Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

TATA-binding Protein-free TAF-containing Complex (TFTC) and p300 Are Both Required for Efficient Transcriptional Activation

2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês

10.1074/jbc.m205860200

ISSN

1083-351X

Autores

Sara Hardy, Marjorie Brand, Gerhard Mittler, Jun Yanagisawa, Shigeaki Kato, Michael Meisterernst, Làszlò Tora,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HAT-containing coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process. Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HAT-containing coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process. TATA-binding protein TBP-associated factor transcription factor TBP-free TAF complex histone acetyltransferase Spt-Ada-GcN5-acetyltransferase complex (alteration/deficiency inactivation human transformation/transcription domain-associated protein CREB-binding protein activation domain antisense human immunodeficiency virus glutathione S-transferase p300/CBP-associated protein Transcription initiation of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, which comprises the TATA-binding protein (TBP)1 and a series of TBP-associated factors (TAFs) (1Bell B. Tora L. Exp. Cell Res. 1999; 246: 11-19Crossref PubMed Scopus (109) Google Scholar, 2Albright S.R. Tjian R. Gene. 2000; 242 (1–2): 1-13Crossref PubMed Scopus (272) Google Scholar, 3Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (193) Google Scholar). However, we have previously shown that initiation of polymerase II transcription can occur in a TFIID-independent manner in the presence of a novel human (h) multiprotein complex, termed TFTC for TBP-free TAF complex (4Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (226) Google Scholar). TFTC is able to direct preinitiation complex assembly from different TATA box-containing and TATA-less promoters in vitro on naked DNA templates. TFTC contains no TBP but is composed of several TAFs and other proteins that have been shown to mediate transcriptional activation or are important in correct initiation site selection (4Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (226) Google Scholar,5Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). The three-dimensional structure of TFTC resembles a macromolecular clamp that contains five globular domains organized around a solvent-accessible groove of a size suitable to bind DNA (6Brand M. Leurent C. Mallouh V. Tora L. Schultz P. Science. 1999; 286: 2151-2153Crossref PubMed Scopus (101) Google Scholar). A large number of recent studies have provided a direct molecular link between histone acetylation and transcriptional activation (reviewed in Refs. 7Kuo M.H. Allis C.D. Bioessays. 1998; 20: 615-626Crossref PubMed Scopus (1068) Google Scholar and 8Brown C.E. Lechner I. Howe I. Workman J.L. Trends. Biochem. Sci. 2000; 25: 15-19Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). In these reports, it has been shown that several previously identified co-activators of transcription possess intrinsic HAT activity. Among these co-activators are yeast Gcn5 (9Brownell J.E. Zhou J. Ranalli T. Kobayashi R. Edmondson D.G. Roth S.Y. Allis C.D. Cell. 1996; 84: 843-851Abstract Full Text Full Text PDF PubMed Scopus (1284) Google Scholar), human GCN5 (10Candau 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), PCAF (11Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar), TATA box-binding protein-associated factor 250 (TAF1; formerly TAFII250) (12Mizzen C.A. Yang X.J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar), p300/CBP (13Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2395) Google Scholar), ACTR (14Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar), and steroid receptor co-activator 1 (SRC-1) (15Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1065) Google Scholar). Many of these chromatin-modifying activities have been found within large multiprotein complexes that also contain several components with homology or identity to known transcriptional regulators. InSaccharomyces cerevisiae the co-activator protein Gcn5 is part of two large multisubunit complexes, the 1.8–2-MDa SAGA complex and the 0.8-MDa ADA (alteration/deficiency inactivation) complex (16Grant P.A. Duggan L. Cote 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 (881) Google Scholar). Yeast SAGA, similar to the TFTC-type complexes, comprises products of at least four distinct classes of genes: (i) the Ada proteins (yAda1, yAda2, yAda3, yGcn5 (yAda4), and yAda5 (ySpt20); (ii) the TBP-related set of Spt proteins (ySpt3, ySpt7, ySpt8, and ySpt 20); (iii) a subset of TAFs, including scTAF5, scTAF6 scTAF9, scTAF10, and scTAF12; and (iv) the product of the essential gene Tra1, which has been shown to be a component of SAGA (8Brown C.E. Lechner I. Howe I. Workman J.L. Trends. Biochem. Sci. 2000; 25: 15-19Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) (for new TAF names see Ref. 3Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (193) Google Scholar). The yeast SAGA complex has been shown to mediate activation by the acidic activators yGcn4 and VP16 and to potentiate transcription activation in an acetyl coenzyme A (acetyl-CoA)-dependent manner on chromatin templates in vitro, whereas the ADA complex failed to do so (17Utley 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, 18Ikeda K. Steger D.J. Eberharter A. Workman J.L. Mol. Cell. Biol. 1999; 19: 855-863Crossref PubMed Scopus (142) Google Scholar, 19Wallberg A.E. Neely K.E. Gustafsson J.A. Workman J.L. Wright A.P. Grant P.A. Mol. Cell. Biol. 1999; 19: 5952-5959Crossref PubMed Scopus (60) Google Scholar, 20Brown C.E. Howe L. Sousa K. Alley S.C. Carrozza M.J. Tan S. Workman J.L. Science. 2001; 292: 2333-2337Crossref PubMed Scopus (293) Google Scholar). Mammalian homologues of yGCN5 include PCAF and GCN5 (11Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 21Xu W. Edmondson D.G. Roth S.Y. Mol. Cell. Biol. 1998; 18: 5659-5669Crossref PubMed Scopus (136) Google Scholar). In human (h) cells a number of GCN5- and PCAF-containing multiprotein complexes have been characterized: such as TFTC (5Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), the PCAF and GCN5 complexes (22Ogryzko V.V. Kotani T. Zhang X. Schlitz 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 the SPT3-TAFII31-GCN5 acetyltransferase complex (STAGA) (23Martinez E. Kundu T.K., Fu, J. Roeder R.G. J. Biol. Chem. 1998; 273: 23781-23785Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 24Martinez E. Palhan V.B. Tjernberg A. Lymar E.S. Gamper A.M. Kundu T.K. Chait B.T. Roeder R.G. Mol. Cell. Biol. 2001; 21: 6782-6795Crossref PubMed Scopus (321) Google Scholar), that all contain either GCN5(L) or PCAF as catalytic HAT subunit, as well as the human ADA proteins hSPTs, hTAFs, and hTRRAP. TRRAP was originally isolated as a Myc-associated transcriptional co-activator (25McMahon S.B. Van Buskirk H.A. Dugan K.A. Copeland T.D. Cole M.D. Cell. 1998; 94: 363-374Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). The SAGA, TFTC, PCAF/GCN5, and STAGA HAT complexes preferentially acetylate histone H3 in both a free and a nucleosomal context (5Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 22Ogryzko V.V. Kotani T. Zhang X. Schlitz 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, 24Martinez E. Palhan V.B. Tjernberg A. Lymar E.S. Gamper A.M. Kundu T.K. Chait B.T. Roeder R.G. Mol. Cell. Biol. 2001; 21: 6782-6795Crossref PubMed Scopus (321) Google Scholar, 26Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Although the human TFTC, PCAF/GCN5, and STAGA complexes share several subunits, they are not identical (5Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 24Martinez E. Palhan V.B. Tjernberg A. Lymar E.S. Gamper A.M. Kundu T.K. Chait B.T. Roeder R.G. Mol. Cell. Biol. 2001; 21: 6782-6795Crossref PubMed Scopus (321) Google Scholar, 27Brand M. Moggs J.G. Oulad-Abdelghani M. Lejeune F. Dilworth F.J. Stevenin J. Almouzni G. Tora L. EMBO J. 2001; 20: 3187-3196Crossref PubMed Scopus (177) Google Scholar), suggesting the existence of overlapping but also different functions between these complexes. Moreover, a TFTC-type HAT complex was shown to be required as a co-factor for nuclear receptor function both in vitro and in vivo (28Yanagisawa J. Kitagawa H. Yanagida M. Wada O. Ogawa S. Nakagomi M. Oishi H. Yamamoto Y. Nagasawa H. McMahon S.B. Cole M.D. Tora L. Takahashi N. Kato S. Mol Cell. 2002; 9: 553-562Abstract