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A Human SPT3-TAFII31-GCN5-L Acetylase Complex Distinct from Transcription Factor IID

1998; Elsevier BV; Volume: 273; Issue: 37 Linguagem: Inglês

10.1074/jbc.273.37.23781

1083-351X

Ernest Martinez, Tapas K. Kundu, Jack Fu, Robert G. Roeder,

DNA Repair Mechanisms

In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP). In particular, yeast SPT3 has been shown to interact directly with TBP. Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18. Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivoeither with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex. However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity. We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex. In yeast, SPT3 is a component of the multiprotein SPT-ADA-GCN5 acetyltransferase (SAGA) complex that integrates proteins with transcription coactivator/adaptor functions (ADAs and GCN5), histone acetyltransferase activity (GCN5), and core promoter-selective functions (SPTs) involving interactions with the TATA-binding protein (TBP). In particular, yeast SPT3 has been shown to interact directly with TBP. Here we report the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. Amino acid sequence comparisons between human SPT3 (hSPT3) and its counterparts in different yeast species reveal three highly conserved domains, with the most conserved 92-amino acid N-terminal domain being 25% identical with human TAFII18. Despite the significant sequence similarity with TAFII18, native hSPT3 is not a bona fide TAFII because it is not associated in vivoeither with human TBP/TFIID or with a TFIID-related TBP-free TAFII complex. However, we present evidence that hSPT3 is associated in vivo with TAFII31 and the recently described longer form of human GCN5 (hGCN5-L) in a novel human complex that has histone acetyltransferase activity. We propose that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is a likely homologue of the yeast SAGA complex. A human SPT3-TAFII31-GCN5-L acetylase complex distinct from transcription factor IID.Journal of Biological ChemistryVol. 273Issue 42PreviewPage 23783, line 14: Substitute the words “immunoprecipitation (IP)” for “inositol phosphate.” Full-Text PDF Open Access suppressor of Ty TATA-binding protein transcription factor IIA FLAG epitope-tagged transcription factor IID SPT-ADA-GCN5 acetyltransferase p300/CBP-associated factor TBP-associated factor TBP-free TAFII complex expressed sequence tag histone acetyltransferase SPT3-TAFII31-GCN5-L acetyltransferase polyacrylamide gel electrophoresis. Yeast SPT (suppressors ofTy)1 genes, including SPT3, encode global transcription regulators and were originally identified in a genetic screen for mutations that suppress transcriptional defects caused by the insertion of the retrotransposon Ty or its long terminal repeat, δ, in the promoter region of several genes (for a review see Ref. 1Winston F. McKnight S.L. Yamamoto K.R. Transcriptional Regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 1271-1293Google Scholar). This approach also identified the gene encoding the yeast TATA-binding protein (TBP) asSPT15 (2Eisenmann D.M. Dollard C. Winston F. Cell. 1989; 58: 1183-1191Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 3Hahn S. Buratowski S. Sharp P.A. Guarente L. Cell. 1989; 58: 1173-1181Abstract Full Text PDF PubMed Scopus (152) Google Scholar). However, in contrast to SPT15, SPT3 is not essential for yeast viability. Genetic and biochemical analyses have shown that SPT3 and TBP interact in yeast (4Eisenmann D.M. Arndt K.M. Ricupero S.L. Rooney J.W. Winston F. Genes Dev. 1992; 6: 1319-1331Crossref PubMed Scopus (190) Google Scholar), and mutations in SPT3, SPT7, and SPT8, as well as particular mutations in TBP/SPT15, all result in a common set of phenotypes that include slow growth and defects in mating and sporulation (2Eisenmann D.M. Dollard C. Winston F. Cell. 1989; 58: 1183-1191Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 5Winston F. Durbin K.J. Fink G.R. Cell. 1984; 39: 675-682Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 6Winston F. Dollard C. Malone E.A. Clare J. Kapakos J.G. Farabaugh P. Minehart P.L. Genetics. 