letters to nature

21. Edison, A. S., Abildgaard, F., Westler, W. M., Mooberry, E. S. & Markley, J. L. Practical introduction to theory and implementation of multinuclear, multidimensional nuclear magnetic resonance experiments. Methods Enzymol. 239, 3–79 (1994). 22. Neri, D., Szyperski, T., Otting, G., Senn, H. & Wuthrich, K. Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28, 7510–7516 (1989). 23. Bru¨nger, A. T. X-PLOR manual (Yale Univ. Press, New Haven, CT, 1993). 24. Kuboniwa, H., Grzesiek, S., Delaglio, F. & Bax, A. Measurement of HN-H␣ J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods. J. Biomol. NMR 4, 871–878 (1994). 25. Spera, S. & Bax, A. Empirical correlation between protein backbone conformation and Ca and C␤ 13C nuclear magnetic resonance chemical shifts. J. Am. Chem. Soc. 117, 5491–5495 (1991). 26. Nilges, M., Clore, G. M. & Gronenborn, A. M. 1H-NMR stereospecific assignments by conformational data-base searches. Biopolymers 29, 813–822 (1990). 27. Laskowski, R. A., Rullman, J. A. C., MacArthur, M. W., Kaptein, R. & Thornton, J. M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–496 (1996). 28. Leonard, D. A., Evans, T., Hart, M., Cerione, R. A. & Manor, D. Investigation of the GTP-binding/ GTPase cycle of Cdc42Hs using fluorescence spectroscopy. Biochemistry 33, 12323–12328 (1994). 29. Carson, M. J. Ribbons 2.0. J. Appl. Crystallogr. 24, 958–961 (1991). 30. Nichols, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet. 11, 281–296 (1990).

Acknowledgements. We thank J. Kelly for participating in the sequential assignment of GDID59; L. E. Kay for discussion and for providing the NMR pulse sequences; B. A. Johnson and F. Delaglio for data analysis and processing software; D. Live for assistance with NMR data acquisition; J. Hubbard for computer system support and assistance with figure preparation; and Y. M. Chook and L. E. Kay for critical reading of the manuscript. This work was supported by a grant from the Society of the Memorial Sloan-Kettering Cancer Center.

Correspondence and requests for materials should be addressed to M.K.R. (e-mail: [email protected] ki.mskcc.org). Coordinates of the minimized average GDID59 structure and of the final ensemble of 20 structures have been deposited in the Brookhaven Protein Data Bank (accession numbers 1gdf and 1ajw, respectively).

Synergistic activation of transcription by CBP and p53

Wei Gu, Xiao-Lu Shi* & Robert G. Roeder Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA ...... The tumour suppressor p53 is a transcriptional regulator whose ability to inhibit cell growth is dependent upon its transactivation function1–3. Here we demonstrate that the transcription factor CBP, which is also implicated in cell proliferation and differentia- tion4–14, acts as a p53 coactivator and potentiates its transcrip- tional activity. The amino-terminal activation domain of p53 interacts with the carboxy-terminal portion of the CBP protein both in vitro and in vivo. In transfected SaoS-2 cells, CBP potentiates activation of the mdm-2 gene by p53 and, reciprocally, p53 potentiates activation of a Gal4-responsive target gene by a Gal4(1–147)–CBP(1678–2441) fusion protein. A double point mutation that destroys the transactivation function of p53 also abolishes its binding to CBP and its synergistic function with CBP. The ability of p53 to interact physically and functionally with a co- activator (CBP) that has histone acetyltransferase activity15,16 and with components (TAFs)17,18 of the general transcription machin- ery indicates that it may have different functions in a multistep activation pathway. CBP functions as a transcriptional coactivator through interac- tions with a number of cellular activators4–14 and, possibly, with components of the basal transcription machinery6. CBP has at least three domains that bind transcription factors: a region containing Figure 1 p53 association with CBP. a, Schematic representations of CBP and its residues 451–662 (designated CBP1) which is required for interac- functional/protein interaction domains, the hybrid activator Gal4–CBP(1678– tions with CREB (refs 6, 7), c-Jun (ref. 7) and c-Myb (ref. 11); a 2441), and the GST–CBP fusion proteins used for affinity chromatography. C/H: region containing residues 1,680–1,891 (designated CBP2) which is cysteine/histidine-rich region. b, Affinity purification of p53 on immobilized GST– essential for interactions with E1a (refs 8, 9), c-Fos (ref. 10) and CBP3. Western blot analysis with anti-p53 (antibody DO-1) of input HeLa nuclear TFIIB (ref. 6); and a carboxy-terminal region containing residues extract (0.5% of 4 mg total protein) and the total 1 M KCl eluates from the indicated 1,990–2,441 (designated CBP3) which is important for interactions GST–CBP affinity columns. c, d, Transactivation domain of 53 interacts with CBP with SRC-1 (ref. 12) and Jun-B (ref. 14) and which contains a and p300, respectively. Western blot analysis with anti-CBP3 (c) or anti-p300 (d) of input HeLa nuclear extract (5% of 1 mg total protein), and half of the total eluates from the GST, GST–p53 affinity columns and from the control column (the GST– * Present address: HHMI/Pharmacology Department, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, Texas 75235-9050, USA. p53 column without HeLa nuclear extract loading).

