Overlapping but Distinct Patterns of Histone Acetylation by the Human Coactivators p300 and PCAF within Nucleosomal Substrates
1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês
10.1074/jbc.274.3.1189
ISSN1083-351X
AutoresR. Louis Schiltz, Craig A. Mizzen, Alex Vassilev, Richard G. Cook, C. David Allis, Yoshihiro Nakatani,
Tópico(s)Ubiquitin and proteasome pathways
ResumoA number of transcriptional coactivators possess intrinsic histone acetylase activity, providing a direct link between hyperacetylated chromatin and transcriptional activation. We have determined the core histone residues acetylated in vitro by recombinant p300 and PCAF within mononucleosomes. p300 specifically acetylates all sites of histones H2A and H2B known to be acetylated in bulk chromatin in vivo but preferentially acetylates lysines 14 and 18 of histone H3 and lysines 5 and 8 of histone H4. PCAF primarily acetylates lysine 14 of H3 but also less efficiently acetylates lysine 8 of H4. PCAF in its native form, which is present in a stable multimeric protein complex lacking p300/CBP, primarily acetylates H3 to a monoacetylated form, suggesting that PCAF-associated polypeptides do not alter the substrate specificity. These distinct patterns of acetylation by the p300 and PCAF may contribute to their differential roles in transcriptional regulation. A number of transcriptional coactivators possess intrinsic histone acetylase activity, providing a direct link between hyperacetylated chromatin and transcriptional activation. We have determined the core histone residues acetylated in vitro by recombinant p300 and PCAF within mononucleosomes. p300 specifically acetylates all sites of histones H2A and H2B known to be acetylated in bulk chromatin in vivo but preferentially acetylates lysines 14 and 18 of histone H3 and lysines 5 and 8 of histone H4. PCAF primarily acetylates lysine 14 of H3 but also less efficiently acetylates lysine 8 of H4. PCAF in its native form, which is present in a stable multimeric protein complex lacking p300/CBP, primarily acetylates H3 to a monoacetylated form, suggesting that PCAF-associated polypeptides do not alter the substrate specificity. These distinct patterns of acetylation by the p300 and PCAF may contribute to their differential roles in transcriptional regulation. The association of DNA with histones in chromatin antagonizes transcription in vitro (1Laybourn P.J. Kadonaga J.T. Science. 1991; 254: 238-245Crossref PubMed Scopus (292) Google Scholar, 2Owen-Hughes T. Workman J.L. Crit. Rev. Eukaryotic Gene Expression. 1994; 4: 403-441PubMed Google Scholar) and molecular genetic analyses in yeast have demonstrated roles for specific, evolutionarily conserved lysine residues within the N termini of the core histones in transcriptional regulation in vivo (for review see Ref. 3Grunstein M. Nature. 1997; 389: 349-352Crossref PubMed Scopus (2352) Google Scholar). Several studies have demonstrated an enrichment of hyperacetylated histones within transcriptionally active/competent chromatin in vivo (for review see Refs. 4Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (456) Google Scholar, 5Kaufman P.D. Curr. Opin. Cell Biol. 1996; 8: 369-373Crossref PubMed Scopus (43) Google Scholar, 6Roth S.Y. Allis C.D. Cell. 1996; 87: 5-8Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 7Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1531) Google Scholar). Strong support for the notion that histone acetylation facilitates transcription is supported by the discovery that transcriptional regulatory proteins, including GCN5 (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar,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 (1273) Google Scholar), PCAF (10Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1306) Google Scholar), p300 (11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2356) Google Scholar), CBP (12Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1518) Google Scholar), TAFII250 (13Mizzen 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 (616) Google Scholar), and the nuclear hormone receptor coactivators 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 (1250) Google Scholar) and 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 (1049) Google Scholar), possess intrinsic histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; HPLC, high pressure liquid chromatography. 