1987; 115: 649-656Crossref PubMed Google Scholar). Accordingly, deletion of the SPT3 gene in yeast results in gene-selective RNA polymerase II transcription defects (5Winston F. Durbin K.J. Fink G.R. Cell. 1984; 39: 675-682Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 6Winston F. Dollard C. Malone E.A. Clare J. Kapakos J.G. Farabaugh P. Minehart P.L. Genetics. 1987; 115: 649-656Crossref PubMed Google Scholar, 7Hirschhorn J.N. Winston F. Mol. Cell. Biol. 1988; 8: 822-827Crossref PubMed Scopus (18) Google Scholar). The mechanisms for the gene-specific functions of SPT3 are still poorly understood but may include core promoter-selective functions of SPT3 in TATA box selection. Indeed, SPT3 has been proposed to facilitate TBP recruitment to weak TATA-containing or TATA-less promoters in yeast (4Eisenmann D.M. Arndt K.M. Ricupero S.L. Rooney J.W. Winston F. Genes Dev. 1992; 6: 1319-1331Crossref PubMed Scopus (190) Google Scholar,8Collart M. Mol. Cell. Biol. 1996; 16: 6668-6676Crossref PubMed Scopus (101) Google Scholar). Consistent with this notion, TFIIA overexpression in yeast partially suppresses an spt3Δ mutation, and spt3Δ/toa1 (TFIIA) double mutants are inviable (9Madison J.M. Winston F. Mol. Cell. Biol. 1997; 17: 287-295Crossref PubMed Scopus (102) Google Scholar). More recently, yeast SPT3 has been shown to be part of the 1.8-MDa multiprotein yeast SAGA (SPT-ADA-GCN5acetyltransferase) complex that also contains SPT7, SPT8, SPT20/ADA5, and the coactivators/adaptors ADA1, ADA2, ADA3, and GCN5 (10Grant P.A. Duggan L. Côté J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (887) Google Scholar, 11Horiuchi J. Silverman N. Piña B. Marcus G.A. Guarente L. Mol. Cell. Biol. 1997; 17: 3220-3228Crossref PubMed Scopus (99) Google Scholar). Altogether these observations suggest an important role for SPT3 (as well as SPT7, SPT8, and SPT20) in linking core promoter-specific functions (e.g. stabilization of TBP/TFIID-DNA interactions) in vivo to upstream activators through an adaptor/coactivator complex(es) with histone acetyltransferase activity. Recently, putative human homologues of components of the yeast SAGA complex have been isolated. These include hADA2 (12Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (159) Google Scholar) and three human GCN5 acetyltransferase family members: PCAF (p300/CBP-associatedfactor) (13Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar), a short 55-kDa hGCN5 (hGCN5-S) (12Candau R. Moore P.A. Wang L. Barlev N. Ying C.Y. Rosen C.A. Berger S.L. Mol. Cell. Biol. 1996; 16: 593-602Crossref PubMed Scopus (159) Google Scholar, 13Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar), and a long 93-kDa hGCN5 (hGCN5-L) (14Smith E.R. Belote J.M. Schiltz R.L. Yang X.-J. Moore P.A. Berger S.L. Nakatani Y. Allis C.D. Nucleic Acids Res. 1998; 26: 2948-2954Crossref PubMed Scopus (92) Google Scholar). The short and long hGCN5 forms are produced from the same gene, presumably by alternatively spliced mRNAs. The longer hGCN5-L contains a 361-amino acid N-terminal domain (the PCAF homology domain) that is absent in hGCN5-S and yGCN5. This domain shares significant homology with the corresponding 351-amino acid N-terminal domain of PCAF that interacts with the coactivator p300/CBP (13Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar, 14Smith E.R. Belote J.M. Schiltz R.L. Yang X.