Nature © Macmillan Publishers Ltd 1997 NATURE | VOL 387 | 19 JUNE 1997 819 letters to nature transactivation domain6. These three domains were fused to GST CBP. Figure 3c confirms that overexpression of wild-type E1a can (Fig. 1a), expressed in Escherichia coli, and used as ligands for inhibit p53-mediated transactivation on the mdm-2 reporter con- affinity chromatography19. To search for proteins that interact with struct, whereas a CR1 region mutant of E1a (E1a(D64–68)), which CBP, the corresponding GST–CBP affinity columns and a GST is expressed at normal levels in the cell but is defective in binding to control column were loaded with equal amounts of HeLa nuclear CBP/p300 (refs 10, 24), is impaired in the repression. Taken extract. Bound proteins were then eluted with high salt, resolved by together, these data indicate that CBP functions as a coactivator SDS–PAGE, and analysed by silver staining and western blot to potentiate p53-dependent activation on a target gene and that analysis. This analysis (Fig. 1b) identified the tumour suppressor E1a-mediated repression of p53 transactivation functions may be p53 as one of the CBP-interacting proteins in nuclear extracts. Thus, mediated, at least in part, through sequestration of endogenous whereas p53 was barely detectable in a crude nuclear extract (Fig. 1b, CBP from p53. lane 1), consistent with its low level of expression in HeLa cells20, it According to our results (Fig. 2), p53 interacts both in vitro and in was enriched in the high-salt fraction from the GST–CBP3 column vivo with the C-terminal portion of CBP (Fig. 1a). This glutamine- (Fig. 1b, lane 3). As relatively little or no p53 was detected in the rich region has a transactivation function when fused to the DNA- eluates from the GST, GST–CBP1 or GST–CBP2 columns (Fig. 1b), binding domain of Gal4 (ref. 6). Cotransfection analysis revealed this result suggests that p53 specifically binds to GST–CBP3 with that p53 increases transactivation by Gal4–CBP(1678–2441) in a high affinity. dose-dependent manner, but has no effect on the transactivation This discovery of a CBP–p53 interaction was corroborated by a function of Gal4–VP16 (Fig. 4). Although conditions in this reciprocal experiment in which we investigated the binding of experiment were not physiological with respect to the way in nuclear extract proteins to a GST–p53 affinity column on which which p53 and CBP are normally anchored to the promoter, the the transactivation domain (residues 1–73) of p53 protein fused to data nevertheless indicate that tethering either CBP or p53 to the GST served as a ligand. The 265K CBP was detected in the high-salt DNA is sufficient for synergism. This also suggests a role for p53 in eluate from the GST–p53 column, but not in that from the GST transcriptional activation that is independent of its role in recruiting column (Fig. 1c). The CBP-related protein p300 (refs 8, 9) also CBP to the promoter. bound to GST–p53 in the same experiment (Fig. 1d), indicating Finally, as the transactivation domain of p53 is important for that there are specific interactions between the transactivation interaction with CBP (Fig. 1c), mutations in this region should help domain of p53 and CBP/p300. To determine whether p53 directly interacts with CBP itself, in vitro binding was assayed with bacterially produced p53 and GST– CBP proteins. As shown in Fig. 2a, 50% of input wild-type p53 was bound to immobilized GST–CBP3 after only a 30-min incubation. In contrast, a p53 mutant protein [p53(22,23)] (ref. 21) containing a double point mutation in the transactivation domain did not bind detectably to CBP. p53 binds tightly to CBP, with an apparent Ϫ 8 dissociation constant, Kd, of 2:5 ϫ 10 M. The same analysis also revealed a weaker interaction between p53 and CBP2, consistent with the affinity-chromatography results (Fig. 1b). To test for interaction between p53 and CBP in vivo, cell extracts from U2OS cells were immunoprecipitated by polyclonal antibodies (anti-CBP1 and anti-CBP3) directed against the CBP-1 and CBP-3 domains shown in Fig. 1a. p53 was detected in the immunopreci- pitate with both crude anti-CBP1 and antigen affinity-purified anti- CBP1 (Fig. 2b, lanes 5 and 6), but not in either of the immunopre- cipitates obtained with the preimmune sera (lane 2) or with another control antiserum (anti-SRB7) (lane 3), nor in the immunopreci- pitate with anti-CBP1 from the p53-negative SaoS-2 cells (lane 7). This indicates that a specific p53–CBP complex is present in U2OS cells (Fig. 2b). Anti-CBP3 only weakly precipitates CBP-p53 immune complexes from U2OS extracts (Fig. 2b, lane 4), which may reflect an overlap between the epitope recognized by the antisera and the domain of CBP bound by p53. The functional consequence of this interaction between p53 and CBP was investigated by cotransfection of SaoS-2 cells with the corresponding expression vectors and a reporter construct contain- ing the mdm-2 promoter, one of the natural targets of p53 (refs 3, 22). As shown in Fig. 3a, overexpression of CBP increased the level of activation by p53 from 5-fold up to 32-fold over the basal activity of the mdm-2 promoter. As SaoS-2 cells do not express p53 endogenously, the synergistic effect is easily detected when as little as 2 ng of CMV–p53 plasmid is transfected into the cell (Fig. 3b). By Figure 2 CBP interacts with p53 both in vitro and in vivo. a, Direct interactions of contrast, overexpression of CBP alone has no effect on this CBP with p53(WT) and p53(22,23) mutant proteins in vitro. Western blot analysis of promoter (Fig. 3b). the indicated GST pull-down complexes with anti-p53 (Ab 421). b, Interactions of It has been shown that E1a downregulates p53-mediated tran- CBP with p53 in U2OS cells. Western blot analysis of U2OS whole-cell extract 23 scriptional function on the synthetic promoter pOLXALuc , (WCE) (ϳ1% of the total protein used for immunoprecipitation) (lane 1), and the although the molecular mechanism of the repression is not under- immune complexes precipitated by preimmune serum (lane 2), control antiserum stood. As E1a binds to CBP and inhibits CBP-dependent coactivator (anti-SRB7) (lane 3), anti-CBP2 (lane 4), anti-CBP1 (lane 5) and anti-CBP1-AP 5–14 activity , we tested whether E1a-mediated repression of transac- (antigen affinity-purified) (lane 6), together with a negative control of immunopre- tivation by p53 is dependent on the interaction with endogenous cipitated complexes by anti-CBP1 from p53-negative cells (SaoS-2).