Full Text PDF PubMed Scopus (146) Google Scholar). CBP and p300 are distinct but functionally related co-activator proteins with intrinsic HAT activities, involved in both proliferative and differentiating pathways (Ref. 29Goodman R.H. Smolik S. Genes Dev. 2000; 14: 1553-1577PubMed Google Scholar and references therein). CBP/p300 efficiently acetylate the N-terminal tails of the four histones, however with a preference for histones H3 and H4 as compared with H2A and H2B (13Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2395) Google Scholar, 30Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1533) Google Scholar). In addition to modifying histones, CBP/p300 proteins have been shown to acetylate non-histone proteins including transcriptional activators, general transcription factors, and chromatin-associated proteins (31Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1402) Google Scholar). The fact that that TFTC (i) mediates transcriptional initiation and activation on naked DNA templates, (ii) contains the hGCN5 HAT as well as several human homologues of yeast SAGA subunits that have been shown to be important for transcriptional activation and correct initiation site selection in different genetic screens, (iii) preferentially acetylates histone H3 on chromatin templates (4Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (226) Google Scholar, 5Brand M. Yamamoto K. Staub A. Tora L. J. Biol. Chem. 1999; 274: 18285-18289Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), and (iv) is required as a co-activator for nuclear receptor function (28Yanagisawa J. Kitagawa H. Yanagida M. Wada O. Ogawa S. Nakagomi M. Oishi H. Yamamoto Y. Nagasawa H. McMahon S.B. Cole M.D. Tora L. Takahashi N. Kato S. Mol Cell. 2002; 9: 553-562Abstract Full Text PDF PubMed Scopus (146) Google Scholar) prompted us to analyze in further details the function of TFTC in activated transcription on chromatin templates. We describe herein the direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, and we show that p300 and CBP interact only with the H2 subdomain of VP16. Using cell transfection experiments we demonstrate the requirement of both CBP/p300 and TFTC for efficient transcriptional activation. Moreover, we report that on an in vitro reconstituted chromatin template human TFTC mediates the transcriptional activity of the VP16 activation domain (AD) in concert with p300 and in an acetyl-CoA-dependent manner. Altogether our results suggest that the two HAT-containing co-activators, p300/CBP and TFTC, play complementary roles during transcriptional activation. The eukaryotic expression plasmids for wild type E1A, E1AΔN mutant, and the E1A-CR2mut, AS-TRRAP, have been described previously (28Yanagisawa J. Kitagawa H. Yanagida M. Wada O. Ogawa S. Nakagomi M. Oishi H. Yamamoto Y. Nagasawa H. McMahon S.B. Cole M.D. Tora L. Takahashi N. Kato S. Mol Cell. 2002; 9: 553-562Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 32Reid J.L. Bannister A.J. Zegerman P. Martinez-Balbas M.A. Kouzarides T. EMBO J. 1998; 17: 4469-4477Crossref PubMed Scopus (108) Google Scholar, 33Kretsovali A. Agalioti T. Spilianakis C. Tzortzakaki E. Merika M. Papamatheakis J. Mol. Cell. Biol. 1998; 18: 6777-6783Crossref PubMed Scopus (154) Google Scholar, 34Paulson M. Press C. Smith E. Tanese N. Levy D.E. Nat. Cell Biol. 2002; 4: 140-147Crossref PubMed Scopus (93) Google Scholar). The 17M/ERE-Glob-Luciferase reporter plasmid has been described elsewhere (35Balaguer P. Boussioux A.M. Demirpence E. Nicolas J.C. Luminescence. 2001; 16: 153-158Crossref PubMed Scopus (85) Google Scholar). The hGCN5 cDNA was cloned into the pcDNA3 vector (Invitrogen) in an antisense orientation to generate pcDNA-AS-GCN5. The expression plasmids to produce either the different GST-VP16 fusion proteins (see Fig.1C) or the mammalian expression plasmids producing GAL-VP16 and its derivatives have been described previously (36Ikeda K. Stuehler T. Meisterernst M. Genes Cells. 2002; 7: 49-58Crossref PubMed Scopus (38) Google Scholar). 