The VP16 Activation Domain Interacts with Multiple Transcriptional Components as Determined by Protein-Protein Cross-linking in Vivo
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m208911200
ISSN1083-351X
Autores Tópico(s)RNA Research and Splicing
ResumoTranscriptional activator proteins recruit the RNA polymerase II machinery and chromatin-modifying activities to promoters. Biochemical experiments indicate that activator proteins can associate with a large number of proteins, and many such proteins have been proposed to be direct targets of activators. However, there is great uncertainty about which biochemical interactions are physiologically relevant. Here, we develop a formaldehyde-based cross-linking procedure to identify protein-protein interactions that occur under physiological conditions. We show that the VP16 activation domain directly interacts with TATA-binding protein (TBP), TFIIB, and the SAGA histone acetylase complex in vivo. Transcriptional activator proteins recruit the RNA polymerase II machinery and chromatin-modifying activities to promoters. Biochemical experiments indicate that activator proteins can associate with a large number of proteins, and many such proteins have been proposed to be direct targets of activators. However, there is great uncertainty about which biochemical interactions are physiologically relevant. Here, we develop a formaldehyde-based cross-linking procedure to identify protein-protein interactions that occur under physiological conditions. We show that the VP16 activation domain directly interacts with TATA-binding protein (TBP), TFIIB, and the SAGA histone acetylase complex in vivo. Transcriptional activator proteins regulate the expression of eukaryotic genes in response to developmental and environmental cues. Such activator proteins contain a DNA-binding domain that recognizes specific promoter DNA sequences and a physically separate transcriptional activation region that stimulates mRNA initiation by RNA polymerase II (1Brent R. Ptashne M. Cell. 1985; 43: 729-736Abstract Full Text PDF PubMed Scopus (435) Google Scholar, 2Hope I.A. Struhl K. Cell. 1986; 46: 885-894Abstract Full Text PDF PubMed Scopus (549) Google Scholar, 3Ma J. Ptashne M. Cell. 1987; 48: 847-853Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 4Hope I.A. Mahadevan S. Struhl K. Nature. 1988; 333: 635-640Crossref PubMed Scopus (220) Google Scholar). Activation domains are functionally autonomous; they function when fused to heterologous DNA-binding domains tethered at different positions in the promoter region. The best characterized activation domains are defined by short acidic regions that show little primary sequence homology (5Ptashne M. Nature. 1988; 335: 683-689Crossref PubMed Scopus (1176) Google Scholar, 6Struhl K. Ann. Rev. Biochem. 1989; 58: 1051-1077Crossref PubMed Scopus (206) Google Scholar). Mutational analysis indicates that acidic and hydrophobic residues within these domains contribute to functional activity, although individual residues make only a minor contribution (4Hope I.A. Mahadevan S. Struhl K. Nature. 1988; 333: 635-640Crossref PubMed Scopus (220) Google Scholar, 7Gill G. Ptashne G. Cell. 1987; 51: 121-126Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 8Cress W.D. Triezenberg S.J. Science. 1991; 251: 87-90Crossref PubMed Scopus (324) Google Scholar, 9Regier J.L. Shen F. Triezenberg S.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 883-887Crossref PubMed Scopus (226) Google Scholar, 10Drysdale C.M. Duenas E. Jackson B.M. Reusser U. Braus G.H. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 1220-1233Crossref PubMed Google Scholar). Acidic activation domains do not have a defined tertiary structure but become structured only upon specific interaction with another protein (11Shen F. Triezenberg S.J. Hensley P. Porter D. Knutson J.R. J. Biol. Chem. 1996; 271: 4827-4837Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 12Uesugi M. Nyanguile O. Lu H. Levine A.J. Verdine G.L. Science. 