-J. Moore P.A. Berger S.L. Nakatani Y. Allis C.D. Nucleic Acids Res. 1998; 26: 2948-2954Crossref PubMed Scopus (92) Google Scholar). Here we describe the molecular cloning of a cDNA encoding a human homologue of yeast SPT3. We present evidence for a specific association in vivo of human SPT3 with TAFII31 (TBP-associatedfactor II 31) and hGCN5-L in a novel human complex that is distinct from TFIID and that has histone acetyltransferase activity with preference for histone H3. Our results together with those just reported by Ogryzko et al. (15Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar) suggest that the human SPT3-TAFII31-GCN5-L acetyltransferase (STAGA) complex is one of perhaps several distinct human homologues of the yeast SAGA complex. A search of the GenBankTM EST division with the yeast SPT3 sequence revealed a human EST sequence (N89343) 36% identical with yeast SPT3 amino acids 7–47 and a mouse EST sequence (W71809) 42% identical with yeast SPT3 amino acids 44–88. A human SPT3 (hSPT3) cDNA fragment was obtained from a Marathon-Ready HeLa cDNA library (CLONTECH) by nested PCR using degenerate primers in the mouse 3′-end EST sequence and primers in the human 5′-end EST sequence. Rapid amplification of cDNA ends and high fidelity PCR with cloned Pfu polymerase (Stratagene) were then used to obtain, from the same library, the full-length hSPT3 cDNA. The sequence was confirmed from at least two independent clones. The hSPT3 cDNA sequence has been deposited in GenBankTM with the accession number AF073930. For efficient expression of full-length recombinant hSPT3 protein in bacteria, hSPT3 cDNA nucleotides 120–128 (GGA AGG AGT; 3 codons for Gly, Arg, and Ser, respectively) were recoded to GGTCGTTCT (the silent changes are underlined) to remove a fortuitous bacterial ribosome binding site. The recoded hSPT3 cDNA, which also contained a newly created NdeI site at the first methionine and a BamHI site insertion after the natural stop codon at position 1031, was inserted between the NdeI and BamHI sites of 6hisT-pET11d (16Hoffmann A. Roeder R.G. Nucleic Acids Res. 1991; 19: 6337-6338Crossref PubMed Scopus (250) Google Scholar) to obtain the bacterial expression vector pET-6His-hSPT3. A human multiple tissue Northern blot (CLONTECH) was probed with32P-labeled cDNA probes. The hSPT3 cDNA probe was a PCR fragment from nucleotides 360–811. Human cDNAs for TAFII150 2E. Martinez, H. Ge, Y. Tao, C.-X. Yuan, V. Palhan, and R. G. Roeder, submitted for publication. and β-actin (CLONTECH) were used as reference probes on the same blot (after stripping it). Human HeLa cell derivatives stably expressing FLAG-tagged human TBP (3Hahn S. Buratowski S. Sharp P.A. Guarente L. Cell. 1989; 58: 1173-1181Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 4Eisenmann D.M. Arndt K.M. Ricupero S.L. Rooney J.W. Winston F. Genes Dev. 1992; 6: 1319-1331Crossref PubMed Scopus (190) Google Scholar, 5Winston F. Durbin K.J. Fink G.R. Cell. 1984; 39: 675-682Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 6Winston F. Dollard C. Malone E.A. Clare J. Kapakos J.G. Farabaugh P. Minehart P.L. Genetics. 1987; 115: 649-656Crossref PubMed Google Scholar, 7Hirschhorn J.N. Winston F. Mol. Cell. Biol. 1988; 8: 822-827Crossref PubMed Scopus (18) Google Scholar, 8Collart M. Mol. Cell. Biol. 1996; 16: 6668-6676Crossref PubMed Scopus (101) Google Scholar, 9Madison J.M. Winston F. Mol. Cell. Biol. 1997; 17: 287-295Crossref PubMed Scopus (102) Google Scholar, 10Grant P.A. Duggan L. Côté J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (887) Google Scholar) (17Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar) and human TAFII1002 have been described. The human cell line stably expressing FLAG-tagged human TAFII135 will be described elsewhere. 3Y. Tao and R. G. Roeder, unpublished data. Nuclear extracts were prepared as described previously (18Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Rabbit polyclonal antibodies against hSPT3 (No. 623) were raised (Covance) against a bacterially expressed insoluble recombinant 6-His-tagged hSPT3 protein fragment (amino acids 1–285) that was purified on Ni2+-NTA-agarose (Qiagen) and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and excision of the protein band. Rabbit polyclonal antibodies against human TBP (19Hoffmann A. Roeder R.G. J. Biol. Chem. 1996; 271: 18184-18202Google Scholar), TAFII31 (20Hisatake K. Ohta T. Takada R. Guermah M. Horikoshi M. Nakatani Y. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8195-8199Crossref PubMed Scopus (70) Google Scholar), the short form of hGCN5 (13Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar, 14Smith E.R. Belote J.M. Schiltz R.L. Yang X.