGA COMPLEX 87

remove the URA3 gene (6). This strain was then used for integration of pRS306-PADH-yGCN5 1-439 and 1-280 by the methods described below.

Rich (YPD), minimal, synthetic complete (SC), 5-FOA, and sporulation me-dia were prepared as described previously (61). Media for testing Gal and Inophenotypes were as described by Gansheroff et al. (20). For caffeine (12 mM)and hydroxyurea (150 mM) plates, SC medium was used. Standard protocols fortransformation were used in strain constructions (61).

Plasmid constructs. For integration of wild-type and bromodomain truncationmutant GCN5 into yeast, constructs were prepared in which the appropriateopen reading frames followed the ADH1 promoter in the vector pRS306 (67). Bystandard recombinant DNA techniques (65), wild-type GCN5 (coding for resi-dues 1 to 439), and the longer of the two mutant genes (with residues 1 to 350)were obtained as KpnI/PvuII fragments from pPC87-yGCN5 plasmids (13) andsubcloned into KpnI/SmaI-cut pRS306. For the other mutant plasmid (withresidues 1 to 280), a vector, pRS306-PADH, was first created by subcloning aKpnI/PvuII fragment of pPC87 (containing the ADH1 promoter and terminator)into pRS306 that had been digested with BstXI, blunted by the exonucleaseactivities of T4 and T7 DNA polymerase, and digested with KpnI. The resultingplasmid was cut with NotI, and truncated GCN5 was inserted as an EagI fragmentof the pSP64-yGCN5 clone for the 1-280 mutant (13). The pRS306-PADH-yGCN5 1-439, 1-350, and 1-280 constructs were then linearized with NsiI andtransformed into the gcn5D mutant FY1370. Clones were selected on Ura2

medium; since pRS306 has no origin of replication, these represent plasmidintegrations into the genome at the URA3 locus.

Plasmids BD1 (SWI1) and FB565 (GAL11), used to recover otherwise lethalprogeny in double-mutant analysis (Table 2), have been described previously (56,59). Plasmid pGEX-3X, for bacterial expression of GST, was from Pharmacia,and pGST-TBP was provided by R. H. Reeder (Fred Hutchinson Cancer Re-search Center, Seattle, Wash.).

HAT complex purification and assays. Nucleosomal HAT complexes wereprepared from ada1D, gcn5D, gcn5D spt3D, gcn5D spt8D, and PSY316 wild-type

cells and from wild-type and bromodomain-truncated GCN5 integrants by thepreviously described purification scheme (24): growth in 4 liters of YPD to anoptical density of 2 to 2.5 at 600 nm, cell breakage with glass beads, incubationof extract with Ni21-agarose and recovery of a 300 mM imidazole eluate, andpurification on a Mono Q column with a 100 to 500 mM NaCl gradient. Forspt3D, spt8D, and accompanying wild-type complex preparation, starting materialwas a 6-liter culture, and an additional Superose 6 column step was employed.Peak Mono Q fractions of SAGA were pooled and concentrated to 0.6 ml in aCentriprep-30 concentrator (Amicon). Samples were loaded on a Superose 6 HR10/30 column (Pharmacia) equilibrated in 250 mM NaCl. The calibration of thiscolumn was as follows: thyroglobulin (669 kDa), fraction 24; ferritin (440 kDa),fraction 27; adolase (158 kDa), fraction 30; and ovalbumin (45 kDa), fraction33/34.

HAT assays were performed by previously described methods (24): 30-mlreaction mixtures contained 2 ml of enzyme sample, 2 mg of oligonucleosomes or10 mg of free histones, and 0.25 mCi of 3H-labeled acetyl coenzyme A in HATbuffer (50 mM Tris-HCl [pH 8.0], 50 mM KCl, 5% glycerol, 0.1 mM EDTA, 1mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium bu-tyrate) and were incubated 30 min at 30°C. Oligonucleosomes were prepared asdescribed previously (16), and free histones (purchased from Sigma) were fromcalf thymus. Half of each reaction was spotted on P81 filter paper (Whatman) forhistone binding, washed, and used for liquid scintillation counting; the other halfwas heated in sodium dodecyl sulfate (SDS) sample buffer with b-mercaptoetha-nol and run on an SDS–18% polyacrylamide gel, which was fluorographed withEnhance (Dupont NEN) and dried. Fluorography was performed with KodakX-Omat film at 270°C, with exposure times of 2.5 days for nucleosome assaysand 1 to 1.5 days for free histone assays. Quantitation of histone H3 and H2Bacetylated by SAGA was achieved by cutting out these bands from the dried gels,pulverizing them, and using them for liquid scintillation counting; five sets ofcounts were averaged for each, and error was 8% or less.