1997; 277: 1310-1313Crossref PubMed Scopus (273) Google Scholar). Taken together, these observations indicate that acidic activation domains are surfaces used to mediate protein-protein interactions. It is presumed that other types of activation domains, such as those rich in glutamine (13Courey A.J. Tjian R. Cell. 1988; 55: 887-898Abstract Full Text PDF PubMed Scopus (1079) Google Scholar) or proline (14Mermod N. O'Neill E.A. Kelly T.J. Tjian R. Cell. 1989; 58: 741-753Abstract Full Text PDF PubMed Scopus (542) Google Scholar) residues, function in a similar manner. Chromatin immunoprecipitation experiments indicate that, in vivo, activator proteins mediate the recruitment of the Pol II machinery and chromatin-modifying activities (e.g. the Swi/Snf nucleosome remodeling complex and the SAGA and NuA4 histone acetylase complexes) to promoters (15Kuras L. Struhl K. Nature. 1999; 399: 609-612Crossref PubMed Scopus (399) Google Scholar, 16Li X.-Y. Virbasius A. Zhu X. Green M.R. Nature. 1999; 389: 605-609Crossref Scopus (206) Google Scholar, 17Cosma M.P. Tanaka T. Nasmyth K. Cell. 1999; 97: 299-311Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 18Kuras L. Kosa P. Mencia M. Struhl K. Science. 2000; 288: 1244-1248Crossref PubMed Scopus (147) Google Scholar, 19Li X.-Y. Bhaumik S.R. Green M.R. Science. 2000; 288: 1242-1244Crossref PubMed Scopus (158) Google Scholar, 20Reid J.L. Iyer V.R. Brown P.O. Struhl K. Mol. Cell. 2000; 6: 1297-1307Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). However, such experiments do not define the direct targets of activator proteins. In yeast cells, individual components of the Pol II machinery associate with promoters in a mutually interdependent manner (15Kuras L. Struhl K. Nature. 1999; 399: 609-612Crossref PubMed Scopus (399) Google Scholar, 16Li X.-Y. Virbasius A. Zhu X. Green M.R. Nature. 1999; 389: 605-609Crossref Scopus (206) Google Scholar), and direct connection of a DNA-binding domain to virtually any component of the Pol II machinery suffices for transcription (21Struhl K. Cell. 1996; 84: 179-182Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 22Ptashne M. Gann A. Nature. 1997; 386: 569-577Crossref PubMed Scopus (941) Google Scholar). Thus, activator-dependent recruitment of the Pol II machinery to promoters in vivo could be due to a direct contact to any component of the Pol II machinery. In addition, activator-dependent recruitment of the Pol II machinery could be an indirect consequence of activator-dependent changes in chromatin structure. Activator-dependent recruitment of Swi/Snf and SAGA can occur even when the Pol II machinery is not associated with promoters (17Cosma M.P. Tanaka T. Nasmyth K. Cell. 1999; 97: 299-311Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar, 23Bhaumik S.R. Green M.R. Genes Dev. 2001; 15: 1935-1945Crossref PubMed Scopus (238) Google Scholar, 24Larschan E. Winston F. Genes Dev. 2001; 15: 1946-1956Crossref PubMed Scopus (244) Google Scholar), consistent with the idea that activators directly interact with these chromatin-modifying complexes. However, chromatin immunoprecipitations experiments are inherently unable to determine which components of the Pol II machinery or which chromatin-modifying activities directly interact with activator proteins in vivo. In vitro, transcriptional activators can interact with TATA-binding protein (TBP) 1The abbreviations used for: TBP, TATA-binding protein; TAFs, TBP-associated factors; HA, hemagglutinin. (25Stringer K.F. Ingles C.J. Greenblatt J. Nature. 1990; 345: 783-786Crossref PubMed Scopus (408) Google Scholar, 26Ingles C.J. Shales M. Cress W.D. Triezenberg S.J. Greenblatt J. Nature. 1991; 351: 588-590Crossref PubMed Scopus (227) Google Scholar, 27Lee W.S. Kao C.C. Bryant G.O. Liu X. Berk A.J. Cell. 1991; 67: 367-376Google Scholar), TBP-associated factors (TAFs) (28Goodrich J.A. Hoey T. Thut C.J. Admon A. Tjian R. Cell. 1993; 75: 519-530Abstract Full Text PDF PubMed Scopus (351) Google Scholar, 29Thut C.J. Chen J.L. Klemm R. Tjian R. Science. 1995; 267: 100-104Crossref PubMed Scopus (407) Google Scholar), TFIIA (30Kobayashi N. Boyer T.G. Berk A.J. Mol. Cell. Biol. 1995; 15: 6465-6473Crossref PubMed Scopus (135) Google Scholar), TFIIB (31Lin Y.-S. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1991; 353: 569-571Crossref PubMed Scopus (261) Google Scholar), TFIIH (32Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Crossref PubMed Scopus (329) Google Scholar), components of the mediator subcomplex of RNA polymerase II holoenzyme (33Hengartner C.J. Thompson C.M. Zhang J. Chao D.M. Liao S.