Nature © Macmillan Publishers Ltd 1997 820 NATURE | VOL 387 | 19 JUNE 1997 letters to nature

Figure 3 CBP enhances p53-mediated transactivation. a, CBP potentiates p53- mediated transactivation of the mdm-2 promoter. SaoS-2 cells were transiently transfected with 10 ␮g Mdm-2–Luc reporter construct22, 10 ng CMV–p53(WT) or CMV–p53(22,23) (ref. 21) and 3–10 ␮g RSV–CBP (ref. 9), as indicated. Both CMV– p53 wild type and CMV–p53(22,23) constructs were gifts from A. Levine and are expressed at comparable levels in the cell21. b, Synergism between CBP and p53. SaoS-2 cells were transiently transfected with 10 ␮g Mdm-2–Luc reporter construct22, 0–10 ng CMV–p53 wild type21, or 5 ␮g RSV–CBP (ref. 9), as indicated. c, CBP-enhanced p53 transcriptional activity is repressed by E1a. SaoS-2 cells were cotransfected with 10 ␮g Mdm-2–Luc reporter construct,1 ␮g CMV–p53(WT), and 5 ␮g 12S E1a or E1a(D64–68) (refs 10, 24) plasmids, as indicated.

Figure 4 Synergism between Gal–CBP(1678–2441) and p53. SaoS-2 cells were transiently cotransfected with 4 ␮g Gal–Luc reporter construct23, 4 ␮g RSV–Gal– CBP(1678–2441) (ref. 6), 50 to 200 ng CMV–p53(WT) or CMV–p53(22,23) mutant plasmids21 or 200 ng SV–Gal–VP16, as indicated. 3 ␮g CMV–LacZ was included in each case as an internal control30.