-J. Moore P.A. Berger S.L. Nakatani Y. Allis C.D. Nucleic Acids Res. 1998; 26: 2948-2954Crossref PubMed Scopus (92) Google Scholar), and the N-terminal domain of PCAF (13Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar, 14Smith E.R. Belote J.M. Schiltz R.L. Yang X.-J. Moore P.A. Berger S.L. Nakatani Y. Allis C.D. Nucleic Acids Res. 1998; 26: 2948-2954Crossref PubMed Scopus (92) Google Scholar) were described previously. Rabbit polyclonal antibodies against human TAFII135 will be described elsewhere.3 Monoclonal anti-FLAG M2 antibody-agarose was from Kodak-IBI. Purification of FLAG epitope-tagged TBP-containing TFIID (eTFIID) from nuclear extracts of the 3-10 cell line was as described previously (17Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). For immunoprecipitations antibodies were cross-linked to protein A-agarose with dimethylpimelimidate (Sigma). The antibody resin (10–50 μl) was mixed with nuclear extracts (400–500 μl) for 5–12 h at 4 °C in binding Buffer C (BC) (20 mm Tris-HCl, pH 7.9, 20% glycerol, 0.2 mmEDTA, 0.05% Nonidet P-40, 8 mm 2-mercaptoethanol, 0.2 mm phenylmethylsulfonyl fluoride) containing 150 mm KCl (BC150) or up to 300 mm KCl (BC300) as indicated. The immune complexes were recovered by low speed centrifugation, and the resin was washed extensively with binding buffer and with BC100 and then eluted with either 20 mmTris-HCl (pH 8.0) containing 2% SDS or with 0.2 mg/ml FLAG peptide as described previously (17Chiang C.-M. Ge H. Wang Z. Hoffmann A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). Western blot analyses were performed by standard procedures and with the ECL detection system (Amersham). HeLa cell nuclear pellets (18Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar) were used to purify core histones. The nuclear pellet (5 ml) was homogenized with a blender in 40 ml of buffer A (0.1m potassium phosphate, pH 6.7, 0.1 mm EDTA, 10% glycerol, 0.1 mm phenylmethylsulfonyl fluoride, 0.1 mm dithiothreitol) containing 0.63 m NaCl and centrifuged in a Ti45 rotor (Beckman) at 25,000 rpm at 4 °C. The supernatant was mixed and incubated at 4 °C with 18 ml of preswollen Bio-Gel-HTP resin (DNA grade, Bio-Rad) for 3 h. The resin was packed into an econo column (Bio-Rad) and washed extensively (0.5 column volume/h, overnight) with buffer A containing 0.63 mNaCl. Core histones were eluted with buffer A containing 2m NaCl and dialyzed first against buffer B (10 mm potassium phosphate, pH 6.7, 150 mm KCl, 10% glycerol) for 3 h and then against a buffer containing 20 mm Tris-HCl (pH 7.9), 100 mm KCl, 20% glycerol, and 0.1 mm dithiothreitol for 3 h. For the inositol phosphate-HAT assays immunoprecipitations were performed in BC200 as described above, except that BC100 was replaced with HAT assay buffer (50 mm Tris-HCl, pH 8.0, 70 mm KCl, 10% glycerol, 0.1 mm EDTA, 0.05% Nonidet P-40, 10 mm sodium butyrate, 1 mm 2-mercaptoethanol, 0.2 mm phenylmethylsulfonyl fluoride) in the final washes of the immune complexes that were then used directly for the HAT assays. The HAT assays were performed at 30 °C for 30 min in HAT assay buffer in a final volume of 25 μl and contained 10–15 μl of resin-immune complexes (or either 10 ng of recombinant PCAF or 50 ng of recombinant p300 HAT domain), 1.6 μg of purified HeLa core histones, and 125 nCi of [3H]acetyl-CoA (3.8 Ci/mmol, 250 μCi/ml). The reactions were then either analyzed by SDS-PAGE and Coomassie staining followed by fluorography for 16–24 h at −70 °C or spotted directly onto Whatman P-81 filters that were then washed with 50 mm sodium carbonate buffer (pH 9.2) and counted in a liquid scintillator. Recombinant FLAG-tagged p300 HAT domain (1195–1810) was expressed in bacteria and purified as reported previously (21Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar). Recombinant human PCAF was a kind gift from Y. Nakatani. Because of the important role of SPT3 in the regulation of TBP/TFIID functions in a core promoter-specific manner