1The abbreviations used are: HAT, histone acetyltransferase; HPLC, high pressure liquid chromatography. activity (for review see Refs. 16Mizzen C.A. Allis C.D. Cell. Mol. Life Sci. 1998; 54: 6-20Crossref PubMed Scopus (186) Google Scholar and 17Wu C. J. Biol. Chem. 1997; 272: 28171-28174Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). Moreover, mutational analyses of yeast GCN5 indicate that GCN5 HAT activity in vitro is correlated with histone acetylation at promoter regions and transcriptional activation of target genes in vivo (18Kuo M.H. Zhou J. Jambeck P. Churchill M.E. Allis C.D. Genes Dev. 1998; 12: 627-639Crossref PubMed Scopus (394) Google Scholar, 19Wang L. Liu L. Berger S.L. Genes Dev. 1998; 12: 640-653Crossref PubMed Scopus (218) Google Scholar). These results are further supported by in vitro transcription experiments from nucleosomal templates showing acetylCoA-dependent activation by the GCN5-containing SAGA complex (20Utley 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 (443) Google Scholar). These lines of evidence demonstrate the requirement of the HAT activity of GCN5 for transcriptional activation in a nucleosomal context. The multiplicity of HATs identified suggests they may serve distinct functions. CBP and p300 are highly homologous coactivator proteins (21Arany Z. Sellers W.R. Livingston D.M. Eckner R. Cell. 1994; 77: 799-800Abstract Full Text PDF PubMed Scopus (370) Google Scholar) that bind a number of sequence-specific transcriptional activators and have been suggested to be central integrators of transcriptional signals from various signal transduction pathways (22Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1915) Google Scholar). Both CBP and p300 interact with the adenoviral E1A oncoprotein, and this interaction results in transcriptional repression of some CBP- and p300-regulated genes (23Bannister A.J. Kouzarides T. EMBO J. 1995; 14: 4758-4762Crossref PubMed Scopus (319) Google Scholar, 24Lundblad J.R. Kwok R.P. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (529) Google Scholar, 25Lee J.S. See R.H. Deng T. Shi Y. Mol. Cell. Biol. 1996; 16: 4312-4326Crossref PubMed Scopus (137) Google Scholar). We have previously described a cellular factor, PCAF (p300/CBP-associatedfactor), which competes both in vitro andin vivo with E1A for binding to p300 and CBP (10Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1306) Google Scholar). The C-terminal half of PCAF bears a high degree of sequence homology to the yeast GCN5 nuclear HAT. We have shown that PCAF (10Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1306) Google Scholar) and p300 (11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2356) Google Scholar) have intrinsic HAT activity. Thus, p300 and PCAF form a histone acetylase complex in vivo. The functional requirements for the HAT activities of PCAF and p300/CBP have recently been examined in the regulation of myogenic differentiation (26Puri P.L. Sartorelli V. Yang X.-J. Hamamori Y. Ogryzko V.V. Howard B.H. Kedes L. Wang J.Y.J. Graessmann A. Nakatani Y. Levrero M. Mol. Cell. 1997; 1: 35-45Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), nuclear receptor-mediated transcriptional activation (27Korzus E. Torchia J. Rose D.W. Xu L. Kurokawa R. McInerney E.M. Mullen T.M. Glass C.K. Rosenfeld M.G. Science. 1998; 279: 703-707Crossref PubMed Scopus (556) Google Scholar), as well as in the cAMP and growth factor-induced signaling pathways (28Xu L. Lavinsky R.M. Dasen J.S. Flynn S.E. McInerney E.M. Mullen T.M. Heinzel T. Szeto D. Korzus E. Kurokawa R. Aggarwal A.K. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1998; 395: 301-306Crossref PubMed Scopus (247) Google Scholar). The HAT activity of PCAF is required for myogenic, nuclear receptor- and growth factor-induced signaling pathways, whereas that of p300/CBP is dispensable. However, the intrinsic HAT activity of p300/CBP is required for cAMP-induced transcriptional activation (27Korzus E. Torchia J. Rose D.W. Xu L. Kurokawa R. McInerney E.M. Mullen T.M. Glass C.K. Rosenfeld M.G. Science. 1998; 279: 703-707Crossref PubMed Scopus (556) Google Scholar, 28Xu L. Lavinsky R.M. Dasen J.S. Flynn S.E. McInerney E.M. Mullen T.M. Heinzel T. Szeto D. Korzus E. Kurokawa R. Aggarwal A.K. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1998; 395: 301-306Crossref PubMed Scopus (247) Google Scholar). These findings indicate distinct functional differences between the HAT activities of PCAF and p300/CBP. To further characterize the functional differences between PCAF and p300, we have examined the specificity of human p300 and PCAF for core histones in mononucleosomes isolated from HeLa cells. To date, the specific residues within core histones targeted by a nuclear HAT have been determined only for yeast GCN5 (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar). In this report, we show that p300 specifically targets all four core histones at sites known to be acetylated in vivo. These sites both overlap and extend the sites used by GCN5 (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar). PCAF, which is homologous to GCN5, targets the same free histones H3 and H4 (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar). Significantly, even though numerous lysine residues are present in the N termini of core histones, only residues known to be acetylated in vivo are acetylated by p300 and PCAF, suggesting that histones are a physiological substrate for these enzymes. A cDNA fragment containing the entire open reading frame of p300 was subcloned into the baculovirus expression vector, pACSG2 (PharMingen), downstream and in-frame with a DNA sequence encoding the FLAG epitope. Individual plaques of recombinant virus that express high levels of FLAG-p300 were isolated. Recombinant FLAG-p300 and FLAG-PCAF were purified as described previously (10Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1306) Google Scholar). HeLa mononuclesomes were prepared as described previously (29O'Neill T.E. Roberge M. Bradbury E.M. J. Mol. Biol. 1992; 223: 67-78Crossref PubMed Scopus (73) Google Scholar). Sucrose density gradient-purified HeLa mononucleosomes (16 μg with respect to DNA) were labeled in a 150-μl reaction containing 50 mmTris-HCl, pH 8.0, 10% glycerol, 10 mm sodium butyrate, 0.1 mm EDTA, 1.0 mm dithiothreitol, and 1.0 mm phenylmethylsulfonyl fluoride. Purified recombinant FLAG-p300 (600 fmol) or FLAG-PCAF (50 pmol) and [3H]acetyl-CoA (1.3 nmol at 50 nCi/μl, Amersham Pharmacia Biotech) were added prior to incubation at 30 °C for 45 min. At the end of the labeling period, an aliquot of each reaction was analyzed by SDS-polyacrylamide gel electrophoresis, and another aliquot was subjected to HAT assay on P81 filters as described previously (30Brownell J.E. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6364-6368Crossref PubMed Scopus (238) Google Scholar). The remainder of each sample was acid precipitated and purified by reverse-phase HPLC and microsequenced as described previously (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar). FLAG epitope-tagged PCAF was expressed in HeLa cells, and a stable high molecular weight PCAF complex was purified as described previously (31Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Nucleosomes and free histones were acetylated as described (31Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar), except that the incubation time of the reaction was increased to 1 h to maximize acetylation. The products of theses reactions were analyzed on an acid-urea gel as described (32West M.H. Bonner W.M. Biochemistry. 1980; 19: 3238-4325Crossref PubMed Scopus (218) Google Scholar), except that Triton X-100 was omitted to avoid separation of H3 isoforms. To date, the specific lysine residues within the core histones targeted by a nuclear histone acetyltransferase have only been examined in detail for yeast GCN5 (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar). Recombinant yGCN5 acetylates Lys14 of H3 and Lys8 and Lys16 of H4 when free (non-nucleosomal) histones are used as substrate. This pattern of acetylation is distinct and nonoverlapping with the acetylation pattern of the cytoplasmic HAT B involved in histone deposition (Refs. 33Sobel R.E. Cook R.G. Allis C.D. J. Biol. Chem. 1994; 269: 18576-18582Abstract Full Text PDF PubMed Google Scholar and 34Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (413) Google Scholar and for review see Refs. 4Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (456) Google Scholar and 5Kaufman P.D. Curr. Opin. Cell Biol. 1996; 8: 369-373Crossref PubMed Scopus (43) Google Scholar). This suggests that acetylation of these residues may correlate with increased transcription. All reports of acetylation site usage by specific HATs to date have utilized free (non-nucleosomal) histone or synthetic peptide substrates (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar, 13Mizzen 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 (616) Google Scholar, 35Smith E.R. Eisen A. Gu W. Sattah M. Pannuti A. Zhou J. Cook R.G. Lucchesi J.C. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3561-3565Crossref PubMed Scopus (262) Google Scholar). Given their nuclear localization, it seems likely that the coactivators PCAF and p300 acetylate nucleosomal substrates in vivo. Because functional differences between PCAF and p300 have been reported (26Puri P.L. Sartorelli V. Yang X.-J. Hamamori Y. Ogryzko V.V. Howard B.H. Kedes L. Wang J.Y.J. Graessmann A. Nakatani Y. Levrero M. Mol. Cell. 1997; 1: 35-45Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 27Korzus E. Torchia J. Rose D.W. Xu L. Kurokawa R. McInerney E.M. Mullen T.M. Glass C.K. Rosenfeld M.G. Science. 1998; 279: 703-707Crossref PubMed Scopus (556) Google Scholar, 28Xu L. Lavinsky R.M. Dasen J.S. Flynn S.E. McInerney E.M. Mullen T.M. Heinzel T. Szeto D. Korzus E. Kurokawa R. Aggarwal A.K. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1998; 395: 301-306Crossref PubMed Scopus (247) Google Scholar), we sought to determine whether the acetylation site profiles of PCAF and p300 on a chromatin substrate were different. Recombinant human p300 and PCAF were used to label HeLa mononucleosomes with [3H]acetyl-coenzyme A. Individual core histones were then separated by reverse-phase HPLC and analyzed by N-terminal microsequencing. Radioactivity eluted during each cycle of the sequencing run was quantitated by scintillation counting for a direct measure of acetylation site usage. Previous studies have shown that the steady-state level of acetylation in unsynchronized growing HeLa cells is relatively low with the majority of the core histones being unacetylated or monoacetylated (36Thorne A.W. Kmiciek D. Mitchelson K. Sautiere P. Crane-Robinson C. Eur. J. Biochem. 1990; 193: 701-713Crossref PubMed Scopus (123) Google Scholar). Moreover, these studies have determined non-random site occupancy on H3 and H4. Lys14 of H3 and Lys16 of H4 are the residues most frequently acetylated in the monoacetylated forms of these histones in bulk chromatin (36Thorne A.W. Kmiciek D. Mitchelson K. Sautiere P. Crane-Robinson C. Eur. J. Biochem. 1990; 193: 701-713Crossref PubMed Scopus (123) Google Scholar). Thus, although the mononucleosome substrates used in these experiments were not completely devoid of acetyl groups, the majority of the core histones were unacetylated or monoacetylated. In interpreting our acetylation data, we have considered that a small fraction of H3 and H4 was pre-acetylated at Lys14 and Lys16, respectively. Acetylation of mononucleosomes with PCAF results in strong acetylation of H3 and weak but reproducible acetylation of H4 (Fig.1). The yeast homolog of PCAF, GCN5, is unable to acetylate mononucleosomes in vitro(37Grant 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 (870) Google Scholar) 2C. D. Allis, unpublished observations. but acetylates H3 and H4 when presented as free histones (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar). Our results indicate that Lys14 of H3 (Fig.2 A) and Lys8 of H4 (Fig. 2 B) are acetylated when purified from a PCAF-acetylated mononucleosome preparation. Little, if any, acetylation occurred at other known sites of in vivo acetylation. In the case of H3, the lack of acetylation at other residues is unlikely to be due to prior acetylation in vivo, because Lys14is the preferred steady-state site in vivo, and this residue is strongly preferred by PCAF under our assay conditions.Figure 2Microsequence analysis of mononucleosomal histones acetylated by PCAF. Histones recovered by reverse-phase HPLC from mononucleosomes acetylated in vitro by PCAF were analyzed by microsequencing. The amount of 3H radioactivity eluted in each cycle of the microsequencing are plotted along the ordinate axis. The amino acid sequence for H3 (A) or H4 (B), as detected during the microsequencing, is indicated along the abscissa axis. Residues in bold and labeled with numbers indicate known in vivo acetylation sites.View Large Image Figure ViewerDownload (PPT) We expected that Lys16 of H4 would be acetylated by PCAF because this residue is acetylated in free histone H4 by