TABLE 1. S. cerevisiae strains used

Strain Genotype

FY2.......................................................................................MATa ura3-52FY37.....................................................................................MATa his3D200 lys2-128d ura3-52FY293...................................................................................MATa spt3D202 his4-917d lys2-173R2 ura3-52FY294...................................................................................MATa spt3D202 his4-917d leu2D1 lys2-173R2 trp1D63 ura3-52FY297...................................................................................MATa spt3D202 his4-917d lys2-173R2 trp1D63 ura3-52FY462...................................................................................MATa spt8D302::LEU2 his4-917d leu2D1 lys2-173R2 trp1D63FY463...................................................................................MATa spt8D302::LEU2 his4-917d leu2D1 lys2-173R2 trp1D63 ura3-52FY602...................................................................................MATa his3D200 leu2D1 lys2-128d trp1D63 ura3-52FY630...................................................................................MATa his4-917d leu2D1 lys2-173R2 trp1D63 ura3-52FY631...................................................................................MATa his4-917d leu2D1 lys2-173R2 trp1D63 ura3-52FY1009.................................................................................MATa spt7-502 his4-917d leu2D1 ura3-52FY1098.................................................................................MATa spt20D100::URA3 his3D200 leu2D1 ura3-52FY1106.................................................................................MATa spt20D100:URA3 his4-917d leu2D1 lys2-173R2 trp1D63 ura3-52FY1253.................................................................................MATa sin4D::TRP1 his3D200 leu2D1 lys2-173R2 trp1D63 ura3-52FY1254.................................................................................MATa swi1D::LEU2 his3D200 leu2D1 lys2-128d ura3-52FY1286.................................................................................MATa gcn5D::HIS3 arg4-12 his3D200 leu2D1 lys2-173R2 ura3-52FY1299.................................................................................MATa spt8D302::LEU2 his3D200 leu2D1 lys2-173R2 trp1D63 ura3-52FY1359.................................................................................MATa srb2D::HIS3 arg4-12 his3D200 lys2-173R2 ura3-52FY1370.................................................................................MATa gcn5D::HIS3 his3D200 leu2D1 ura3-52FY1440.................................................................................MATa gcn5D::HIS3 spt3D202 arg4-12 his3D200 leu2D1 lys2-173R2 ura3-52FY1441.................................................................................MATa gcn5D::HIS3 spt3D202 his3D200 leu2D1 lys2-173R2 ura3-52FY1542.................................................................................MATa ada3D::HIS3 his3D200 leu2D1 lys2-173R2 trp1D63 ura3-52FY1545.................................................................................MATa ada3D::HIS3 arg4-12 his3D200 lys2-173R2 trp1D63 ura3-52FY1547.................................................................................MATa ada3D::HIS3 his3D200 lys2-173R2 trp1D63 ura3-52FY1548.................................................................................MATa ada2D::HIS3 his3D200 leu2D1 lys2-128d ura3-52FY1553.................................................................................MATa ada2D::HIS3 his3D200 his4-917d leu2D1 lys2-173R2 ura3-52FY1554.................................................................................MATa ada2D::HIS3 his3D200 lys2-173R2 trp1D63 ura3-52FY1557.................................................................................MATa ada1D::HIS3 his3D300 leu2D1lys2-128d ura3-52FY1559.................................................................................MATa ada1D::HIS3 his3D200 his4-917d leu2D1 lys2-173R2 trp1D63 ura3-52FY1599.................................................................................MATa gcn5D::HIS3 arg4-12 his3D200 his4-917d leu2D1 lys2-173R2 trp1D63 ura3-52FY1656.................................................................................MATa snf2D::LEU2 his3D200 leu2D1 ura3-52FY1657.................................................................................MATa gal11D::TRP1 arg4-12 his3D200 lys2-173R2 trp1D63 ura3-52FY1658.................................................................................MATa snf5D2 his3D200 lys2-128d ura3-52FY1719.................................................................................MATa gcn5D::HIS3 spt8D302::LEU2 arg4-12 his3D200 leu2D1 lys2-173R2 trp1D63 ura3-52FY1720.................................................................................MATa gcn5D::HIS3 spt8D302::LEU2 his4-917d leu2D1 lys2-173R2 ura3-52L937......................................................................................MATa srb5D::URA3hisG arg4-12 his3D200 leu2 trp1D63 ura3-52PSY316.................................................................................MATa ade2-101 his3D200 leu2-3, 2-112 lys2 ura3-52SB11......................................................................................MATa ada1D::HIS3 ade2-101 his3D200 leu2-3, 2-112 lys2 trp1D ura3-52SB326....................................................................................MATa gcn5D spt7-502 his4-917d leu2D1 ura3-52

88 STERNER ET AL. MOL. CELL. BIOL.

Antibodies and Western blotting. For Western blot experiments, samples wereboiled in SDS–b-mercaptoethanol sample buffer, electrophoresed on SDS–8 or10% polyacrylamide gels, electroblotted to nitrocellulose, and visualized immu-nochemically by standard methods (32). Anti-Ada2, anti-Gcn5, and anti-Spt3antisera and anti-Spt20 affinity-purified antibodies were as described previously(24); primary antibody dilutions used were typically 1:4,000, 1:4,000, 1:500, and1:2,000, respectively. Anti-TafII90, anti-TafII68, and anti-TafII60 antisera (25)were used at dilutions of 1:3,000, 1:6,000, and 1:6,000, respectively. Rabbitanti-Spt8 antibodies were raised against a synthetic peptide spanning the amino-terminal 20 amino acids (aa) of Spt8 (MDEVDDILINNQVVDDEEDD) byCocalico Biologicals; this antiserum was used at a dilution of 1:2,000. Immuno-detection was performed with a secondary antibody (goat anti-rabbit immuno-globulin G-horseradish peroxidase conjugate; Bio-Rad) and an enhanced chemi-luminescence kit (Amersham). Some blots were stripped of antibody beforereprobing; this was accomplished with 62.5 mM Tris (pH 6.8)–2% SDS–100 mMb-mercaptoethanol at 50°C for 30 min.

In vivo RNA analysis. Yeast strains used for analyzing transcriptional effects ofthe bromodomain were FY1370 (gcn5D) and GCN5 wild-type and bromodomaindeletion integrants as described above; for HAT domain experiments, strainswere FY1370 plasmid integrants containing wild-type GCN5, empty vector(gcn5D), or GCN5 with HAT domain substitution mutations (78). Total RNAwas isolated from cultures grown in SC medium to an optical density at 600 nmof 0.8 to 1.2 by the hot phenol method as described previously (40). To derepressHIS3 transcription, cells were grown in SC medium lacking histidine, and 3-ami-notriazole was added to 40 mM 2 h (for bromodomain study) or 6 h (for HATdomain study) prior to RNA isolation. RNA concentration was quantitatedspectrophotometrically at 260-nm wavelength; 50 mg of each RNA sample washybridized to a completion with an excess of 32P-end-labeled HIS3 and tRNAw

oligonucleotides and treated with S1 nuclease as described elsewhere (40, 54).HIS3 RNA levels were quantitated on a PhosphorImager (Molecular Dynamics).S1 assays were performed in duplicate several times (exception for mutants LKN,IFQ, and YIA, performed once), and PhosphorImager quantitation showed lessthan 15% error. tRNAw probe (bromodomain study) and PMA1 RNA (HATdomain study) were used as internal controls for equal loading.