in yeast, and because of the core promoter-specific functions of both yeast and human TFIID/TAFIIs (for reviews see Refs. 22Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 23Verrijzer C.P. Tjian R. Trends Biochem. Sci. 1996; 21: 338-342Crossref PubMed Scopus (319) Google Scholar, 24Tansey W.P. Herr W. Cell. 1997; 88: 729-732Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), we searched for a potential human homologue of yeast SPT3. A Blast alignment (25Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60216) Google Scholar) of GenBankTM data base sequences with the yeast (Saccharomyces cerevisiae) SPT3 (ySPT3) protein sequence identified two overlapping mouse and human EST sequences that together encoded a hypothetical protein with significant identity to ySPT3 amino acids 7–88 (see “Experimental Procedures”). This information allowed us to clone by PCR a full-length human cDNA of 1,165 nucleotides, including part of the poly(A) tract, that encodes a 317-amino acid protein with a calculated molecular weight of 35,790 and 27% overall sequence identity to ySPT3 (Fig. 1, A and C). This suggests that this cDNA encodes a human homologue of ySPT3 that will be referred to hereafter as hSPT3. A multiple tissue Northern blot analysis revealed a specific 1.4-kilobase hSPT3 mRNA that is approximately the size of the cloned cDNA and is expressed in all human tissues tested in a manner similar to the ubiquitously expressed β-actin and hTAFII150 mRNAs (Fig. 1 B). Interestingly, a longer and less abundant 2.5-kilobase mRNA with a more restricted tissue distribution was also detected (Fig. 1 B, SPT3 2.5 kilobases), suggesting the possible existence of an additional longer tissue-specific variant of hSPT3. A Blast alignment of the hSPT3 protein sequence with the protein sequences in the data bases retrieved all the cloned SPT3genes of various yeast species as well as hTAFII18 and itsS. cerevisiae homologue FUN81/yTAFII19 (Fig. 1 C). The interspecies SPT3 alignments presented in Fig. 1 C and schematized in Fig. 1 D reveal a high degree of conservation between human and yeast SPT3 in three domains (A, B, and C) that most likely reflects functional conservation. Interestingly, the 92-amino acid domain A of hSPT3 is 38% identical to the yeast SPT3 A domains and 25% identical to human TAFII18 and its yeast homologue FUN81 (Fig. 1 D). This strongly suggests that the SPT3 domain A may fold in a structure similar to TAFII18 and may have related functions. Since hTAFII18 has been shown to interact directly with hTAFII28 and hTAFII30 (26Mengus G. May M. Jacq X. Staub A. Tora L. Chambon P. Davidson I. EMBO J. 1995; 14: 1520-1531Crossref PubMed Scopus (108) Google Scholar), the hSPT3 domain A also may serve as a TAFII-interacting surface, possibly also contacting hTAFII28 and/or hTAFII30. No homologies with other known proteins in the data bases were found for the less conserved domains B and C, suggesting that these regions may perform SPT3-specific functions. Interestingly, the SPT3 glutamic acid that is mutated to a lysine in the strongest yeast SPT3 suppressor mutant of thespt15-21 (TBP mutant) phenotype (4Eisenmann D.M. Arndt K.M. Ricupero S.L. Rooney J.W. Winston F. Genes Dev. 1992; 6: 1319-1331Crossref PubMed Scopus (190) Google Scholar) is conserved in domain B between human and all yeast SPT3 proteins (Fig. 1 C, E→K). This may indicate a possible function of domain B in direct interactions with TBP. The above observations suggested that hSPT3 may interact with TFIID through direct contacts with either TBP, as in the case of its yeast counterpart, or TAFIIs. We addressed this by testing for the presence of hSPT3 in highly purified human TFIID and by analyzing the physiological interacting partners of human SPT3 in HeLa cells. Highly purified eTFIID was shown to lack any detectable hSPT3 by immunoblot analyses (Fig. 2 A, lane 2), whereas a specific 37-kDa hSPT3 protein was detected in the crude HeLa nuclear extract (lane 1). Immunopurification of eTFIID through its FLAG-tagged TBP subunit was performed after