-M. Koleske A.J. Okamura S. Young R.A. Genes Dev. 1995; 9: 897-910Crossref PubMed Scopus (189) Google Scholar, 34Natarajan K. Jackson B.M. Zhou H. Winston F. Hinnebusch A.G. Mol. Cell. 1999; 4: 657-664Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 35Lee Y.C. Park J.M. Min S. Han S.J. Kim Y.J. Mol. Cell. Biol. 1999; 19: 2967-2976Crossref PubMed Scopus (133) Google Scholar, 36Myers L.C. Kornberg R.D. Annu. Rev. Biochem. 2000; 69: 729-749Crossref PubMed Scopus (321) Google Scholar), Swi/Snf (34Natarajan K. Jackson B.M. Zhou H. Winston F. Hinnebusch A.G. Mol. Cell. 1999; 4: 657-664Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 37Yudkovsky N. Logie C. Hahn S. Peterson C.L. Genes Dev. 1999; 13: 2369-2374Crossref PubMed Scopus (188) Google Scholar, 38Neely K.E. Hassan A.H. Wallberg A.E. Steger D.J. Cairns B.R. Wright A.P. Workman J.L. Mol. Cell. 1999; 4: 649-655Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), SAGA (39Drysdale C.M. Jackson B.M. Klebanow E.R. Bai Y. Kokubo T. Swanson M. Nakatani Y. Weil A. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 1711-1724Crossref PubMed Scopus (84) Google Scholar, 40Utley R.T. Ikeda K. Grant P.A. Cote J. Steger D.J. Eberharter A. John S. Workman J.L. Nature. 1998; 394: 498-502Crossref PubMed Scopus (446) Google Scholar), and NuA4 (40Utley R.T. Ikeda K. Grant P.A. Cote J. Steger D.J. Eberharter A. John S. Workman J.L. Nature. 1998; 394: 498-502Crossref PubMed Scopus (446) Google Scholar). However, it is generally not understood which of these interactions occur under physiological conditions and are relevant for transcriptional activation in vivo. In many cases, activator-target interaction experiments are performed under very artificial conditions. For example, standard GST pulldown experiments involve very high concentrations of activation domains and potential targets, and the potential targets are often assayed as isolated proteins rather than multiprotein complexes that occur in cells. GST pulldowns, and other techniques such as co-immunoprecipitation and far-Western blotting, are prone to binding artifacts, and this is particularly likely for acidic activation domains, which are largely unstructured and have an abundance of negative and hydrophobic residues. For example, while the strength of biochemical interactions between activation domains and several potential targets strongly correlates with the transcriptional potency of the activation domain, this correlation is equally strong for activator binding to lysozyme, a protein that is clearly not a physiologically relevant target (41Melcher K. J. Mol. Biol. 2000; 301: 1097-1112Crossref PubMed Scopus (54) Google Scholar). Biochemical interactions between activators and potential targets have also been identified by photo-cross-linking. This approach has identified the Tra1 subunit of the SAGA complex (42Brown C.E. Howe L. Sousa K. Alley S.C. Carrrozza M.J. Tan S. Workman J.L. Science. 2001; 292: 2333-2337Crossref PubMed Scopus (293) Google Scholar), several subunits of the Swi/Snf complex (43Neely K.E. Hassan A.H. Brown C.E. Howe L. Workman J.L. Mol. Cell. Biol. 2002; 22: 1615-1625Crossref PubMed Scopus (144) Google Scholar), and the Srb4 subunit of the mediator complex (44Koh S.S. Ansari A.Z. Ptashne M. Young R.A. Mol. Cell. 1998; 1: 895-904Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) as direct targets of activation domains in vitro. In the case of Tra1, mutations that reduce the interaction with the activation domain without compromising the integrity of the SAGA complex show transcriptional activation defects in vivo. These experiments provide strong evidence that the Tra1 subunit of SAGA is a physiologically relevant target. However, there is no direct physical evidence for these activator-target interactionsin vivo. We wished to develop a new procedure that can detect protein-protein interactions inside living cells under physiological conditions. To that end, we utilized formaldehyde, which rapidly permeates the cell and generates protein-protein and protein-DNA cross-links. Proteins that are cross-linked to transcriptional activators are co-immunoprecipitated under