GST-TBP binding study. Escherichia coli DH5a, grown in LB medium with100 mg of ampicillin per ml, was used for induction of GST and GST-TBP.Expression was induced with 1 mM isopropyl-b-D-thiogalactopyranoside for 4 hat 37°C, and cell lysates were prepared by sonication in phosphate-buffered saline(65) containing protease inhibitors and rotated for 30 min at 4°C with glutathi-one-Sepharose beads (Pharmacia). Samples of these beads (about 15 ml contain-ing several mg of GST or GST-TBP) were then rotated alone or with Superose6-purified wild-type (fraction 20), spt3D (fraction 21), or spt8D (fraction 21)SAGA complexes for 1 h at 4°C. Amounts of SAGA samples used (30 ml forwild-type and spt8D strains and 20 ml for spt3D strains) were adjusted to havesimilar amounts of protein, based on prior Western blotting. Each reaction (150ml, final volume) was adjusted to a final NaCl concentration of approximately 60mM and contained 100 ml of binding buffer (20 mM HEPES [pH 7.3], 5 mMMgCl2, 0.5 mM EDTA, 15% glycerol, 1 mM dithiothreitol, 1 mM phenylmeth-ylsulfonyl fluoride). The resulting beads were washed three times with 500 ml ofbinding buffer with 50 mM NaCl, boiled in SDS sample buffer, and loaded ontoan SDS–10% polyacrylamide gel, which was used for Western blot analysis withanti-Ada2, anti-Spt20, and anti-Spt3 antibodies.

RESULTS

Loss of Spt3 or Spt8 has moderate effects on SAGA struc-ture. Our previous results have indicated that severe pheno-types correspond to disruption in SAGA structure; i.e., themajor growth defects seen in spt20D and spt7D mutants corre-late with a complete loss of the SAGA complex. In contrast,the mild gcn5D phenotype corresponds to a subtly alteredSAGA complex. To test further the hypothesis that severity inmutant phenotype relates directly to biochemical integrity ofthe SAGA complex, we examined SAGA structure in the mod-erately defective spt3D and spt8D and the severe ada1D mu-tants and then compared their mutant phenotypes.

First, extracts were prepared from both SPT3 and SPT8disruption strains to determine whether the SAGA complexexists in the absence of these subunits. The extracts were chro-matographed through Ni21-agarose and Mono Q resins toseparate four previously identified nucleosomal HAT com-plexes (24). Western blotting using Ada2 and Gcn5 antibodiesdemonstrated that the mutant SAGA complexes were chro-matographically distinct from wild-type SAGA: spt3D andspt8D SAGA complexes elute from Mono Q approximately twoand seven fractions earlier, respectively (data not shown).

To investigate these altered complexes further, larger-scalepreparations of SAGA from spt3D and spt8D mutants werefractionated with an additional Superose 6 column step (24).Size determination via anti-Ada2 Western blots of the Super-ose fractions indicated that the SAGA complexes derived fromboth mutants were only slightly smaller and eluted in a broaderpeak than wild-type SAGA (Fig. 1A), suggesting that few, ifany, additional subunits were lost along with either Spt3 orSpt8. In addition, Spt3 is present in spt8D SAGA (Fig. 1A), andSpt8 is present in spt3D SAGA (Fig. 1B). Furthermore, dele-tion of Spt3 or Spt8 has no effect on TafII90, TafII68, or TafII60interaction with SAGA (Fig. 1B).

Ada1 is integral to SAGA structure. In contrast to the mod-est effects on structure from loss of either Gcn5 (24), Spt3, orSpt8, our previous biochemical analyses of Spt20 and Spt7suggested that these proteins are critical to the structural in-tegrity of the SAGA complex (24). The large size of SAGA(1.8 MDa) indicates that there may be additional proteinshaving a similar role in holding the complex together. Theobservation that a strain bearing an ADA1 disruption exhibitssevere phenotypes similar to those of SPT20 mutants, alongwith the indication that Ada1 may exist in a large complex withAda2, Ada3, Gcn5, and Spt20 (5, 37), suggested that Ada1might be present in SAGA and required for its integrity.

To test whether Ada1 has a role in SAGA, cell extracts wereprepared from wild-type and ada1D strains and tested for thepresence and activity of SAGA. Nucleosomal HAT assays wereperformed on Mono Q column fractions, following Ni21 che-

FIG. 1. Western blot characterization of Superose 6-purified spt3D and spt8DSAGA complexes. (A) SAGA complexes derived from wild-type (FY631), spt3D(FY294), and spt8D (FY462) strains were pooled, concentrated, and run on aSuperose 6 column for separation based on molecular weight. Western blots ofthese fractions were performed to compare the sizes of the complexes. Blots werevisualized with dilutions of antisera raised against the Ada2 or Spt3 proteins asindicated. (B) Western blots of wild-type (fraction 20), spt3D (fraction 21), andspt8D (fraction 21) SAGA, visualized with antisera specific to Spt8, TafII90,TafII68, or TafII60.


late affinity chromatography (24). Extract from ada1D cellsspecifically lacked the nucleosomal HAT activity of SAGA(around fraction 42), while the activity of the Ada complex(peak in fractions 24 to 26) was intact (Fig. 2A). This profile isidentical to that previously observed for both SPT20 and SPT7disruptions.

To confirm that Ada1 is directly involved in the SAGAcomplex, Western blot analysis was performed. An anti-Ada1blot revealed that wild-type SAGA, but not the Ada complex,does contain Ada1 (Fig. 2B). Furthermore, immunoblottinganalyses using anti-Ada2 and anti-Gcn5 antibodies indicatethat SAGA itself is lost in the ada1D mutant preparation, whilethe Ada complex was unaffected (Fig. 2B). Ada1 is thereforesimilar to Spt20 and Spt7 in that it is required for the overallstructural integrity of SAGA.

Three phenotypic classes of SAGA components. Thus, struc-tural analysis of SAGA provides a biochemical correlation withprevious observations that disruptions of SAGA componentscause phenotypes with a range of severity. We further testedthe hypothesis that loss of components that have little detect-able effect on SAGA complex integrity result in modest phe-notypes, and in contrast, loss of components that are com-pletely disruptive to the complex result in severe phenotypes.