two chromatographic steps, including a high salt (0.85 m KCl) elution from phosphocellulose P11. Therefore, it remained possible that the resins or high salt could have disrupted a potential interaction between hSPT3 and TFIID and/or that the FLAG epitope at the N terminus of TBP might have interfered with hSPT3 association with eTFIID. To further address this issue we performed direct immunoprecipitations both from nuclear extracts of cells expressing FLAG-tagged TAFII100 (f:TAFII100) and TAFII135 (f:TAFII135) and from nuclear extracts of normal HeLa cells with, respectively, antibodies against the FLAG epitope and against native TAFII31 and TBP. Anti-TBP (Fig. 2 A, lane 6), anti-f:TAFII100 (lane 3), and anti-f:TAFII135 (lane 4) immunoprecipitations all failed to co-precipitate hSPT3, in agreement with the above results, whereas they efficiently precipitated TBP and TAFIIs. These results demonstrate that hSPT3 is not a component of the human TFIID complex and that it is unlikely to be part of any other TAFIIcomplex containing TAFII100 and/or TAFII135, such as, for instance, the recently described TBP-free TAFII complex TFTC (27Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (229) Google Scholar). While these results do not exclude direct physical interactions between hSPT3 and TBP/TFIID, as suggested previously by co-immunoprecipitations of overexpressed ySPT3 and yTBP in yeast cells and by corresponding genetic interactions (4Eisenmann D.M. Arndt K.M. Ricupero S.L. Rooney J.W. Winston F. Genes Dev. 1992; 6: 1319-1331Crossref PubMed Scopus (190) Google Scholar), they do emphasize that in human cells native hSPT3 does not efficiently interact with TBP/TFIID in solution. Thus, a more efficient interaction may require the presence of additional components, such as promoter DNA and associated general transcription factors and/or activators. Interestingly, however, under the same conditions anti-TAFII31 antibodies efficiently immunoprecipitated hSPT3 in addition to TFIID components (Fig. 2 A, lane 5; Fig. 2 B, lane 3), suggesting that in human cells hSPT3 and TAFII31 are in association in a complex distinct from TFIID (and TFTC). This was further confirmed by immunoprecipitations with anti-hSPT3 antibodies that also coprecipitated TAFII31 but not TBP (Fig. 2 B, lane 4), TAFII18, TAFII80, TAFII100, or TAFII135 (data not shown). Because yeast SPT3 is associated with GCN5 histone acetyltransferase in the SAGA complex (10Grant P.A. Duggan L. Côté J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (887) Google Scholar), we tested whether the hSPT3-TAFII31 complex also has histone acetyltransferase (HAT) activity. Fig. 3 A shows that immune complexes obtained with both anti-hSPT3 and anti-TAFII31 have significant HAT activity when compared with mock (protein A resin alone) or anti-TBP immunoprecipitates. To address the type of HAT involved we compared the pattern of core histone acetylation by the hSPT3-TAFII31 complex with that of PCAF and p300. The results presented in Fig. 3 B indicate that immune complexes obtained with anti-hSPT3 (lane 5) and anti-TAFII31 (lane 6) both preferentially acetylate histone H3. This suggests that the HAT associated with hSPT3 and TAFII31 is different from p300, which acetylates all core histones with a preference for H3 and H4 (21Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar) (lane 7), and more related to the human GCN5 family member PCAF (lane 2). In accord with this, and while this manuscript was being prepared, we learned that immunoprecipitations of ectopic FLAG-tagged PCAF and FLAG-tagged hGCN5-S from HeLa cell lines stably overexpressing these HAT factors also coprecipitated hSPT3 and TAFII31, as well as TAFII20, TAFII30, and additional proteins that include novel TAFII-related factors (15Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Interestingly, however, we did not find significant amounts of PCAF (Fig. 3 C, lane 6 in top panel) or hGCN5-S (lane 6 in bottom panel) in our immunoprecipitated complexes. Instead, we detected predominantly the