Nature © Macmillan Publishers Ltd 1997 NATURE | VOL 387 | 19 JUNE 1997 821 letters to nature to elucidate the specificity of this interaction. As predicted, a p53 incubated at 4 ЊC for 30 min with 1 ␮g of each of the different GST fusion mutant (p53(22,23)) that is defective in CBP binding (Fig. 2a) is proteins in 500 ␮l total volume. This was followed by incubation with 10 ␮l severely impaired in functional synergy with CBP. Thus, as shown in glutathione–Sepharose beads for another 15 min. Beads were then washed six Fig. 3a and Fig. 4, p53(22,23) fails to show synergy either with CBP times in 1 ml BC0 buffer containing 200 KCl and 0.2% N-P40. Bound proteins on the mdm-2 promoter (Fig. 3a) or with Gal4–CBP on the Gal4- were eluted with an equal volume of SDS loading buffer, resolved by SDS– target promoter (Fig. 4). Therefore, the interaction of these two PAGE, and analysed by western blot. proteins correlates with their functional synergy. Coimmunoprecipitation assay. About 2 ϫ 108 human osteosarcoma U2OS Our results indicate that CBP is required for optimal p53- (or SaoS-2) cells were extracted with 5 ml lysis buffer (25 mM HEPES-KOH, mediated transactivation. CBP participates in preventing the G0/ pH 8.0, 150 mM KCl, 2 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 ␮g ml Ϫ 1 G1 transition during the cell cycle, by activating certain enhancers aprotinin, 10 ␮g ml Ϫ 1 leupeptin, 1 ␮g ml Ϫ 1 pepstatin A, 20 mM NaF, 0.1% NP- and stimulating differentiation pathways4–14, whereas the tumour- 40). After centrifuging twice, the supernatants were incubated with different suppressor gene product p53 is a key regulator of cell proliferation, antisera (as indicated), crosslinked to protein A-Sepharose (Pharmacia) for 4 h differentiation and apoptosis1–3,25. p53 may be one of the targets for at 4 ЊC. The beads were washed six times with 1 ml lysis buffer, after which the CBP in mediating its cellular functions. As sequence-specific tran- associated proteins, including p53, were eluted with buffer BC0 containing 1 M scriptional activation by p53 correlates with its ability to suppress KCl and 1% deoxycholate. Elution was for 30 min at 0 ЊC to avoid significant cell growth1–3, cell-growth suppression by CBP may be mediated, at elution of antibodies and associated antigens, and possible interference in the least in part, though synergistic activation with p53 on its target western blot result as a result of the similar size of the immunoglobulin proteins genes. CBP is also a target of adenovirus E1a (refs 5–14 and this and p53. Anti-CBP1 and anti-CBP3 antisera were prepared by immunizing work) and our results indicate that the binding of E1a to CBP could rabbits with the corresponding CBP-derived recombinant proteins (Fig. 1a). suppress the synergistic effect of CBP and p53 on transcriptional Transient transfection assays. SaoS-2 cells (1 ϫ 106) were transfected by activation, which could also be essential for the transformation calcium phosphate precipitation as described30, and ␤-galactosidase and activity of this oncoprotein. luciferase were assayed 30 h later. All transfections were done in duplicate; CBP/p300 is involved in transcriptional activation by several representative experiments depict the average of three experiments with cellular factors, although the precise mechanisms by which these standard deviations indicated. activators stimulate the