Phenotypic effects of disruptions of representatives of eachputative phenotypic class are presented in Fig. 3. Growth wastested under various conditions known to cause growth defectsin some yeast mutant strains (31). As shown previously andhere, Ada2/Gcn5 and Spt3/Spt8 each have modest phenotypiceffects. In contrast, ada1D causes genetic phenotypes similar inseverity to those caused by spt7D and spt20D mutations andmore severe than those of the others (spt3D, spt8D, gcn5D,ada2D, and ada3D). In particular, as reported previously (37),ada1D is like spt20D in that it is an inositol auxotroph anddisplays an Spt2 phenotype; we show here that both mutantsalso have the additional phenotype of failure to grow on media

containing caffeine or the DNA synthesis inhibitor hydroxy-urea.

We tested another phenotype that differed for the subgroupsof SAGA mutants: double-mutant lethality in combinationwith mutations in either SNF/SWI genes or Srb/mediatorgenes. These results (Table 2) also demonstrate that ada1Dmutations cause the same phenotypes as spt20D and spt7Dmutations (59): ada1D is lethal in combination with every snf/swi or Srb/mediator mutation tested. In contrast, ada2D andada3D mutations did not cause inviability in similar double

FIG. 2. Presence of Ada1 in the SAGA complex. HAT complexes were derived from wild-type (PSY316) and ada1D (SB11) yeast strains through the Mono Qcolumn step of the previously described purification procedure. (A) Fluorographs of nucleosome acetylation assays of the even-numbered column fractions. The arrowsdenote the relative positions of the four histone proteins as separated on SDS–18% polyacrylamide gels; visualized species contain acetyl groups labeled with 3H.Wild-type fractions displaying the H3/H2B-acetylating activities of the Ada and SAGA complexes are indicated at the top. (B) Western blots of the fractions, visualizedwith dilutions of antisera raised against Ada1, Ada2, or Gcn5 protein.

FIG. 3. Phenotypic comparison of ada1D and other SAGA deletion mutants.Strains FY630, FY1106, FY1559, FY297, FY463, FY1599, and FY1553 weregrown overnight in liquid YPD medium. For each strain, approximately 5 3 103

cells were spotted on the indicated plates and grown for 2 days at 30°C. Allstrains contain the his4-917d insertion mutation. Otherwise, wild-type strainscontaining this allele are His2, while strains containing a mutation able tosuppress this allele are His1 (Spt2 phenotype).


mutants (Table 2), as was also previously seen for spt3D, spt8D,and gcn5D mutations (59), indicating a less critical role forthese subunits in cellular growth. These results contrast withthose of Pollard and Peterson (58), who observed lethality indouble mutants of gcn5D, ada2D, or ada3D combined withswi1D, albeit in a different yeast strain background.

Thus, both biochemical and genetic evidence places Ada1 inthe Spt20/Spt7 class; i.e., it is required for SAGA complexintegrity, and loss of it is strongly debilitating to function invivo. In contrast, either Gcn5, Spt3, or Spt8 has modest effectson SAGA structure and function in vivo. In summary, wedetect three phenotypic and structural classes of SAGA com-ponents: Gcn5/Ada2/Ada3, Spt3/Spt8, and Ada1/Spt7/Spt20.

Double mutants gcn5D spt3D and gcn5D spt8D are similar inphenotype to spt20D and spt7D mutants. Based on the abovedata, it appears that each of the two moderate phenotypicclasses contributes distinct functions to SAGA. Because mod-erate phenotypes result from deletions of genes of either class,we postulated that in the absence of one class, the function ofthe second class remains intact. However, in the cases ofada1D, spt7D, and spt20D mutations, where the complex iscompletely disrupted, both functions are lost and severe phe-notypic defects result. Based on these observations and ideas,we hypothesized that double mutants that disrupt the functionof each moderate phenotypic class simultaneously would causesevere phenotypes similar to those of ada1D, spt7D, and spt20Dmutations.

To test this idea, gcn5D spt3D and gcn5D spt8D mutants wereconstructed. As had been seen previously in other phenotypicassays (reference 59 and Fig. 3), neither spt3D, spt8D, norgcn5D cells were severely defective (Fig. 4). In fact, spt3D andspt8D strains grew well in the absence of inositol, in the pres-ence of hydroxyurea or caffeine, or with galactose as a solecarbon source; gcn5D cells also grew well under all conditionsexcept caffeine. In contrast, the double mutants gcn5D spt3Dand gcn5D spt8D were significantly more defective, failing togrow under the latter three conditions, and gcn5D spt8D grewless vigorously than the single mutants in media lacking inosi-tol (Fig. 4A). These levels of impairment are similar to what isseen in an spt20D mutant tested under the same conditions(Fig. 4A). These results show that the double mutants havesevere phenotypes similar to those of single mutations thatdisrupt SAGA altogether. Importantly, SAGA is intact in thedouble mutants and can be purified by standard methods, asshown by immunoblot analysis of SAGA from these strains(Fig. 4B). Overall, the results support the idea that the twophenotypically moderate subgroups of components contributedistinct biochemical functions to SAGA.

Gcn5 bromodomain is important for normal levels of nu-cleosome acetylation by SAGA. We wished to characterize thebiochemical functions of the Gcn5/Ada2/Ada3 and Spt3/Spt8subgroups in SAGA. Each member of the Gcn5/Ada2/Ada3subgroup previously was shown to be required for histone

TABLE 2. Phenotypes of ada mutants

Double mutanta Phenotypee

ada1D swi1Db,d Deadada1D snf5D2c Deadada1D srb5Dc Deadada1D gal11Db,d Dead

ada2D swi1D Alive, sickada2D snf2Dd Alive, sickada2D srb5D Alive, slow growingada2D srb2D Alive, slow growingada2D gal11D Alive

ada3D swi1Dd Alive, sickada3D snf2D Alive, sickada3D srb5D Alive, slow growingada3D srb2D Alive, slow growingada3D gal11Dd Aliveada3D sin4D Alive

a In each cross, genetic markers allowed us to unambiguously identify allrelevant markers in every tetrad. The parents for the crosses in the order listedare as follows: FY602 3 FY1254, FY1658 3 FY1557, FY1557 3 L937, FY602 3FY1657, FY1548 3 FY1254, FY602 3 FY1656, FY1548 3 L937, FY1548 3FY1359, FY1554 3 FY1657, FY602 3 FY1254, FY1542 3 FY1656, FY1542 3L937, FY1542 3 FY1359, FY602 3 FY1657, and FY1253 3 FY1545.

