Overlapping but Distinct Patterns of Histone Acetylation by the Human Coactivators p300 and PCAF within Nucleosomal Substrates

Abstract

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. 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. Sci. S. A. 1995; PubMed Scopus Google Scholar). The of each was and purified by and 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 PCAF was in HeLa and a stable high molecular PCAF complex was purified as described previously V.V. T. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. J. Nakatani Y. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). and free histones were acetylated as described V.V. T. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. J. Nakatani Y. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), that the incubation of the reaction was to to The of were analyzed on an gel as described M.H. PubMed Scopus (218) Google Scholar), that was to of H3 To date, the specific lysine residues within the core histones targeted by a nuclear histone acetyltransferase have only been examined in 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 acetylates of H3 and and of H4 free histones are used as of acetylation is distinct and with the acetylation of the HAT in histone R.E. Cook R.G. Allis C.D. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar and R.E. Cook R.G. C.A. Allis C.D. Proc. Natl. Sci. S. A. 1995; PubMed Scopus 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). suggests that acetylation of these residues may with of acetylation by specific HATs to have free histone or (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, 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, A. M. A. Zhou J. Cook R.G. Allis C.D. Proc. Natl. Sci. S. A. 1998; PubMed Scopus Google Scholar). their nuclear that the coactivators PCAF and p300 nucleosomal in vivo. functional differences between PCAF and p300 have been (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, 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 to the acetylation of PCAF and p300 on a chromatin substrate were Recombinant human p300 and PCAF were used to HeLa mononucleosomes with A. Individual core histones were by and analyzed by each of the was by for a direct of acetylation studies have shown that the of acetylation in HeLa is with the of the core histones or monoacetylated D. K. P. C. J. PubMed Scopus Google Scholar). Moreover, these studies have determined on H3 and H4. of H3 and of H4 are the residues acetylated in the monoacetylated of these histones in bulk chromatin D. K. P. C. J. PubMed Scopus Google Scholar). Thus, the used in these experiments were not of the of the core histones were or In acetylation we have that a of H3 and H4 was at and of mononucleosomes with PCAF results in acetylation of H3 and but acetylation of H4 The yeast of PCAF, GCN5, is to mononucleosomes in P.A. L. Cote J. Brownell J.E. R. R. Owen-Hughes T. Allis C.D. Berger Workman J.L. Genes Dev. 1997; PubMed Scopus Google Scholar) D. but acetylates H3 and H4 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). results indicate that of H3 and of H4 are acetylated purified from a acetylation at known sites of in vivo In the of the of acetylation at residues is to be to prior acetylation in the in and this is by PCAF assay of histones acetylated by by from mononucleosomes acetylated in vitro by PCAF were analyzed by The of in each of the are the The sequence for H3 or H4 as the is the in and labeled with indicate known in vivo acetylation We that of H4 be acetylated by PCAF this is acetylated in free histone H4 by yeast 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 that may have been for acetylation in this is a known acetylation in HeLa P.A. L. Cote J. Brownell J.E. R. R. Owen-Hughes T. Allis C.D. Berger Workman J.L. Genes Dev. 1997; PubMed Scopus Google Scholar). We the of bulk and lysine in and from the of the lysine residues in 5 and were acetylated not that the of acetylation at these residues by PCAF was not to in at these We that of H4 was acetylated not even though was at this may have been a to to this to of H4 in by HATs may be to the association of the H4 with nucleosomal DNA K. Nature. 1997; 389: PubMed Scopus Google Scholar) or its to the of H4 K. Nature. 1997; 389: PubMed Scopus Google G. Wang Proc. Natl. Sci. S. A. 1991; PubMed Scopus Google Scholar). In this be to acetylation is in the of chromatin as and (for review see Ref. P.D. Curr. Opin. Cell Biol. 1998; PubMed Scopus Google Scholar). We have that PCAF in its native form is present in a complex containing polypeptides V.V. T. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. J. Nakatani Y. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). not all of these have been the PCAF complex human of the yeast and a of and with the that the interaction of p300 and CBP with PCAF in vivo is not 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 p300 or CBP V.V. T. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. J. Nakatani Y. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the half of PCAF, the required for p300 and CBP is for complex the form of which homologous to the N of PCAF, is in an complex V.V. T. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. J. Nakatani Y. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is with the notion that the N of PCAF may be in with p300/CBP, or sequence-specific transcription The PCAF recombinant PCAF, preferentially acetylates H3 but acetylates H4 V.V. T. Schiltz R.L. Howard T. Yang X.-J. Howard B.H. J. Nakatani Y. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). We examined the PCAF complex and recombinant PCAF a of acetylation of H3 and H4. and free core histones were acetylated by the native PCAF complex or recombinant PCAF, and the degree of acetylation was determined by gel by In to p300, which acetylates H3 and H4 on lysine residues and both recombinant PCAF and the PCAF complex primarily H3 to a monoacetylated form free histones and or and are used as H4 is also primarily the complex a of H4 on free histone H4 the of H4 monoacetylated at is is that this form is from of monoacetylated H4 at a Thus, we that the PCAF complex and the recombinant PCAF have a substrate specificity in that they both preferentially a of H3 and are only to a of H4 within nucleosomal in We have previously shown that p300 is of all four core histones in HeLa mononucleosomes 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 of nucleosomal H3 acetylated by p300 a for and with a of labeling on A of was also at Thus, p300 is of four of the lysines known to be acetylated in a for and Significantly, p300 not residues and of at these sites been correlated with of H3 in chromatin R.E. Cook R.G. Allis C.D. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, R.E. Cook R.G. C.A. Allis C.D. Proc. Natl. Sci. S. A. 1995; PubMed Scopus Google Scholar), and the of acetylation at these sites by p300 is evidence for the of a distinct activity in acetylation of both PCAF and p300, which 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 for The of both of these HATs at this may contribute to the high of acetylation of this in vivo D. K. P. C. J. PubMed Scopus Google Scholar). nucleosomal H4 was acetylated by p300, a for and was less acetylation at and high levels of acetylation at or of this within may in levels of acetylation at this in of and been correlated with histone R.E. Cook R.G. Allis C.D. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, R.E. Cook R.G. C.A. Allis C.D. Proc. Natl. Sci. S. A. 1995; PubMed Scopus Google the results that acetylation of these sites may also a in transcriptional activation. The of these acetylation within these distinct may be by the in which they nucleosomal and nuclear and and also the for in by of H2A demonstrated an specificity of p300 for the acetylated in H2A in vivo D. K. P. C. J. PubMed Scopus Google Scholar), the of lysines specificity was in to the specificity of p300 for H2B 10 lysine residues within its 30 of which and have been to be acetylated in vivo D. K. P. C. J. PubMed Scopus Google Scholar and four of these sites are acetylated by p300 in vitro with an of p300 for and Significantly, acetylation was not at lysine residues that are not known to be acetylated in vivo. that we the signal in of this to of a of the signal at to These findings indicate that the HAT activity of p300 is highly for known in vivo acetylation sites and acetylates all the lysines in H2A and H2B that are acetylated in vivo. The acetylation specificity of p300 and PCAF with nucleosomal assay is in of p300 and PCAF nucleosomal acetylation acetylation sites of nucleosomal core histones acetylated by p300 or PCAF as identified by are The known in vivo acetylation sites of each of the core histones is residues are acetylated by p300, the sites are in a The acetylation sites of nucleosomal core histones acetylated by p300 or PCAF as identified by are The known in vivo acetylation sites of each of the core histones is residues are acetylated by p300, the sites are The results demonstrate that both PCAF and p300 known in vivo acetylation been that these are to and the activities of proteins histones in vitro A. Yang X.-J. Ogryzko V.V. Nakatani Y. H. Curr. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, R.G. Cell. 1997; 90: Full Text Full Text PDF PubMed Scopus Google Scholar, K. J.E. S. T. M. A. E. Genes Dev. 1998; 12: PubMed Scopus Google Scholar), support to the that core histones are of p300 and PCAF in in to that histones were recently shown to be targeted by the HAT activity of yeast 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 differences in the acetylation sites by p300 and PCAF in nucleosomal histones, with PCAF showing specificity for sites p300 the 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 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 with and these requirements for the HAT activity of PCAF that of p300 is that acetylation are in transcriptional regulation. The by which acetylation facilitates transcription to be are with the that acetylation at sites by PCAF in may serve to required for transcription that are not by acetylation at sites by is that differences in the in which PCAF and p300 are to the differential functional PCAF may be to a interaction with p300/CBP may with that to the transcription binding the HAT activity of p300/CBP may be to direct acetylation within the promoter or the of the to its at the transcription binding of the sites acetylated by these HATs in nucleosomal as of the of activation by these and transcriptional that possess HAT activity. We Ogryzko for in of the p300 into the expression and Russanova for for the gel of labeled