stringent conditions and then identified by Western blotting after reversal of the formaldehyde cross-links. Kinetic experiments using formaldehyde cross-linking to measure protein-DNA association in vivo strongly suggest that formaldehyde inactivates cellular enzymes almost immediately upon addition to the growing cells and that the 20-min incubation time merely increases the cross-linking in fixed and metabolically inert cells (45Katan-Khaykovich Y. Struhl K. Genes Dev. 2002; 16: 743-752Crossref PubMed Scopus (169) Google Scholar, 46Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodriguez-Navarro S. Rondon A.G. Aguilera A.A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (641) Google Scholar, 47Proft M. Struhl K. Mol. Cell. 2002; 9: 1307-1317Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). As such, formaldehyde cross-linking is likely to provide a snapshot of protein-protein interactions at the particular time point. Here we use this technique to address whether TFIID, TFIIA, and TFIIB and the SAGA histone acetylase complex interact directly with activation domains in yeast cells. The plasmids listed in Fig. 1 were constructed by PCR amplification of the various segments and insertion into the indicated restriction sites of theLEU2 vector YCplac111 (48Gietz R.D. Sugino A. Gene. 1988; 74: 527-534Crossref PubMed Scopus (2522) Google Scholar). In addition, ApaI andSalI sites and a His6 tag were introduced between the NcoI site and the CYC1 termination domain. The activation domains described in Fig. 7 were cloned into this construct between the BamHI and NcoI sites (Hap4, Ace1, Adr1) or between the NcoI and SalI sites (Gcn4, Put3). The GAL-LacZ reporter plasmid pRY131 contains a 2μ origin of replication and a URA3marker (49Yocum R.R. Hanley S. West R. Ptashne M. Mol. Cell. Biol. 1984; 4: 1985-1998Crossref PubMed Scopus (233) Google Scholar). All yeast strains were derived from a Research Genetics strain (record number 11044) with a gal4 deletion (MATα his3−Δ1 leu2-Δ0lys2-Δ0 ura3-Δ0gal4::KAN. Yeast strains expressing 3-Myc-tagged proteins from the normal genomic locus were obtained by using gene-specific PCR primers to amplify derivatives of pMPY-3xMYC DNA, introducing the resultant PCR fragment into yeast cells by one-step integration, followed by looping out of the URA3marker (50Schneider B.L. Seufert W. Steiner B. Yang Q.H. Futcher A.B. Yeast. 1995; 11: 1265-1274Crossref PubMed Scopus (290) Google Scholar). pMPY-3xMYC DNA was modified by inserting theCYC1 termination region (246 bp) into the BamHI site (pDH035 for C-terminal tagging) or insertion of theADH1 (1200 bp) or TEF1 (400 bp) promoter into theEcoRI site (pDH036 and pDH037 for N-terminal tagging); these modifications maintain stable expression of the target yeast protein before looping out of the URA3 marker. All proteins were tagged at the C terminus except for TBP, TAF8, Spt7, Spt20, and Tra1. Yeast strains bearing SAGA deletions (ada2, no. 4282;spt3, no. 4228; spt20, no. 7390) were obtained from Research Genetics and were derived from BY4741 (MAT a his3-Δ1 leu2-Δ0met15-Δ0 ura3-Δ0). Strains containing the desired Myc-tagged protein, SAGA deletion, and gal4 deletion were generated by mating and tetrad dissection.Figure 7Cross-linking of various activation domains to TAF12 and TBP. Immunoprecipitations were performed with the α-HA antibody on cross-linked samples from strains expressing Gal4 derivatives containing the indicated activation domains (and control proteins lacking the epitope tag or activation domain) as well as Myc-tagged TBP. The resulting samples were analyzed as described in Fig. 2. The levels of transcriptional activation (β-galactosidase units) for each derivative are indicated.View Large Image Figure ViewerDownload (PPT) Cells were grown in 200 ml of synthetic complete medium lacking uracil and leucine to OD = 0.4, and then CuSO4 (1 mm) was added for 1.5 h. A 37% solution of formaldehyde (5.4 ml) was added directly to the culture to bring the final concentration to 1%. After 20 min, cross-linking was quenched by addition of 2 m glycine (60 ml). The cells were harvested, washed with 400 ml of cold Tris-buffered saline followed by 40 ml of cold lysis buffer (50 mm HEPES-KOH, pH 7.5, 150 mm NaCl, 