b Viability was scored by the ability of the double mutant to lose a URA3-marked wild-type SWI1 (BD1) or GAL11 (FB565) plasmid as determined bygrowth on 5-FOA medium (alive, growth on 5-FOA; dead, no growth on5-FOA).

c Viability was determined by failure to recover any double mutants afterdissecting 20 tetrads.

d For all crosses in which FY602 is listed as the MATa parent, the doublemutant was constructed by first creating a diploid between FY602 and thecorresponding MATa parent. This diploid was then transformed with a PCRfragment containing the relevant ADA ORF completely disrupted by the HIS3gene. Those single purified His1 diploid transformants that displayed correctADA knockout integration (as described in Materials and Methods) were used intetrad analysis.

e Sick, extremely small colonies after 2 to 3 days of growth at 30°C. Slowgrowing, colonies were moderately smaller than those of wild-type strains after 2to 3 days of growth at 30°C.

FIG. 4. Comparison of gcn5D spt3D and gcn5D spt8D double mutants with anspt20D mutant. (A) Strains FY2, FY293, FY1299, FY1286, FY1440, FY1719, andFY1098 were grown overnight in liquid YPD medium. For each strain, approx-imately 2 3 103 cells were spotted on the indicated plates. Growth is shown after2 days of incubation at 30°C, except in the case of the caffeine plates, which werephotographed after 3 days. (B) Double-mutant SAGA complexes are intact.Mono Q fractions from gcn5D spt3D (FY1441) and gcn5D spt8D (FY1720) HATcomplex preparations were analyzed by Western blotting. SAGA subunits werevisualized with dilutions of antisera raised against Ada2 or Spt20 protein asindicated.


acetylation, and Gcn5 was identified as the HAT catalytic sub-unit. Recombinant Gcn5 acetylates free core histones and notnucleosomes; however, both the Ada and SAGA complexesacetylate nucleosomes (24). Thus, an important question is thedeterminant(s) of physical interaction with nucleosomes.

One clue to a possible determinant of nucleosome access iswithin the Gcn5 protein itself. The bromodomain at the car-boxyl terminus of Gcn5 (aa 349 to 422 [22]) is a conservedsequence that is found in a variety of transcription-relatedproteins but has no clear function (33, 41, 72). In particular,the bromodomain is found in several transcription cofactorsand coactivators that possess HAT activity or other nucleo-some remodeling activity, thus making the bromodomain acandidate domain to mediate interaction with nucleosomes. Itwas shown previously that the Gcn5 bromodomain is not re-quired for acetylation of free histones by recombinant Gcn5 invitro or for transcriptional activation by strong activators, butits deletion does result in minor growth defects and reducedactivation by weak activators (13, 21, 48). To investigate therole of the bromodomain in nucleosome acetylation, we pre-pared Ada and SAGA complexes from extracts of gcn5D cellscontaining integrated copies of wild-type or bromodomain-deleted (DBrD) GCN5. To control for potential construct-specific differences in stability or expression, two independentbromodomain mutants (13), containing the sequences for ei-ther the first 350 or 280 aa of the 439-aa full-length Gcn5, wereused for these experiments.

Wild-type Ada and SAGA complexes (Mono Q peak frac-tions 20 and 40, respectively) showed comparable levels ofhistone H3-acetylating activity on nucleosomal substrates (Fig.5A). Ada complex fractions derived from the bromodomainmutants had nucleosome-acetylating activities similar to that ofthe wild-type Ada complex (fraction 20), but both mutantsdisplayed significantly reduced activity by SAGA (fraction 40).A different situation was observed when free histones wereused as a substrate for acetylation: the levels of acetylation bythe DBrD SAGA fractions were only moderately lower thanthat of wild-type SAGA. Quantitation of the H3/H2B speciesin the HAT assays shown for fraction 40 revealed that thenucleosomal substrates were acetylated approximately half aswell as free histones by DBrD SAGA compared to wild-typeSAGA (Fig. 5A, bar graph). In addition, in each case acetyla-tion of free histones by SAGA was higher than free histoneacetylation by Ada and nucleosome acetylation by eitherSAGA or Ada. Anti-Ada2 and anti-Gcn5 Western blots (Fig.5B) confirmed that these free histone acetylation results accu-rately reflected the amount of complex in the fractions, sinceSAGA contained more protein than Ada. The immunoblotsalso show that the bromodomain mutants had nearly wild-typeamounts of both Ada and SAGA.

Overall, the results show that despite the fact that SAGAfractions derived from wild-type and Gcn5 DBrD strains havecopious amounts of Gcn5 and innate HAT activity, they acet-ylate nucleosomes less effectively than the less concentratedAda complex. Furthermore, the observed defects may be dueto absence of the bromodomain and not due to loss of otherassociated protein factors, since no major differences in sizewere observed in Superose 6 fractionations of wild-type andbromodomain mutant SAGA complexes (data not shown). Inaddition, Western analysis showed that TafII60, TafII68, andTafII90 were present in the DBrD SAGA complexes, as well asin gcn5D SAGA (Fig. 5B).

Since Spt7 is another SAGA component harboring a bro-modomain, we also analyzed a similar deletion within thisprotein. Ada and SAGA complexes prepared from SPT7 DBrDcells (20) or from a double mutant with GCN5 DBrD were

indistinguishable from their wild-type SPT7 counterparts interms of nucleosome acetylation or growth phenotypes undervarious conditions (data not shown). Thus, the Spt7 bromodo-main has no discernible functional effect or synthetic interac-tion with the Gcn5 bromodomain in these assays.

Gcn5 bromodomain and HAT domain mutants display sim-ilar HIS3 transcriptional defects. In vitro, nucleosomal acety-lation by Gcn5 was specifically lowered by deletion of thebromodomain. The effect of Gcn5 bromodomain deletion invivo was investigated by S1 analysis of RNA produced from theHIS3 gene, which has been shown to be regulated by Gcn5’snucleosome acetylation activity (44). There are two RNA startsites in HIS3 (Fig. 6A), and in wild-type cells under basalconditions these sites are used equally (Fig. 6C and reference70). In strains lacking Gcn5 or bearing Gcn5 DBrD, usage ofthe 113 start site was reduced (Fig. 6C). Also similar to theabsence of Gcn5, the Gcn5 DBrD strains show diminishedactivation potential (Fig. 6D).