transcriptional machinery through CBP/ Immunoblotting. Proteins were resolved on either 6% SDS–PAGE (for CBP) p300 are not yet clear. A number of activators that interact with CBP or 10% SDS–PAGE (for p53), followed by transfer to nitrocellulose membranes (refs 4–14, and this work) interact both physically and functionally for 14 h at 4 ЊC. After incubation with the indicated antibodies for 1 h at room with other coactivators: these coactivators include specific TAFs in temperature, blots were subsequently incubated with secondary antibodies and the case of p53 (refs 17, 18) and CREB (ref. 26), members of the Src visualized by ECL as suggested by the manufacturer (Amersham). All proce- family12,13,27,28 and potentially other factors (reviewed in refs 12, 13) dures were basically the same as for the coimmunoprecipitation experiments, in the case of nuclear receptors, and a special set of coactivators except that the blot was incubated with anti-p53 (DO-1) for 12 h at 4 ЊC. 29 (TRAPs) in the case of the thyroid hormone receptor . Optimal Received 30 October 1996; accepted 17 March 1997. function of the activators requires both CBP/p300 and the other 1. Ko, L. J. & Prives, C. p53: puzzle and paradigm. Genes Dev. 10, 1054–1072 (1996). coactivators, suggesting that these are important but have distinct 2. El-Deiry, W. S. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825 (1993). roles. This idea is supported by the discovery that both p300/CBP 3. Wu, X., Bayle, J. H., Olson, D. & Levine, A. J. The p53-mdm-2 autoregulatory feedback loop. Genes Dev. 7, 1126–1132 (1993). and an interacting factor (P/CAF) have histone acetyltransferase 4. Chrivia, J. C. et al. Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365, 5,15,16 activity , which could modify chromatin structure. The fact that 855–859 (1993). p53 exists as a tetramer in vivo should allow it to interact simulta- 5. Yang, X. J., Ogryzko, V. V., Nishikawa, J., Howard, B. H. & Nakatani, Y. A p300/CBP-associated factor that competes with the adenoviral oncoprotein E1a. Nature 382, 319–324 (1996). neously with CBP and other coactivators (TAFs). This may be 6. Kwok, R. P. et al. Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370, essential for optimal activation in vivo and also explains the synergy 223–226 (1994). 7. Arias, J. et al. Activation of cAMP and mitogen responsive gene relies on a common nuclear factor. between CBP and p53, even when CBP is tethered to the promoter Nature 370, 226–229 (1994). by an alternative mechanism. Ⅺ 8. Arany, Z., Newsome, D., Oldread, E., Livingston, D. M. & Eckner, R. A family of transcriptional ...... adaptor proteins targeted by the E1a oncoprotein. Nature 374, 81–84 (1995). 9. Lundblad, J. R., Kwok, R. P., Laurance, M. E., Harter, M. L. & Goodman, R. H. Adenoviral E1a- Methods associated protein p300 as a functional homologue of the transcriptional coactivator CBP. Nature 374, Plasmids and fusion proteins. To construct GST fusion proteins, DNA 85–88 (1995). 10. Banister, A. J. & Kouzarides, T. CBP-induced stimulation of c-Fos activity is abrogated by E1a. EMBO sequences corresponding to the indicated regions of CBP were amplified by J. 14, 4758–4762 (1995). PCR and subcloned into pGEX-2T (Pharmacia). GST fusion proteins were 11. Dai, P. et al. CBP as a transcriptional coactivator of c-Myb. Genes Dev. 10, 528–540 (1996). expressed in E. coli, extracted with buffer BC0 (20 mM Tris-HCl, pH 8.0, 12. Kamei, Y. et al. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85, 403–414 (1996). 0.5 mM EDTA, 20% glycerol, 1 mM DTT and 0.5 mM PMSF) containing 13. Chakravati, D. et al. Roles of CBP/p300 in nuclear receptor signalling. Nature 383, 99–103 (1996). 500 mM KCl and 1% NP-40, and purified on glutathione–Sepharose 14. Lee, J. S., See, R. H., Deng, T. & Shi, Y. Adenovirus E1a downregulates c-Jun and JunB-mediated transcription by targeting their coactivator p300. Mol. Cell. Biol. 16, 4312–4326 (1996). (Pharmacia). Plasmids encoding p53 wild-type or mutant proteins were 15. Ogryzko, V. V., Schiltz, R. L., Russanova, V., Howard, B. H. & Nakatani, Y. The transcriptional created by amplification of the appropriate DNA fragments, including the Flag coactivator p300 and CBP are histone acetyltransferase. Cell 87, 953–959 (1996). sequence, followed by subcloning into pET-11d (Novagene). The p53 proteins 16. Bannister, A. J. & Kouzarides, T. The CBP coactivator is a histone acetyltransferase. Nature 384, 641– 643 (1996). were expressed in BL21 (Lys) cells and purified on an M2 agrose affinity column 17. Thut, C., Chen, J. L., Klemm, R. & Tjian, R. p53 transcriptional activation mediated by coactivators (IBI). TAFII40 and TAFII60. Science 267, 100–104 (1995). 18. Lu, H. & Levine, A. J. Human TAF31 protein is a transcriptional coactivator of the p53 protein. Proc. Affinity chromatography. Affinity columns were prepared by immobilizing Natl Acad. Sci. USA 92, 5154–5158 (1995). GST or GST–CBP proteins on glutathione–Sepharose beads as described19. 19. Xiao, H. et al. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 400-␮l aliquots of HeLa nuclear extract (ϳ4 mg protein) were loaded onto 20- and p53. Mol. Cell. Biol. 14, 7013–7024 (1994). 20. Seto, E. et al. Wild-type p53 binds to the TATA-binding protein and represses transcription. Proc. Natl ␮l affinity columns containing different GST fusion proteins. After washing the Acad. Sci. USA 89, 12028–12032 (1992). beads with 200 ␮l buffer BC0 containing 100 mM KCl and 0.1% N-P40, bound 21. Lin, J., Chen, J., Elenbaas, X. & Levine, A. J. Several hydrophic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1b 55- proteins were eluted with the BC0 buffer containing 1M KCl, and the total kD protein. Genes Dev. 8, 1235–1246 (1994). eluates and 0.5% of the input nuclear extract were subjected to western 22. Haupt, Y., Rowan, S., Shaulian, E., Vousden, K. & Oren, M. Induction of apoptosis in HeLa cells by blotting. For the GST–p53 affinity columns the procedures were basically the transactivation-deficient p53. Genes Dev. 9, 2170–2183 (1995). 23. Steegenga, W. T. et al. Adenovirus E1a proteins inhibit activation of transcription by p53. Mol. Cell. same, except that 100-␮l aliquots of HeLa nuclear extract were loaded on the Biol. 16, 2101–2109 (1996). affinity columns, and only half of the total proteins eluted from the columns 24. Wong, H. K. & Ziff, E. B. Complementary functions of E1a conserved region 1 cooperate with conserved region 3 to activate adenovirus serotype 5 early promoters. J. Virol. 68, 4910–4920 (1994). were used for western blot analysis. 25. Godley, L. A. et al. Wild-type p53 transgenic mice exhibit altered differentiation of the ureteric bud In vitro binding assays. 100 ng of p53(WT) or p53 mutant proteins were and possess small kidneys. Genes Dev. 10, 836–850 (1996).