recently described long form (hGCN5-L) of hGCN5 (Fig. 3 C, lane 6 in third panel from thetop; and Fig. 3 D, lanes 2–5). The reason for the apparent absence of PCAF and hGCN5-S in our human SPT3-TAFII31-GCN5-L acetylase (STAGA) complex is not clear. However, this most likely results from the different immunoprecipitation approaches used here and in the recent study by Ogryzko et al. (15Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). One possibility is that our anti-hSPT3 and anti-TAFII31 antibodies, including different antibodies against the N-terminal and C-terminal regions of TAFII31 (data not shown), all dissociated PCAF and hGCN5-S, but not hGCN5-L, from the STAGA complex(es). Another interesting and more likely possibility is that the PCAF, hGCN5-S, and STAGA complexes are distinct and differ with respect to their associated HAT subunits (and perhaps other components as well) and their relative abundance in HeLa cells. Indeed, this is also suggested by the clear indication that the composition of the PCAF and hGCN5-S complexes are indistinguishable except for the corresponding overexpressed HAT subunits (15Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). Related to this and in accord with a recent report (14Smith E.R. Belote J.M. Schiltz R.L. Yang X.-J. Moore P.A. Berger S.L. Nakatani Y. Allis C.D. Nucleic Acids Res. 1998; 26: 2948-2954Crossref PubMed Scopus (92) Google Scholar), our results indicate that PCAF is apparently not very abundant in HeLa cells, given the difficulty in detecting it in crude nuclear extracts by immunoblot analyses that efficiently detect the recombinant PCAF protein (Fig. 3 C, lane 2 in top panel). It also is important to note that in contrast to the analyses of Ogryzko et al. (15Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), our immunoprecipitation analyses were performed with antibodies against two different native subunits of the STAGA complex that were not overexpressed. Hence, we propose from these results that hSPT3 and TAFII31 are predominantly associated with hGCN5-L in HeLa cells. In conclusion, our finding that human SPT3 exists in a novel in vivo complex (STAGA) with TAFII31 and the recently described hGCN5-L histone acetyltransferase (14Smith E.R. Belote J.M. Schiltz R.L. Yang X.-J. Moore P.A. Berger S.L. Nakatani Y. Allis C.D. Nucleic Acids Res. 1998; 26: 2948-2954Crossref PubMed Scopus (92) Google Scholar) demonstrates the existence of TAFIIs in complexes distinct from TFIID and the recently described TFTC (27Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (229) Google Scholar). This is in accord with the very recent complementary findings of TAFIIs within the yeast SAGA complex (28Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates III, J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar) and in the human PCAF and hGCN5-S complexes (15Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). It also suggests a possible diversity of human homologues of the yeast SAGA complex that differ (at least in part) by the associated HAT subunit and perhaps also by their relative abundance/activity in different tissues. This is also supported by the very recent study on PCAF and hGCN5-S complexes (15Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar) and by the observed higher steady state PCAF mRNA levels in muscle as compared with other tissues (13Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar). The future structural and functional characterizations of these human complexes, in vitro and in vivo, will provide important new insights into the mechanisms that control promoter-targeted chromatin modifications and that coordinate the transcription regulation of a selected group of genes during development, cell proliferation, and differentiation. We thank Y. Nakatani for the p300 HAT domain expression vector, recombinant PCAF, antibodies against PCAF and GCN5-S, and for sharing unpublished data. We also thank Y. Tao for antibodies and extracts from the f:TAFII135 cell line and M. Guermah for antibodies against hTAFII31.