Under activated conditions in wild-type cells, there is astrong preference for the 113 start site over the 11 site (Fig.6B and D and reference 70). In gcn5D cells, 113 and 11 startsites were used equally in induced conditions (Fig. 6B and D).To examine the potential role of acetylation in start site usage,we tested the effects of HAT domain and bromodomain mu-tations. HAT domain substitution mutations, which exhibit acorrelation between HAT competence and transcriptional ac-tivation at a model promoter (78), also show a clear correlationwith start site usage (Fig. 6B). The 113 start site was preferredin yeast strains bearing the LKN, IFQ, or YDR mutation(HAT-competent mutants), and hence these were similar tothe wild type. KQL and PKM (HAT-defective mutants) hadmuch reduced start site preference for 113 and were similar tothe gcn5D strain. Finally, YIA (HAT-intermediate mutant)showed an intermediate preference for start sites betweenGCN51 and gcn5D. Thus, the results demonstrate a strongcorrelation between HAT activity and start site preference,indicating that HAT activity of Gcn5 is critical for normal startsite selection at the HIS3 promoter.

We then tested the effects of the two DBrD mutations on the113/11 start site ratio under activated conditions. Interest-ingly, these mutants also showed a change in start site usage(Fig. 6D), and the effect was intermediate between those of thewild type and HAT domain mutants that result in completeloss of HAT activity (compare Fig. 6D with Fig. 6B). Thus,several aspects of transcriptional control of HIS3 are altered inthe bromodomain mutants in a manner resembling a defectspecifically in Gcn5 HAT catalytic function.

TBP binding by SAGA in vitro requires Spt8 but not Spt3.We then characterized SAGA prepared from strains bearingdeletions of the other moderate class, Spt3 or Spt8, to deter-mine whether the biochemical functions of the complexes werealtered. SAGA complexes purified from spt3D and spt8D cellswere tested for HAT activity, and no major differences inacetylation were observed (data not shown). Thus, in contrastto Gcn5, neither Spt3 nor Spt8 is crucial for the HAT activityof SAGA.

SAGA complexes prepared from SPT3 or SPT8 mutantstrains were therefore intact and possessed HAT activity, butas described above, genetic evidence had indicated that theseSpt proteins are important for normal function. Previous re-sults suggested that Spt3 and Spt8 interact functionally, andpossibly physically, with TBP. Although TBP does not cofrac-tionate with SAGA (23), pull-downs of GST-Spt20 from yeastextracts recovered TBP along with the other Spt proteins (59).Therefore, we tested whether SAGA interacts with TBP,


and if so, whether Spt3 and/or Spt8 was required for thisinteraction.

GST-TBP was used for pull-down experiments with theSuperose-fractionated SAGA complexes derived from wild-type, spt3D, and spt8D strains, and the binding of complexeswas monitored by Western analysis. Wild-type SAGA inter-acted with GST-TBP but not with the GST negative controlprotein, as visualized by using antibodies to Ada2, Spt3, andSpt20 (Fig. 7). Interestingly, while SAGA lacking Spt3 alsobound to GST-TBP, SAGA lacking Spt8 showed a very lowlevel of binding (Fig. 7). The finding that Spt3 was not acritical determinant of SAGA interaction with TBP wassupported by the observation that Spt3 was present inSAGA prepared from the Spt8 disruption, which bound

poorly to TBP (Fig. 7). Similarly, the potential participationof Spt8 in TBP binding is bolstered by the finding that Spt8is present in the spt3D SAGA sample (Fig. 1B). Altogether,these data indicate that the Spt3/Spt8 subgroup is not crit-ical for SAGA structure, but at least Spt8 is required forSAGA interaction with TBP in vitro.

DISCUSSION

A number of interconnected processes are involved in theactivation of transcription, and the recently discovered SAGAcomplex apparently combines several of them: activator inter-action (through Ada2, Gcn5, and Ada3), histone acetylation(by Gcn5), and TBP interaction (through Spt8 and Spt3). We

FIG. 5. Effect of Gcn5 bromodomain deletion on nucleosomal HAT activity. Cells containing wild-type (w.t.; residues 1 to 439) or DBrD (residues 1 to 350 or 1to 280) GCN5 were used to prepare Mono Q fractions by the standard purification method for the HAT complexes. (A) HAT assays with nucleosomal or free histonesubstrates were performed with even-numbered fractions surrounding the Ada and SAGA peaks; fluorographs of the resulting gels are presented. The arrows denotethe relative positions of the four histone proteins as separated on SDS–18% polyacrylamide gels; visualized species contain acetyl groups labeled with 3H. The bar graphat right presents the ratio of nucleosomal to free histone H3/H2B acetylation for SAGA fraction 40, normalized to the wild-type ratio. (B) Samples of fractions 20 (Adapeak), 30 (negative control; no HAT complex), and 40 (SAGA peak) were also analyzed by Western blotting. Five microliters of each was run on SDS-polyacrylamidegels and electroblotted to nitrocellulose, which underwent immunodetection with dilutions of anti-Ada2, anti-Gcn5, or anti-TafII antibodies. Markers on the Gcn5panels indicate the Gcn5 species visualized on equivalent portions of the blots, and their positions of migration agreed with their predicted sizes (51, 40, and 32 kDafor the 1-439, 1-350, and 1-280 constructs, respectively). The peak SAGA fraction from a preparation of gcn5D (FY1370) cells was also tested for TafIIs (right).


have sought to characterize the latter two of these functions invivo and in vitro and to analyze the structure of SAGA bystudying mutant complexes and individual subunits in vitro.These studies indicate that while neither the Gcn5/Ada2/Ada3

nor Spt3/Spt8 subgroup is critical for SAGA structure, eachconfers a distinct and important biochemical function. Eithermay be partially dispensable, but loss of both is highly detri-mental to overall SAGA function.