10 mm EDTA, 5 mm EGTA, and 1% Triton X-100). Cells were resuspended in 1 ml of lysis buffer containing 1 mm phenylmethylsulfonyl fluoride, 2 mmbenzamidine, 1× CompleteTM protease inhibitor mixture (Roche Molecular Biochemicals). To this suspension, 1.5 ml of zirconia/silica beads (0.5 mm, BioSpec Products) was added, and then the cells were disrupted on a Mini-Beadbeater (BioSpec Products) with 6 pulses (1 min each) at full power with icing between cycles. The mixture was transferred to a Falcon tube and separated from the beads by centrifugation through a needle hole into a 30-ml tube. The beads were further washed with 3 ml of lysis buffer plus protease inhibitors. The lysate was then sonicated with 4 × 30-s pulses (Branson Ultrasonics Sonifier Model 450, 50% pulses, power = 7) and centrifuged for 20 min at 32,000 × g. The supernatant was transferred to a 15-ml tube taking care that no solid material from the pellet was dislodged. α-HA monoclonal antibody (12CA5) was coupled to protein A-Sepharose beads (Amersham Biosciences) with dimethyl pimelimidate. In the case of Western blots involving proteins larger than 100 kDa, the antibody was not covalently coupled to the resin due to high molecular mass background. For each reaction, 30 μl of ascites fluid and 60 μl of beads were incubated with the supernatant for 1 h at 4 °C. The beads were transferred to a 2-ml column and washed 4 × 3 ml with lysis buffer with 0.1% sodium deoxycholate and 0.1% SDS, 2 × 3 ml of the same buffer but with 500 mm NaCl, 2 × 3 ml of 10 mm Tris-HCl, pH 8.0, 0.25 m LiCl, 1 mm EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, and 2 × 3 ml of TE (10 mm Tris-HCl, pH 8.0, 1 mm EDTA). The beads were then transferred to a 1.5-ml microcentrifuge tube with TE and pelleted. The TE was removed to dryness with a 25-gauge needle. The immunoprecipitated material was removed by incubating the beads in 60 μl of 50 mmTris-HCl, pH 7.5, 10 mm EDTA, 1% SDS at 65 °C for 10 min. The beads were removed by filtration, and 5× SDS-PAGE buffer was added to the eluted material. After reversing the cross-links by boiling for 20 min, SDS-PAGE gels were run using 3 μl of immunoprecipitated material for α-HA Western blots, 15 μl for α-TAF61, and 30 μl for α-Myc. The α-TAF61 and α-Myc Western blots were detected with SuperSignal® West Femto Substrate (Pierce). The yeast strains in these experiments express the chimeric activator Gal4-VP16, which consists of the Gal4 DNA-binding domain fused to the VP16 transcriptional activation domain, or related proteins that contain mutated forms of either domain (Fig.1 A). To facilitate immunopurification, each protein also contains three copies of the HA epitope at the N terminus (51Field J. Nikawa J.-I. Broek D. MacDonald B. Rodgers L. Wilson I.A. Lerner R.A. Wigler M. Mol. Cell. Biol. 1988; 8: 2159-2165Crossref PubMed Scopus (731) Google Scholar). As high levels of Gal-VP16 are toxic to yeast cells (52Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Crossref PubMed Scopus (986) Google Scholar, 53Berger S.L. Pina B. Silverman N. Marcus G.A. Agapite J. Regier J.L. Triezenberg S.J. Guarente L. Cell. 1992; 70: 251-265Abstract Full Text PDF PubMed Scopus (355) Google Scholar), expression is controlled by a copper-inducible promoter that is essentially inactive in the absence of copper (54Klein C. Struhl K. Science. 1994; 266: 280-282Crossref PubMed Scopus (106) Google Scholar). The yeast strains also contain a multicopy LacZ reporter plasmid to monitor the transcriptional activity as well as to provide additional DNA binding sites from which to activate transcription. Within the range of copper tolerated by the cells, the level of transcription continuously increased, and no toxicity was observed (Fig. 1 B). To generate protein-protein cross-linking in vivo, copper-induced cells were treated with 1% formaldehyde for 20 min. Gal4-VP16 derivatives and cross-linked proteins were immunopurified from cell-free extracts with antibodies against the HA epitope under stringent conditions. A control Western blot with the antibody against the HA epitope verifies that equal amounts of each fusion protein were immunoprecipitated (Fig. 1 C). Western blots of the immunoprecipitated samples using an antibody against