yeast GCN5in vitro (8Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1997; 383: 269-272Crossref Scopus (500) Google Scholar). We considered that Lys16 may have been unavailable for acetylation in our experiments, because this is a known preferred acetylation site in vivoin HeLa cells (37Grant 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 (870) Google Scholar). We measured the amounts of bulk acetyllysine and lysine eluted in cycles 5, 8, 12, and 16 from the microsequencer. Less than 5% of the lysine residues in cycles 5 and 12 were acetylated (data not shown), indicating that the lack of acetylation at these residues by PCAF was not due to in vivoacetylation at these sites. We found that Lys16 of H4 was 59% acetylated (data not shown), even though little, if any, [3H]acetate was incorporated at this position (Fig. 2 C). Thus site occupancy may have been a contributing factor to our inability to acetylate this site. Alternatively, access to Lys16 of H4 in nucleosomes by HATs may be restricted either due to the association of the H4 tail with nucleosomal DNA (38Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6725) Google Scholar) or its proximity to the globular domain of H4 (38Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6725) Google Scholar,39Arents G. Burlingame R.W. Wang B.C. Love W.E. Moudrianakis E.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10148-10152Crossref PubMed Scopus (599) Google Scholar). In this regard, it would be interesting to determine whether acetylation site usage is altered in the presence of ATP-dependent chromatin remodeling complexes, such as Swi/SNF, NURF, and CHRAC (for review see Ref. 40Varga-Weisz P.D. Becker P.B. Curr. Opin. Cell Biol. 1998; 10: 346-353Crossref PubMed Scopus (109) Google Scholar). We have reported that PCAF in its native form is present in a multisubunit complex containing more than 20 polypeptides (31Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Although not all of these subunits have been identified, the PCAF complex contains human counterparts of the yeast ADA2, ADA3, and SPT3 proteins; a subset of TAFs (TBP-associatedfactors); and TAF-related factors. Consistent with the observation that the interaction of p300 and CBP with PCAF in vivo is not stoichiometric or stable (10Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1306) Google Scholar), the PCAF complex contains no p300 or CBP (31Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). Importantly, the N-terminal half of PCAF, the region required for p300 and CBP interaction, is apparently dispensible for complex formation, because the short form of hGCN5, which lacks sequences homologous to the N terminus of PCAF, is found in an indistinguishable complex (31Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). This finding is consistent with the notion that the N terminus of PCAF may be involved in transient interactions with p300/CBP, other coactivators, or sequence-specific transcription factors. The PCAF complex, like recombinant PCAF, preferentially acetylates H3 but weakly acetylates H4 (31Ogryzko V.V. Kotani T. Zhang X. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). We examined whether the PCAF complex and recombinant PCAF exhibit a similar pattern of acetylation of H3 and H4. Mononucleosomes and free core histones were acetylated by the native PCAF complex or recombinant PCAF, and the degree of acetylation was determined by acid-urea gel analysis followed by autoradiography. In contrast to p300, which acetylates H3 and H4 on multiple lysine residues (Fig. 3, A,lane 2, and B, lane 2), both recombinant PCAF and the PCAF complex primarily acetylate H3 to a monoacetylated form when either free histones (Fig. 3 A,lanes 3 and 4, respectively) or nucleosomes (Fig.3 A, lanes 6 and 7, respectively) are used as substrates. Histone H4 is also primarily monoacetylated; however, the complex does yield a significant level of diacetylated H4 on free histone H4 (Fig. 3 B, lane 4). Because the steady-state level of H4 monoacetylated at Lys16 is approximately 60%, it is likely that this diacetylated form is derived from modification of endogenously monoacetylated H4 at a residue other than Lys16. Thus, we conclude that the PCAF complex and the recombinant PCAF catalytic subunit have a similar substrate specificity in that they both preferentially acetylate a single