FIG. 6. HIS3 transcriptional defects observed in GCN5 null, HAT domain substitution, and DBrD mutants. (A) Diagram of HIS3 promoter function. Weak andstrong TBP-binding sequences (TATA boxes) direct transcription from the 11 and 113 start sites, respectively. The activator Gcn4 binds to multiple upstream sites(UAS). (B) Start site usage for activated HIS3 transcription in wild-type (w.t.), gcn5D, and GCN5 HAT domain substitution mutant strains. Activating conditions wereachieved with 3-aminotriazole. PhosphorImager quantitation of the S1 assays is presented as the ratio of 113 to 11. Shown at the bottom are in vitro HAT activities(as a percentage of wild-type activity) of native SAGA complexes purified from the indicated strains (27). (C) Start site preference for wild-type, gcn5D, and DBrDstrains under noninducing conditions (no 3-aminotriazole). (D) Activated HIS3 transcription in wild-type, gcn5D, and DBrD strains. PMA1 RNA and tRNA wereincluded as internal controls for sample loading.


Components necessary for SAGA structure. In this study, wehave identified Ada1 as a component of the SAGA complex,and we have further shown that its loss leads to the physicaldisruption of SAGA and severe phenotypic defects. This placesit in a class with two other previously described SAGA com-ponents with similar qualities, Spt20 and Spt7. These threesubunits each appear to be necessary for the structure of thecomplex, with the critical role of holding it together. The pre-cise interactions among these proteins and with other SAGAcomponents remain to be determined, but their function couldbe to link the Spt and Ada subunits within SAGA.

We have also characterized spt3D and spt8D mutants andfind that in contrast to loss of Spt20/Spt7/Ada1, they maintainoverall SAGA integrity, as was seen with gcn5D. Moreover,SAGA integrity is maintained in the double disruption of Gcn5and either Spt3 or Spt8. Based on these data, we conclude thatthe Gcn5/Ada2/Ada3 and Spt3/Spt8 groups of proteins arelikely to be peripheral within the complex, i.e., exposed and ina position to form protein contacts with other factors, includ-ing histones and TBP. Overall, the biochemical characteriza-tion of structure nicely mirrors the genetic analysis of function:disruptions of the SAGA components that are integral forstructure are extremely debilitating in vivo, while disruptions ofthe more structurally peripheral components are less severe.

Nucleosomal acetylation by the SAGA complex. The mostthoroughly characterized function within the SAGA complexhas been the HAT activity of Gcn5, and recent studies indicatethat the HAT domain is required for Gcn5’s role in transcrip-tion (13, 44, 78). The experiments presented here suggest thatanother region of Gcn5, the bromodomain, may have signifi-cant effects on the function of the SAGA complex in acetyla-tion. Specifically, removal of the bromodomain reduces theHAT activity of SAGA on nucleosomal substrates, even thoughthe complex and its activity on free histones are otherwiselargely intact. One interpretation of these data is that thebromodomain is required for physical interaction either withhistones or with other components in SAGA that bind tohistones (and other proteins would fulfill such a function in theAda complex). Relevant to this is the observation that the sizeof SAGA prepared from the DBrD strain was not grosslyaltered. However, small changes in size or shape of SAGAwould not be apparent due to the low sensitivity of the Super-

ose 6 sizing column near the void volume (where SAGAelutes). In this view, Gcn5 possesses separate domains forcatalytic HAT function and for interaction with nucleosomalsubstrates. This is consistent with two previous observations.First, recombinant Gcn5 acetylates free core histones but notnucleosome-assembled histones (24); second, mutations inTafII68 similarly reduce nucleosomal histone acetylation with-out reducing free histone acetylation (25). However, theSAGA complexes bearing Gcn5 deletions of the bromodomainare not altered for TafII60, TafII68, or TafII90, indicating thatthere may be multiple determinants for nucleosome interac-tion.

A second interpretation of the data is that the bromodomaindefect could be related to the fact that nucleosome acetylationby wild-type SAGA is already somewhat inhibited relative tothat of the 0.8-MDa Ada complex, as shown in Fig. 5. Thisunidentified inhibitory activity, not present in the Ada com-plex, may reside with the Spt, TafII, or unknown subunits ofSAGA and may act by interfering with Gcn5’s interaction withnucleosomes. Thus, the function of the Gcn5 bromodomain inSAGA could be to counteract this inhibition partially, so thatdeletion of the domain leads to a more complete inhibition.Such a process would be relevant only in the context of SAGA,since DBrD Ada complex acetylates nucleosomes very well.Further study will be required to determine which of these twomodels is correct and how regulation of acetylation activitymay be achieved through the bromodomain.

In either case, it is important to note that the deletion of thebromodomain affected transcription of the HIS3 gene in vivo,causing both a slight reduction in activation potency and analteration in start sites. These effects were also seen in a totaldisruption of Gcn5 or in HAT-defective substitution mutantsof Gcn5. Thus, the effect of the bromodomain deletion in vivoclearly ties this domain to acetylation function of Gcn5.

Bromodomains occur in numerous proteins having a role inchromatin remodeling, including the acetyltransferases CBPand TAFII250, and Snf2/Swi2, a component of the ATP-de-pendent remodeling complex Swi-Snf (41). Our data do notnecessarily indicate that in general, bromodomains are re-quired for nucleosome association. First, the deletion of thebromodomain in Spt7 did not affect acetylation; second, thebromodomain of human Gcn5 interacts with a nonhistoneDNA binding protein complex, called Ku70/80 (4). The bro-modomain-Ku physical interaction results in phosphorylationof human GCN5 and repression of its HAT activity, apparentlyvia a Ku-dependent recruitment of DNA-dependent proteinkinase. Thus, we believe that there may not be a unified mech-anism for bromodomains, but rather, there may be a multiplic-ity of roles involved in modulation of activity of the residentcomplex.

TBP interaction by the SAGA complex. TBP interaction isanother function of SAGA that had been suggested by previ-ous studies but has been directly demonstrated here. In vitrobinding experiments show specific interaction between purifiedSAGA and GST-TBP. Thus, these data agree with previousevidence that TBP interacts with Ada2 (3), Ada3 (64), or Spt20(59) in the context of yeast whole-cell extract.