2 × 105 HeLa cells were cotransfected by calcium phosphate precipitation in 6-well dishes. Total amounts of DNA were adjusted by supplementing with an empty vector up to 5 μg/well. Routinely 300 ng reporter plasmid was used with 20 ng of GAL-VP16 expression vector, or with its derivatives, and with the indicated amounts of the other expression vectors (see also the legend to Fig.2). Cell culture and growth conditions as well as the luciferase assay have been described (37Bertolotti A. Bell B. Tora L. Oncogene. 1999; 18: 8000-8010Crossref PubMed Scopus (70) Google Scholar). For the luciferase assays, the same amount of protein was taken from each transfection. Similar results were obtained in at least three independent transfections. GST-pull down assays were carried out as described (36Ikeda K. Stuehler T. Meisterernst M. Genes Cells. 2002; 7: 49-58Crossref PubMed Scopus (38) Google Scholar). Routinely proteins were boiled in SDS sample buffer and separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with the indicated primary antibodies. Chemiluminescence detection was performed according to manufacturer's instructions (Amersham Biosciences, Inc.). The anti-TRRAP antisera (25McMahon S.B. Van Buskirk H.A. Dugan K.A. Copeland T.D. Cole M.D. Cell. 1998; 94: 363-374Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 38Herceg Z. Hulla W. Gell D. Cuenin C. Lleonart M. Jackson S. Wang Z.Q. Nat. Genet. 2001; 29: 206-211Crossref PubMed Scopus (105) Google Scholar), anti-SPT3, anti-GCN5, and anti-SAP130 antibodies (27Brand M. Moggs J.G. Oulad-Abdelghani M. Lejeune F. Dilworth F.J. Stevenin J. Almouzni G. Tora L. EMBO J. 2001; 20: 3187-3196Crossref PubMed Scopus (177) Google Scholar), anti-TBP, anti-TAF10, anti-TAF5, and anti-TAF6 monoclonal antibodies (4Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (226) Google Scholar), anti-Med-6 (kind gift from R. Kornberg), anti-Med 7 (kind gift from D. Reinberg), anti-CBP, anti-p300, anti-TRAP240, and anti-TRAP95, anti-SPT6 (Santa Cruz) have been described previously. For the chromatin assembly the pIC-2085S/G5-E4R plasmid (39Steger D.J. Workman J.L. Methods. 1999; 19: 410-416Crossref PubMed Scopus (33) Google Scholar) was digested with HaeIII and Asp718 to generate a 1241-bp fragment and incubated with HeLa histones octamers (1:1 molar ratio between one histone octamer and one nucleosome binding site). The chromatin template was assembled by salt dilution (39Steger D.J. Workman J.L. Methods. 1999; 19: 410-416Crossref PubMed Scopus (33) Google Scholar). Nucleosomal assembly was confirmed by MNase digestion. Chromatin template (250 ng of DNA) was digested with 2 milliunits of micrococcal nuclease (Sigma) in buffer B (10 mm Hepes, pH 7.6, 50 mm KCl, 5% glycerol, 10 mm sodium butyrate, 5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 0.25 mg/ml bovine serum albumin) containing 3 mm CaCl2 for 20 s to 3 min at 25 °C. DNA was precipitated, run on a 1.3% agarose gel, and either visualized by ethidium bromide staining or transferred to a nylon membrane (Hybond, Amersham Biosciences, Inc.) and hybridized with a 32P-labeled probe (Fig. 3B). Nuclear extract preparations, in vitro transcription reactions, and primer extensions were carried out as described previously (40Brou C. Chaudhary S. Davidson I. Lutz Y., Wu, J. Egly J.M. Tora L. Chambon P. EMBO J. 1993; 12: 489-499Crossref PubMed Scopus (151) Google Scholar, 41Steger D.J. Owen-Hughes T. John S. Workman J.L. Methods. 1997; 12: 276-285Crossref PubMed Scopus (17) Google Scholar). GAL1–147, GAL-VP16, TFTC, and p300 were purified as described previously (4Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (226) Google Scholar, 42Brou C., Wu, J. Ali S. Scheer E. Lang C. Davidson I. Chambon P. Tora L. Nucleic Acids Res. 1993; 21: 5-12Crossref PubMed Scopus (59) Google Scholar, 43Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (202) Google Scholar). About 20 ng of chromatin-assembled E4 template was preincubated with the indicated factors (Fig. 3) in the presence or absence of 2 μmacetyl-CoA in a 20-μl volume of buffer B. After 40 min of preincubation at 30 °C, 30 μl of buffer T (30 mmHepes, pH 7.8, 60 mm KCl, 12 mmMgCl2, 4% PVA (30–70), 10 mm sodium butyrate, 12 ng/μl poly(dI·dC)) was added to the reactions together with 2 ng of pHIV-1 plasmid as an internal control (41Steger D.J. Owen-Hughes T. John S. Workman J.L. Methods. 