Nature © Macmillan Publishers Ltd 1997 822 NATURE | VOL 387 | 19 JUNE 1997 letters to nature

26. Ferreri, K., Gill, G. & Montminy, M. The cAMP-regulated transcription factor CREB interacts with a p300/CBP complexes to be detected. Complex formation was component of the TFIID complex. Proc. Natl Acad. Sci. USA 91, 1210–1213 (1994). R 3 27. Onate, S. A., Tsai, S. Y., Tsai, M-J. & O’Malley, B. W. Sequence and characterization of a coactivator for assessed in ts20TG cells , which are temperature sensitive in the the steroid hormone receptor superfamily. Science 270, 1354–1357 (1995). E1 ubiquitin-activating function, and accumulate stable p53 at 28. Voegel, J. J., Heine, M. J. S., Zechel, C., Chambon, P. & Gronemeyer, H. TIF2, a 160 kd transcriptional Њ mediator for the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J. 15, 3667– 39.5 C. Endogenously labelled p400 and CBP co-immunoprecipitated 3675 (1996). with p53 (Fig. 1a, compare lanes 4 and 8 with 2; also data not 29. Fondell, J. D., Ge, H. & Roeder, R. G. Ligand induction of a transcriptionally active thyroid hormone shown), and p53 co-immunoprecipitated with p300/CBP family receptor coactivator complex. Proc. Natl Acad. Sci. USA 93, 8329–8333 (1996). 30. Gu, W., Bhatia, K., Magrath, I. T., Dang, C. V. & Dalla-Favera, R. Binding and suppression of the Myc members (Fig. 1a, lanes 6 and 7) at the non-permissive temperature. transcriptional activation domain by p107. Science 264, 251–254 (1994). Unlike CBP, p300 co-immunoprecipitated with p53 at both tem-

Acknowledgements. We thank R. H. Goodman, A. Levine, M. Oren, A. G. Jochemsen, N. C. Jones, peratures by p300-specific immunoblotting (Fig. 1b, lanes 4 and 6), A. J. Banister and T. Kouzarides for plasmids; H. Xiao, Y. Tao, L. Wang, S. Stevens and J. D. Fondell for suggesting that detection of p300/p53 binding is not dependent on discussions and for critical comments on the manuscript; and Y. Nakatani for sharing unpublished observations. This work was supported by a postdoctoral fellowship from Life Science Foundation for p53 metabolic stabilization. Taken together, the data show that Advanced Cancer Studies to W.G., and by grants from the NIH to R.G.R. stable p53–p300/CBP family-member complexes form in the

