From the Laboratory of Molecular Growth Regulation,
National Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 20892, the ¶ Department
of Biology, University of Rochester, Rochester, New York 14627, and the ** Department of Microbiology and Immunology, Baylor
College of Medicine, Houston, Texas 77030
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ABSTRACT |
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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 (1, 2) 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. 3). Several studies have demonstrated an enrichment of hyperacetylated histones within transcriptionally active/competent chromatin in vivo (for review see Refs. 4-7). Strong support for the notion that histone acetylation facilitates transcription is supported by the
discovery that transcriptional regulatory proteins, including GCN5 (8,
9), PCAF (10), p300 (11), CBP (12), TAFII250 (13), and the
nuclear hormone receptor coactivators ACTR (14) and SRC-1 (15), possess
intrinsic histone acetyltransferase (HAT)1 activity (for review
see Refs. 16 and 17). 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 (18, 19). These results are further
supported by in vitro transcription experiments from
nucleosomal templates showing acetylCoA-dependent activation by the GCN5-containing SAGA complex (20). 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 (21)
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 (22). Both CBP and
p300 interact with the adenoviral E1A oncoprotein, and this interaction
results in transcriptional repression of some CBP- and p300-regulated
genes (23-25). We have previously described a cellular factor, PCAF
(p300/CBP-associated
factor), which competes both in vitro and
in vivo with E1A for binding to p300 and CBP (10). The
C-terminal half of PCAF bears a high degree of sequence homology to the
yeast GCN5 nuclear HAT. We have shown that PCAF (10) and p300 (11) 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 (26), nuclear receptor-mediated transcriptional activation (27), as well as in the cAMP and growth
factor-induced signaling pathways (28). 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 (27, 28). 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 (8). 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 (8). PCAF, which is homologous to GCN5, targets the
same free histones H3 and H4 (8). 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.
Preparation of Recombinant p300 and PCAF--
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 (10).
Acetylation of Nucleosomal Histones in Vitro--
HeLa
mononuclesomes were prepared as described previously (29). Sucrose
density gradient-purified HeLa mononucleosomes (16 µg with respect to
DNA) were labeled in a 150-µl reaction containing 50 mM
Tris-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 (30).
The remainder of each sample was acid precipitated and purified by
reverse-phase HPLC and microsequenced as described previously (8).
Acid Urea Gel Analysis of Nucleosomal Histones Acetylated by the
PCAF Complex--
FLAG epitope-tagged PCAF was expressed in HeLa
cells, and a stable high molecular weight PCAF complex was purified as
described previously (31). Nucleosomes and free histones were
acetylated as described (31), 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 (32), 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 (8). 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. 33 and 34 and for review see Refs. 4 and 5). 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
(8, 13, 35). 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 (26-28), 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 (36). 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 (36). 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
(37)2 but acetylates H3 and
H4 when presented as free histones (8). Our results indicate that
Lys14 of H3 (Fig.
2A) and Lys8 of H4
(Fig. 2B) 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 Lys14
is the preferred steady-state site in vivo, and this residue is strongly preferred by PCAF under our assay conditions.
INTRODUCTION
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Abstract
Introduction
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Results & Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
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RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References
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Fig. 1.
In vitro acetylation of HeLa
mononucleosomes with recombinant human p300 and PCAF. Sucrose
gradient-purified HeLa mononucleosomes labeled in vitro with
[3H]acetyl-coenzyme A by recombinant p300 (lane
2) or PCAF (lane 3) were resolved on a 12%
SDS-polyacrylamide gel electrophoresis gel and fluorographed as shown.
Control reactions lacking histone acetylase enzyme were incubated with
bovine serum albumin (lane 1).
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Fig. 2.
Microsequence 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.
We expected that Lys16 of H4 would be acetylated by PCAF because this residue is acetylated in free histone H4 by yeast GCN5 in vitro (8). We considered that Lys16 may have been unavailable for acetylation in our experiments, because this is a known preferred acetylation site in vivo in HeLa cells (37). 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 vivo acetylation 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. 2C). 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 (38) or its proximity to the globular domain of H4 (38, 39). 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. 40).
We have reported that PCAF in its native form is present in a multisubunit complex containing more than 20 polypeptides (31). 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-associated factors); and TAF-related factors. Consistent with the observation that the interaction of p300 and CBP with PCAF in vivo is not stoichiometric or stable (10), the PCAF complex contains no p300 or CBP (31). 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 (31). 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 (31). 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. 3A, lanes 3 and 4, respectively) or nucleosomes (Fig. 3A, 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. 3B, 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.
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We have previously shown that p300 is capable of acetylating all four core histones in HeLa mononucleosomes (Ref. 11 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. 4C). 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 vivo but 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 (33, 34), 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 (10), demonstrate a strong preference for Lys14 of 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 (36).
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When nucleosomal H4 was acetylated by p300, a strong preference for Lys5 and Lys8 was detected (Fig. 4D). 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 (33, 34); 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 (36), despite the presence of three additional lysines (Fig. 4A). 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. 36 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. 3B). 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.
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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 (41-43), 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 GCN5 in vivo (18).
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 (26), as well as nuclear hormone receptor (27)
and growth factor-dependent signaling (28), 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
Present address: Dept. of Biochemistry and Molecular Genetics,
University of Virginia Health Sciences Center, Charlottesville, VA 22908.
To whom correspondence should be addressed: NIH, Bldg. 6, Rm.
416, Bethesda, MD 20892-2753. Tel.: 301-402-4904; Fax: 301-496-7823; E-mail: yoshi{at}helix.nih.gov.
The abbreviations used are: HAT, histone acetyltransferase; HPLC, high pressure liquid chromatography.
2 C. D. Allis, unpublished observations.
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REFERENCES |
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