1997; 12: 276-285Crossref PubMed Scopus (17) Google Scholar) and 30 μg of HeLa nuclear extract. Transcription reactions were started by the addition of the four rNTPs (10 mm) at time 0. Reactions were stopped after a 45-min incubation at 30 °C with the S buffer (300 mm NaCl, 20 mm EDTA, 1% SDS, 50 ng/μl tRNA). Correctly initiated transcripts were detected by primer extension using a 32P-labeled probe corresponding to the complementary positions of +86 to +110 of the E4 transcript (39Steger D.J. Workman J.L. Methods. 1999; 19: 410-416Crossref PubMed Scopus (33) Google Scholar). First we wanted to examine whether VP16 AD could directly recruit TFTC. Thus, we tested whether TFTC would bind to the activation domain of VP16 when fused to GST. The GST-VP16 fusion protein was immobilized on glutathione-agarose resin in parallel with GST alone, as a control (Fig. 1A). Highly purified TFTC was able to bind to the GST-VP16 fusion protein but not to the GST alone (compare lanes 2 and 3). This is in accordance with the fact that a TFTC-like complex was previously purified by its direct association with the liganded estrogen receptor (28Yanagisawa J. Kitagawa H. Yanagida M. Wada O. Ogawa S. Nakagomi M. Oishi H. Yamamoto Y. Nagasawa H. McMahon S.B. Cole M.D. Tora L. Takahashi N. Kato S. Mol Cell. 2002; 9: 553-562Abstract Full Text PDF PubMed Scopus (146) Google Scholar) and that the related STAGA complex was able to interact directly with the VP16 activator (24Martinez E. Palhan V.B. Tjernberg A. Lymar E.S. Gamper A.M. Kundu T.K. Chait B.T. Roeder R.G. Mol. Cell. Biol. 2001; 21: 6782-6795Crossref PubMed Scopus (321) Google Scholar). As it has also been shown that the VP16 AD functions in in vitro transcription systems through direct interactions with p300/CBP and the Mediator complex (TRAP/DRIP/SMCC/ARC) (36Ikeda K. Stuehler T. Meisterernst M. Genes Cells. 2002; 7: 49-58Crossref PubMed Scopus (38) Google Scholar, 43Kraus W.L. Manning E.T. Kadonaga J.T. Mol. Cell. Biol. 1999; 19: 8123-8135Crossref PubMed Scopus (202) Google Scholar, 44Kundu T.K. Palhan V.B. Wang Z., An, W. Cole P.A. Roeder R.G. Mol. Cell. 2000; 6: 551-561Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and that these factors can interact directly with the VP16 AD from crude extracts, we tested whether TFTC could also bind directly to the GST-VP16 in parallel with the Mediator complex and/or p300/CBP from a HeLa cell nuclear extract. Similar to the results obtained with purified TFTC (Fig. 1A), TFTC components such as TRRAP, GCN5, SAP130, TAF5 (formerly TAFII100 (3Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (193) Google Scholar), TAF6 (formerly TAFII80), and TAF10 (formerly TAFII30) were detected in the high salt elution from the VP16 column (Fig1B, lane 6). Moreover, as previously reported, other known VP16-interacting proteins such as CBP, p300, MED6 and MED7, TRAP240, and TRAP95 were also present in the elution, whereas hSPT6, another nuclear protein, did not bind to the VP16 column (Fig.1B, lane 6, and data not shown). It has also been reported that the activation domain of VP16 can be subdivided into two regions, the N-terminal region, H1 (amino acids 411–452), and the C-terminal, region H2 (amino acids 453–490), both of which independently activate transcription (45Triezenberg S.J. Kingsbury R.C. McKnight S.L. Genes Dev. 1988; 2: 718-729Crossref PubMed Scopus (593) Google Scholar, 46Walker S. Greaves R. O'Hare P. Mol. Cell. Biol. 1993; 13: 5233-5244Crossref PubMed Scopus (63) Google Scholar). The Mediator complex binds to both regions, whereas CBP only binds to the H2 subregion (36Ikeda K. Stuehler T. Meisterernst M. Genes Cells. 2002; 7: 49-58Crossref PubMed Scopus (38) Google Scholar). These observations prompted us to further analyze the regions of VP16 that interact with TFTC and p300. The VP16 H1 and H2 regions were expressed separately as GST fusion proteins, and as controls functional mutations of VP16/H1 (F442P) and VP16/H2 regions (F473A, F475A and F479A) were also expressed as GST fusions (calledGST-H1mut and GST-H2mut in Fig. 1C). TFTC subunits, i.e. TRRAP, GCN5, SAP130, TAF5, TAF6, and TAF10, were specifically recruited by both the GST-H1 and the GST-H2 regions, similar to the Mediator complex subunits MED6, MED7, TRAP240, and TRAP95. These interactions are specific because none of these factors were bound to either the GST-H1mut or GST-H2mut columns (Fig.1B, lanes 2–4). Interestingly, p300 was recruited by the GST-H2 region, but not by GST-H1, in good agreement with the previous finding that CBP binds only to the VP16 H2 region (Fig. 1B, lanes 2–4) (36Ikeda K. Stuehler T. Meisterernst M. Genes Cells. 