GST-TBP interaction analysis of two mutant complexes in-dicated that Spt8 is required for SAGA-TBP interaction invitro, but Spt3 is not. We do not yet know whether Spt8 makesdirect contact with TBP. While the mutant SAGA is not obvi-ously smaller than wild-type SAGA, the sensitivity of Superose6 sizing is low, as discussed above. The three largest TafIIs inSAGA are not sufficient for TBP interaction, because they arepresent in complex prepared from the spt8D mutant strain,which is impaired in TBP binding. In addition, SAGA pre-

FIG. 7. In vitro binding of wild-type, spt3D, and spt8D SAGA complexes toTBP. Glutathione-Sepharose beads containing bacterially expressed GST orGST-TBP were incubated alone or with similar amounts of wild-type, spt3D, orspt8D SAGA complexes at 4°C under conditions of approximately 60 mM NaCl.After washing, Western blotting was performed with these beads to analyze theproteins that had bound to GST (G lanes) or GST-TBP (T lanes). The input (I)lanes contained half of the amount of SAGA used for the binding experiments.The position of the antibody-visualized Ada2, Spt20, and Spt3 bands on theWestern blots are indicated by arrows. An unidentified species in the GST-TBPpreparation (T lanes) had a mobility similar to but slightly slower than that ofSpt3 and cross-reacted with anti-Spt3 antibodies; the band seen in the spt8D Tlane is a combination of this species and a small amount of Spt3.


pared from the tafII68 mutant strain maintains binding to TBPbut has lost Spt3 (25), which underscores that Spt3 is notrequired for TBP interaction in vitro.

The requirement of Spt8, as opposed to Spt3, for TBP bind-ing was unexpected in light of previous in vivo results, whichsuggested a direct interaction between Spt3 and TBP and anauxiliary role for Spt8 (17, 18). It is possible that Spt3 contrib-utes significantly to the interaction under physiological condi-tions within the cell. Thus, there may be multiple ways thatSAGA recruits TBP, including through TafIIs, and our in vitrobinding assay detects only a subset of them.

Model for multiple functionality of SAGA. The spt3D gcn5Dand spt8D gcn5D double mutants (Fig. 4) apparently disrupttwo in vivo functions, namely, TBP binding and nucleosomeacetylation, leading to major phenotypic defects. The com-bined losses cause defects which approximate those of a mu-tant which disrupts SAGA altogether (spt20D). This providesfurther evidence that multiple functions are incorporated inthe SAGA complex in vivo, and that the two discussed aboveare needed for its full functionality but are partially redundant.Thus, one hypothesis that emerges from these data is that twofunctions of SAGA are directed at the same objective: regu-lation of TBP binding to the TATA box. Activation domaininteraction is a third presumptive function of the SAGA com-plex. This was indicated in earlier studies of Ada2, Ada3, andGcn5 function and has been shown directly by recent studieswith SAGA itself (75).

These results, along with additional findings presented inthis and previous studies, suggest an overall model for thestructure and function of the SAGA complex, shown in Fig. 8.In this model, SAGA consists of two major parts with discretefunctions: adaptor related and Spt related. A knockout of anyof the identified adaptor-related components (Gcn5, Ada2, orAda3) leads to moderate growth defects and resistance to thetoxicity of overexpressed GAL4-VP16, while knockouts of theshown Spt proteins result in defective phenotypes of varyingseverity and in suppression of Ty element insertion in a pro-moter. At the interface between these two parts would beAda1, Spt20, and Spt7 proteins, identified as being importantto SAGA structural integrity; knockouts of any of these havecombined Ada2 and Spt2 phenotypes. A number of unknown

proteins, as well as the several TafIIs mentioned above, are alsopresent in SAGA.

As the complex approaches a promoter, the Ada2 subunitwould interact with the activation domain of an activatorbound to an upstream activating sequence, bringing Gcn5 andthe Spt components in proximity with the promoter. We pro-pose that after this recruitment, the Gcn5 moiety of SAGAfunctions to acetylate histones within the nucleosome(s) of thepromoter region, with the assistance of the bromodomain,thereby remodeling the chromatin structure in a localized re-gion. This makes the TATA box available to TBP, and its DNAbinding and/or interaction with general transcription factorsmay be regulated through contact with Spt8, Spt3, TafIIs, orundetermined SAGA subunits. In this model, loss of eitheracetylation of the nucleosomes at the TATA box (e.g., gcn5D)or loss of TBP regulation (e.g., spt8D) is tolerated, but loss ofboth functions (e.g., gcn5D spt3D and ada1D) is highly detri-mental.

This model assumes that SAGA is an independent complexwith multiple functions, but the exact relationship betweenAda and SAGA has not yet been established. The two HATcomplexes are known to have Gcn5, Ada2, and Ada3 in com-mon, but other subunit identities await the direct characteriza-tion and comparison of purified samples—Ada and SAGAmay well be functionally distinct complexes that only share afew subunits. Further characterization of SAGA will shed lighton this issue.

The present functional analyses of SAGA illustrate an im-portant concept, i.e., in large complexes, individual compo-nents have distinct biochemical functions. The combined bio-chemical and genetic analysis of SAGA yields unique insightsinto function. In general, these studies are a paradigm forfurther studies of this multicomponent complex, as well asother large acetylation and deacetylation complexes now underinvestigation.

ACKNOWLEDGMENTS

S. M. Roberts and P. A. Grant contributed equally to this work.We thank R. Candau for preparation of the ADA1 disruption strain

SB11, Z. Yang for technical assistance in the preparation of Spt7DBrDHAT complexes, and N. Barlev for advice on binding assays. We thankR. Reeder for the gift of GST-TBP plasmid, J. Reese and M. Green forTafII antibodies, and A. Navas, Z. Zhou, and S. Elledge for informingus that spt20 mutants have an HUS phenotype. We thank G. Moore forhelpful discussions and critical comments on the manuscript.

This research was supported by grants from the National Institutesof General Medical Sciences to S.L.B., F.W., and J.L.W. and from theNational Science Foundation and the Council for Tobacco Research toS.L.B. P.A.G. was supported by a postdoctoral fellowship from theAmerican Cancer Society. An NIH Cancer Core training grant to theWistar Institute supported D.E.S. and L.J.D.; D.E.S. was also sup-ported by an NIH postdoctoral fellowship.

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