residue of H3 and are only able to weakly acetylate a single residue of H4 within nucleosomal substrates in vitro. We have previously shown that p300 is capable of acetylating all four core histones in HeLa mononucleosomes (Ref. 11Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2356) Google Scholar and Fig. 1, lane 2). Sequence analysis of nucleosomal H3 acetylated by p300 revealed a strong preference for Lys14 and Lys18 with a significant amount of labeling on Lys23 (Fig. 4 C). A low level of [3H]acetyllysine was also detected at Lys4. Thus, p300 is capable of acetylating four of the six lysines known to be acetylated in vivobut has a preference for Lys14 and Lys18. Significantly, p300 did not acetylate residues Lys9 and Lys27 of H3. Acetylation at these sites has been correlated with deposition of H3 in replicating chromatin (33Sobel R.E. Cook R.G. Allis C.D. J. Biol. Chem. 1994; 269: 18576-18582Abstract Full Text PDF PubMed Google Scholar, 34Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (413) Google Scholar), and the lack of acetylation at these sites by p300 is strong evidence for the involvement of a distinct activity in deposition-related acetylation of H3. Interestingly, both PCAF and p300, which can associate under certain conditions in vivo (10Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1306) Google Scholar), demonstrate a strong preference for Lys14of H3. The combined action of both of these HATs at this residue may contribute to the high steady-state level of acetylation of this residue observed in vivo (36Thorne A.W. Kmiciek D. Mitchelson K. Sautiere P. Crane-Robinson C. Eur. J. Biochem. 1990; 193: 701-713Crossref PubMed Scopus (123) Google Scholar). When nucleosomal H4 was acetylated by p300, a strong preference for Lys5 and Lys8 was detected (Fig.4 D). Considerably less acetylation occurred at Lys12 and Lys16. As mentioned above, relatively high steady-state levels of acetylation at Lys16 or restricted accessibility of this residue within nucleosomes may result in low levels of acetylation at this site in vitro. Acetylation of Lys5 and Lys 12 has been correlated with histone deposition (33Sobel R.E. Cook R.G. Allis C.D. J. Biol. Chem. 1994; 269: 18576-18582Abstract Full Text PDF PubMed Google Scholar, 34Sobel R.E. Cook R.G. Perry C.A. Annunziato A.T. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1237-1241Crossref PubMed Scopus (413) Google Scholar); however, the results presented here suggest that acetylation of these sites may also play a role in transcriptional activation. The role of these particular acetylation events within these distinct processes may be distinguished by the context in which they occur (e.g. nucleosomal and nuclear versus non-nucleosomal and cytoplasmic) and also the potential for additional acetylations in combinatorial fashion by transcription-associated HATs. Sequence analysis of H2A demonstrated an absolute specificity of p300 for Lys5, the predominant site acetylated in H2A in vivo (36Thorne A.W. Kmiciek D. Mitchelson K. Sautiere P. Crane-Robinson C. Eur. J. Biochem. 1990; 193: 701-713Crossref PubMed Scopus (123) Google Scholar), despite the presence of three additional lysines (Fig.4 A). This single residue specificity was in stark contrast to the multi-residue specificity of p300 for H2B. H2B contains 10 lysine residues within its first 30 amino acids, 4 of which (Lys5, Lys12, Lys15, and Lys20) have been reported to be acetylated in vivo (Ref. 36Thorne A.W. Kmiciek D. Mitchelson K. Sautiere P. Crane-Robinson C. Eur. J. Biochem. 1990; 193: 701-713Crossref PubMed Scopus (123) Google Scholar and Table I). All four of these sites are acetylated by p300 in vitro with an apparent preference of p300 for Lys12 and Lys15(Fig. 3 B). Significantly, acetylation was not detected at lysine residues that are not known to be acetylated in vivo. (Note that we attribute the [3H]acetyllysine signal detected in cycle 16 of this analysis to carryover of a portion of the strong signal at Lys15 due to sequencing lag). These findings indicate that the HAT activity of p300 is highly selective for known in vivo acetylation sites and acetylates essentially all the lysines in H2A and H2B that are acetylated in vivo. The acetylation site specificity of p300 and PCAF with nucleosomal substrates under our assay conditions is summarized in Table I.Table ISummary of p300 and PCAF