2002; 7: 49-58Crossref PubMed Scopus (38) Google Scholar). Strikingly, under the same conditions, TBP did not interact significantly, or only very weakly, with either the H1 or H2 subdomain of VP16. Altogether these results show that the two subdomains of VP16 AD are able to specifically recruit the TFTC and the Mediator complexes from nuclear extracts, whereas CBP and p300 interact only with the H2 region. The fact that p300 and CBP bind only to the H2 subdomain but TFTC and the Mediator bind to both H1 and H2 subdomains, suggests also that the binding of p300 and/or CBP to the H2 subdomain can occur in the absence of TFTC and/or the Mediator, and vice versa, TFTC and the Mediator binding to an activation (sub)domain does not necessarily require p300 and/or CBP. To study the role of TFTC and p300/CBP in the activation of transcription mediated by GAL-VP16, we used mammalian cell-based transfection experiments. Consistent with previous reports (47Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (889) Google Scholar), GAL-VP16 strongly stimulated transcription from the rabbit β-globin promoter, which contains one GAL4 binding site fused to a luciferase reporter in HeLa cells (Fig.2A). When a vector expressing an antisense region of either TRRAP or GCN5 mRNA (TRRAP AS and GCN5 AS, respectively), we observed an important (dose-dependent) decrease in the GAL-VP16 activation potential (Fig. 2A, and data not shown). This decrease was paralleled by the reduction of the amount of endogenous cellular TRRAP or GCN5 protein levels as detected by Western blot (Fig.1D). These results indicate that in HeLa cells normal levels of TRRAP or GCN5 proteins are needed for full activation by GAL-VP16. Interestingly, the co-transfection of TRRAP AS with GCN5 AS did not further reduce activation by GAL-VP16, suggesting that both AS constructions inhibit the same step in the activation pathway. The expression of the transcriptional repressor E1A (12S) (48Jones N. Curr. Top. Microbiol. Immunol. 1995; 199: 59-80PubMed Google Scholar, 49Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Crossref PubMed Scopus (490) Google Scholar, 50Bannister A.J. Kouzarides T. EMBO J. 1995; 14: 4758-4762Crossref PubMed Scopus (319) Google Scholar), which has been proposed to inhibit the activity of CBP/p300, PCAF, or GCN5, also efficiently diminished the transcriptional activation by GAL-VP16 (Fig. 2A), in accordance with Ikeda et al. (36Ikeda K. Stuehler T. Meisterernst M. Genes Cells. 2002; 7: 49-58Crossref PubMed Scopus (38) Google Scholar). In contrast to the results obtained by co-transfection of TRRAP AS and GCN5 AS, co-transfection of TRRAP AS together with the E1A expression plasmid cooperatively reduced activation by GAL-VP16, suggesting that TRRAP AS and E1A affect independent interactions (or processes) in the mechanism of VP16-mediated activation of transcription. As TFTC binds to both the H1 and H2 VP16 activation domains (Fig.1C), and as it has been described that E1A inhibits only the activation by the H2 activation domain of VP16 (36Ikeda K. Stuehler T. Meisterernst M. Genes Cells. 2002; 7: 49-58Crossref PubMed Scopus (38) Google Scholar), we tested whether TRRAP AS and GCN5 AS would inhibit the activity of H1 or H2 domains of VP16. As shown in Fig. 2, B and C, both TRRAP AS and GCN5 AS inhibit activation by H1 as well as by H2; however, again no cooperativity in the inhibition was observed. These data further underline the above observations, suggesting that both AS constructions may impair the function of the same complex and thus inhibit the same step in the VP16 activation pathway. The N-terminal 20 amino acids of E1A and a portion of conserved region 1 (CR1) where shown to be responsible for p300/CBP and PCAF binding (11Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1317) Google Scholar, 48Jones N. Curr. Top. M

Referência(s)