Correspondence and requests for materials should be addressed to R.G.R. (e-mail: roeder@rockvax. absence of T antigen. A comparison of the quantities of p53- rockefeller.edu). coprecipitated p300 and total p300 available (Fig. 1b) suggests that р1% of total cellular p300 coprecipitated with p53 under the conditions used. E1A is known to inhibit p53 transcription-activation function4, Binding and modulation of p53 and binds to a specific domain (C/H3) of p300/CBP5. Given that p53 also binds p300/CBP family members, we investigated whether by p300/CBP coactivators E1A inhibits p53 transcription by binding C/H3 and downregulat- ing p300/CBP-mediated p53 coactivation. U-2 OS cells, which Nancy L. Lill*, Steven R. Grossman, Doron Ginsberg*, synthesize wild-type p53, were transfected with a consensus p53 James DeCaprio & David M. Livingston binding site-containing CAT reporter (PG13-CAT), or its mutant The Dana-Farber Cancer Institute and Harvard Medical School, 44 Binney Street, counterpart (MG15-CAT), which is not responsive to p53 (ref. 6). Boston, Massachusetts 02115, USA Wild-type 12S E1A specifically inhibited p53-mediated activation of * Present addresses: Department of Rheumatology and Immunology, Brigham and PG13-CAT (Fig. 2a). E1A mutant D2-36 failed to repress p53- Women’s Hospital, 514 Seeley G. Mudd Building, 250 Longwood Avenue, Boston, dependent transcription. This mutant binds to pRB, but not to Massachusetts 02115, USA (N.L.L.); Department of Molecular Cell Biology, p300/CBP family members7. The E1A mutant, CXd1, which binds Weizmann Institute of Science, Rehovot, 76100, Israel (D.G.). p300/CBP but not proteins of the retinoblastoma family, fully ...... repressed promoter activity. These data indicate that one or more The adenovirus E1A and SV40 large-T-antigen oncoproteins bind p300/CBP family members may coactivate p53. Mutant E1A D26– to members of the p300/CBP transcriptional coactivator family. 35 is defective in binding to p400 but not p300 (ref. 8), yet E1A Binding of p300/CBP is implicated in the transforming mechan- D26–35 actively repressed p53-dependent transcription. Hence isms of E1A and T-antigen oncoproteins. A common region of the p400 is not the only p300 family member responsible for p53 T antigen is critical for binding both p300/CBP and the tumour coactivation. suppressor p53 (ref. 1), suggesting a link between the functions of PG13-CAT and two other p53-responsive reporter plasmids, p53 and p300. Here we report that p300/CBP binds to p53 in the pWWP-luc, which carries the p21WAF1/cip1 promoter9, and absence of viral oncoproteins, and that p300 and p53 colocalize pTM667-3, which carries the bax promoter10, were transfected within the nucleus and coexist in a stable DNA-binding complex. with a p53 expression plasmid into p53-null Saos-2 cells (Fig. 2b– Consistent with its ability to bind to p300, E1A disrupted d). Again, E1A repression of p53-mediated transcription activation functions mediated by p53. It reduced p53-mediated activation correlated with its ability to bind p300/CBP family members. Hence of the p21 and bax promoters, and suppressed p53-induced cell- the same genetics of E1A repression of p53 transcription activation cycle arrest and apoptosis. We conclude that members of the p300/ apply to two naturally occurring p53-activated promoters. There- CBP family are transcriptional adaptors for p53, modulating its fore, p300/CBP family members are implicated as modulators of checkpoint function in the G1 phase of the cell cycle and its p53-dependent transcription-activation function. induction of apoptosis. Disruption of p300/p53-dependent Overproduction of wild-type p300 overrode E1A-mediated growth control may be part of the mechanism by which E1A repression of PG13-CAT (Fig. 2e). The mutant p300 species, del33, induces cell transformation. These results help to explain how p53 which lacks an intact C/H3 domain and cannot bind to E1A5, failed mediates growth and checkpoint control, and how members of the to override repression. This is consistent with the view that E1A p300/CBP family affect progression from G1 to the S phase of the represses p53-mediated transcription activation by binding p300/ cell cycle. CBP family member(s). The data are also consistent with a model in When stabilized by Tantigen, p53 was found to bind members of which the C/H3 domain of p300/CBP contributes to p53 coactiva- the p300/CBP family (p300, CBP and p400)1,2. We investigated tion, and E1A inactivation of this domain is relieved through whether non-T antigen-mediated stabilization of p53 enables p53– sequestration of E1A by overexpressed p300. C/H3 is also the

Table 1 E1A override of p53-induced G1 cell-cycle arrest and apoptosis

Transfected cells (%)

Experiment code Transfected DNA sub-G1 (apoptotic) G1 S G2/M ...... A pCMV 7.24 21.4 26.5 52.1 p53 17.72 43.9 14.3 41.9 p53 þ wt E1A 7.30 28.8 17.1 54.2 p53 þ E1A D 2–36 14.08 41.4 19.3 39.4 ...... B pCMV 7.66 31.5 36.4 32.0 p53 18.04 47.2 16.7 36.0 p53 þ wt E1A 10.80 34.5 29.3 36.2 p53 þ E1A D 2–36 16.10 45.3 21.5 33.3 ...... Results are for Saos-2 cells. The G1, S and G2/M values are percentages of the non-apoptotic transfected cell population.

Nature © Macmillan Publishers Ltd 1997 NATURE | VOL 387 | 19 JUNE 1997 823