TAF12 (previously known as TAF68 or TAF61) (55Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (193) Google Scholar), a component of both TFIID and SAGA, reveals that TAF12 cross-links to Gal4-VP16 but not to the Gal4 DNA-binding domain alone (Fig. 1 C). VP16-TAF12 cross-linking depends on formaldehyde treatment (see Fig.6), and the level of cross-linking is proportional to the amount of activator expressed (data not shown). DNase I treatment of the cell-free extract destroys the DNA but does not affect the level of the observed VP16-TAF12 cross-link (data not shown), indicating that observed interactions in vivo reflect protein-protein cross-linking that are not mediated through the DNA. Under the conditions shown here, we estimate that ∼1% of the total TAF12 is cross-linked to the VP16 activation domain. The level of cross-linking is correlated with the functional quality of the VP16 activation domain, because the Gal4-VP16-F442P derivative shows reduced levels of activation and TAF12 cross-linking. However, VP16-dependent cross-linking occurs at the same level even upon deletion of the N-terminal 31 residues of Gal4, which disrupts the zinc-finger domain essential for DNA binding and hence prevents Gal4-dependent transcription in vivo. Thus, although cross-linking depends on the presence of the VP16 activation domain, it does not depend on a functional DNA-binding domain. To determine whether the VP16 activation domain cross-links to other potential target proteins in vivo, we generated an isogenic set of yeast strains in which individual proteins were tagged with the Myc epitope. In each case, following cross-linking, the immunoprecipitated material was analyzed with antibodies against the Myc epitope as well as the antibody against TAF12, which serves as an internal control to verify the cross-linking and co-immunoprecipitation procedure. As shown in Fig. 2, we observed a VP16-dependent cross-link to TBP and TFIIB, but to neither subunit of TFIIA. Next, we examined all 14 TAF components of TFIID (56Sanders S.L. Garbett K.A. Weil P.A. Mol. Cell. Biol. 2002; 16: 6000-6013Crossref Scopus (89) Google Scholar) for their ability to cross-link with the VP16 activation domain in vivo (Fig. 3). VP16-dependent cross-linking is observed with a number of TAFs, but only TAFs that are also present in the SAGA complex are cross-linked (TAF5, TAF6, TAF9, and TAF12; weak cross-linking to TAF10 is also observed).Figure 3The VP16 activation domain cross-links to multiple TAFs in the SAGA complex but not to TFIID-specific TAFs. Immunoprecipitations were performed with the α-HA antibody on cross-linked samples from strains expressing the Gal (–) or Gal-VP plasmid (+) as well as Myc-tagged versions of the indicated TAFs. The resulting samples were analyzed as described in Fig. 2.View Large Image Figure ViewerDownload (PPT) These results suggest that the VP16 activation domain interacts directly with TBP, TFIIB, and SAGA in vivo. Further support for the VP16 interaction with SAGA comes from the observation that many non-TAF components of SAGA (Ada1, Spt3, Spt7, Spt20, Tra1) also co-purify with VP16 following cross-linking. Some SAGA subunits (Ada2, Ada3, and Gcn5, which is responsible for the histone acetylase activity of the SAGA complex), are present in the related ADA complex (57Eberharter A. Sterner D.E. Schieltz D. Hassan A. Yates 3rd, J.R. Berger S.L. Workman J.L. Mol. Cell. Biol. 1999; 19: 6621-6631Crossref PubMed Scopus (149) Google Scholar). However, we did not detect VP16-dependent cross-linking to Ahc1, a subunit that is specific to the ADA complex (Fig. 4), suggesting that VP16 does not interact with ADA in vivo. The above results strongly suggest that the VP16 activation domain directly interacts with the SAGA complex and not TFIID in vivo. To demonstrate this directly, cross-linking was examined in strains deleted for individual genes encoding SAGA subunits. SAGA subunits have been categorized into three functional types based on genetic and biochemical observations (23Bhaumik S.R. Green M.R. Genes Dev. 2001; 15: 1935-1945Crossref PubMed Scopus (238) Google Scholar, 24Larschan E. Winston F. Genes Dev. 2001; 15: 1946-1956Crossref PubMed Scopus (244) Google Scholar, 58Roberts S.M. Winston F. Genetics. 