nucleosomal acetylation sitesCore histoneH2AH2BH3H4PCAFNDND148p30055,12,15,2014,18,235,8,12In vivo55,12,15,204,9,14,18,235,8,12,16The acetylation sites of nucleosomal core histones acetylated by p300 or PCAF as identified by microsequence analysis are summarized. The known in vivo acetylation sites of each of the core histones is indicated. Where multiple residues are acetylated by p300, the preferred sites are underlined. ND, none detected. Open table in a new tab The acetylation sites of nucleosomal core histones acetylated by p300 or PCAF as identified by microsequence analysis are summarized. The known in vivo acetylation sites of each of the core histones is indicated. Where multiple residues are acetylated by p300, the preferred sites are underlined. ND, none detected. The results presented here demonstrate that both PCAF and p300 acetylate known in vivo acetylation sites. Therefore, although it has been reported that these enzymes are able to acetylate and modulate the activities of proteins other than histones in vitro (41Imhof A. Yang X.-J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 42Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2144) Google Scholar, 43Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1010) Google Scholar), our data lend strong support to the idea that core histones are bona fide substrates of p300 and PCAF in vivo, possibly in addition to other proteins. Note that histones were recently shown to be targeted by the HAT activity of yeast GCN5in vivo (18Kuo M.H. Zhou J. Jambeck P. Churchill M.E. Allis C.D. Genes Dev. 1998; 12: 627-639Crossref PubMed Scopus (394) Google Scholar). We show striking differences in the acetylation sites preferred by p300 and PCAF in nucleosomal histones, with PCAF showing specificity for fewer sites than p300 under the conditions employed here. Recent studies have demonstrated that the HAT activity of PCAF is required for myogenic differentiation (26Puri P.L. Sartorelli V. Yang X.-J. Hamamori Y. Ogryzko V.V. Howard B.H. Kedes L. Wang J.Y.J. Graessmann A. Nakatani Y. Levrero M. Mol. Cell. 1997; 1: 35-45Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), as well as nuclear hormone receptor (27Korzus E. Torchia J. Rose D.W. Xu L. Kurokawa R. McInerney E.M. Mullen T.M. Glass C.K. Rosenfeld M.G. Science. 1998; 279: 703-707Crossref PubMed Scopus (556) Google Scholar) and growth factor-dependent signaling (28Xu L. Lavinsky R.M. Dasen J.S. Flynn S.E. McInerney E.M. Mullen T.M. Heinzel T. Szeto D. Korzus E. Kurokawa R. Aggarwal A.K. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1998; 395: 301-306Crossref PubMed Scopus (247) Google Scholar), whereas that of p300 is dispensible. One hypothesis consistent with our data and these unique requirements for the HAT activity of PCAF over that of p300 is that acetylation site-specific phenomena are involved in transcriptional regulation. The mechanism(s) by which acetylation facilitates transcription remains to be defined, yet current data are consistent with the possibility that acetylation at sites preferred by PCAF in nucleosomes positioned about promoters may serve to recruit factors required for transcription that are not recruited by acetylation at sites preferred by p300. Alternatively it is conceivable that differences in the manner in which PCAF and p300 themselves are recruited to promoters underlie the differential functional requirements. For example, PCAF may be recruited to promoters through a transient interaction with p300/CBP and, once recruited, may associate with other factors that allow more extensive accessibility to nucleosomes beyond the immediate transcription factor binding site. That is, the HAT activity of p300/CBP may be unable to direct extensive acetylation within the promoter region or throughout the coding region of the gene due to its sequestration at the transcription factor binding site. Identification of the sites acetylated by these HATs in nucleosomal substrates, as reported here, should facilitate experimental investigation of the mechanisms of activation by these and other transcriptional regulators that possess HAT activity. We thank Vasily Ogryzko for assistance in subcloning of the full-length p300 coding region into the baculoviral expression vector and Valya Russanova for technical advice for the acid-urea gel analysis of labeled nucleosomes.
Referência(s)