1997; 147: 451-465Crossref PubMed Google Scholar, 59Sterner D.E. Grant P.A. Roberts S.M. Duggan L.J. Beloserkovskaya R. Pacella L.A. Winston F. Workman J.L. Berger S.L. Mol. Cell. Biol. 1999; 19: 86-98Crossref PubMed Scopus (289) Google Scholar, 60Dudley A.M. Rougeulle C. Winston F. Genes Dev. 1999; 13: 2940-2945Crossref PubMed Scopus (173) Google Scholar). Some subunits, such as Spt20, are required for the integrity of the SAGA complex, and hence all SAGA functions. Subunits such as Ada2 are required for histone acetylase activity and hence chromatin structure but are not required for certain transcriptional functions of SAGA. Conversely, subunits such as Spt3 are important for transcriptional functions that connect SAGA to the general transcription machinery, particularly TBP. We examined cross-linking of TBP, TFIIB, Ada1, Tra1, and TAF12 in wild-type and mutant strains representing each class of SAGA subunit (Fig. 5). For this experiment, the Gal4 and Gal4-VP16 proteins were expressed from the EFT2promoter, which is less sensitive to SAGA mutations than the copper-inducible promoter. VP16-dependent cross-linking of TAF12, which is present in both TFIID and SAGA, is essentially eliminated in the spt20 strain, in which the SAGA complex is completely disrupted. However, the amount of Gal4-VP16 cross-linking to TAF12 is only slightly reduced in the ada2 andspt3 strains. Similar results are observed for cross-linking to Ada1 and Tra1, although cross-linking to Ada1 is more sensitive to loss of Ada2 and Tra1 is more sensitive to deletion of Spt3. In contrast to these results with SAGA subunits, cross-linking to TBP and TFIIB is only slightly reduced in any of the mutant strains. The total cellular levels of these SAGA subunits as well as TBP and TFIIB are similar in wild-type and all three mutant strains (data not shown). These results strongly suggest that the VP16 activation interacts with SAGA but not TFIID in vivo. As formaldehyde is a rather nonspecific cross-linking agent and as SAGA is a very stable complex, our experimental procedure is likely to generate significant (and perhaps extensive) cross-linking between SAGA subunitsin vivo. Thus, it is difficult to determine whether an observed VP16-dependent interaction in vivoreflects a direct cross-link with the protein examined or is due to a network of protein-protein cross-links in which the protein examined is not a direct target. Because a majority of the SAGA subunits co-purify with Gal4-VP16 following cross-linking, we suspected that some of them might not be directly cross-linked to the VP16 activation domain. As an attempt to investigate this possibility, we carried out experiments at reduced concentrations of formaldehyde. We reasoned that reduced cross-linking efficiency would have less of an effect on direct targets of the VP16 activation domain (i.e. those requiring a single protein-protein cross-link) as opposed to SAGA components that do not contact the VP16 activation domain (i.e. those requiring multiple protein-protein cross-links). As expected, the level of cross-linking decreases as the formaldehyde concentration is reduced. However, as shown by a comparison to TAF12 in each case, a decrease in relative cross-linking efficiency was not observed for any of the six proteins tested (Fig. 6). We analyzed other activation domains for their ability to cross-link to TBP and TAF12 in vivo (Fig.7). Specifically, we analyzed these activation domains in the context of a Gal4 fusion protein in the same manner described for Gal4-VP16. While the activator proteins are expressed at different levels (assayed by the Western blotting with the HA antibody), the relative cross-linking of TBP and TAF12 can be compared among the activators. In this regard, it is interesting that the VP16, Gcn4, Put3, and Adr1 activation domains cross-link to both TBP and TAF12, whereas the Hap4 and Ace1 activation domain cross-link preferentially to TAF12. Protein-protein interactions are responsible for a great deal of biological specificity, but it has been difficult to identify such interactions under physiological conditions. Biochemical assays, by definition, are not performed under
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