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INTRODUCTION |
Chromatin structure plays a major role in all aspects of DNA
function in eukaryotes (1, 2). The nucleosome core particle, the
fundamental repeating unit of chromatin, consists of a core histone
octamer surrounded by 146 bp of DNA wrapped in a left-handed superhelix
(3-5). Recent studies have revealed that chromatin structure is highly
dynamic, with structural changes that result in alterations in gene
activity (6, 7). These structural alterations are mediated largely by
postsynthetic modifications of the flexible N-terminal amino acids of
the core histones and by ATP-dependent transitions in
nucleosome structure and position (8, 9).
Covalent modifications of nucleosomal histone tails have proved to be
of major importance for various nuclear processes (10-13), and the
best characterized of these modifications is the post-translational acetylation of specific lysine residues (14). Transcriptionally active
regions of chromatin have long been correlated with the presence of
hyperacetylated histones, whereas silent regions are correlated with
the presence of hypoacetylated forms (1, 15). Recent studies have
indicated that steady-state levels of histone acetylation are
maintained by a balanced equilibrium of histone acetylation and
deacetylation (16-18). Acetylation may alter internucleosomal interactions and thus alter higher order chromatin structure (4). However, the histone code hypothesis proposes that different
combinations of postsynthetic modifications may function as recognition
signals for proteins that regulate transcription more directly (10, 11,
19).
p300 and its paralog, CBP, are well known multifunctional
coactivators that mediate the action of a variety of transcription factors (20) and possess intrinsic histone acetyltransferase (HAT)1 activities (21, 22).
They can be recruited to promoters by direct interactions with DNA
binding transcription factors (20) and function with other cofactors
such as p300/CBP-associated factor (PCAF) and the
p160/SRC family of proteins (20). p300 contains a highly
conserved bromodomain that is found in many chromatin-associated
proteins (23-25). Although functional correlations between
bromodomains and histone modifications remain to be firmly established,
recent studies (26-30) have shown that bromodomains can serve as
histone recognition motifs for cofactor association with histone tails.
Our recent work (31) has indicated that the removal of histone H3 and
H4 tails or lysine-to-arginine substitutions at major acetylation sites
impairs p300-dependent transcriptional activation by
Gal4-VP16. Given that HAT-containing coactivators also can functionally
modify various (non-histone) regulatory proteins (32), these
observations provide the first proof of a direct link between
coactivator function and histone acetylation per se and thus
extend earlier studies showing only correlations between these events.
In this study, we generated a variety of histone-containing substrates
and systematically examined functional interactions with p300. Our
results reveal context-dependent differences in histone
acetylation that are relevant to an understanding of the mechanisms by
which p300-mediated histone acetylation affects transcription. In
addition, the free histone tails, notably those of histone H3 and H4,
are shown to modulate p300 activities in a manner that is largely
independent of their acetylation state. p300 is shown to bind directly
to unacetylated H3 and H4 tails but not to H2A and H2B tails. These
results support the idea of an activator-independent, histone H3 and H4
tail-mediated chromatin binding activity of p300, which may contribute
to functional effects of H3 and H4 tails in transcriptional regulation.
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EXPERIMENTAL PROCEDURES |
Preparation of Xenopus laevis Recombinant Histone--
Mutations
in the histone N termini were generated by PCR-directed mutagenesis as
described previously (31). The expression and purification of histones
were essentially as described by Luger et al. (33). Histone
preparations were analyzed by 15% SDS-PAGE and Western blot.
Construction and Expression of X. laevis Core Histone
Tails--
Glutathione S-transferase (GST)-histone tail
fusion proteins were made as follows: the N-terminal tail sequences
corresponding to residues 1-38 for H2A, 1-33 for H2B, 1-41 for H3,
and 1-36 for H4 were PCR-amplified and inserted into the
EcoRI and BamHI sites of the plasmid pGEX-2T.
Fusion proteins were expressed in E. coli strain
BL21 and purified on glutathione-Sepharose 4B (Amersham Biosciences)
following the manufacturer's protocol. Bound material was subjected to
thrombin digestion at 22 °C for 5 h and the supernatant, containing free tails, was dialyzed overnight against BC100. Tail integrity was checked by 18% SDS-PAGE (Fig. 3A) and tail
concentrations were determined both by Coomassie Blue staining and by
Bradford assay. Molar concentrations of tails were determined assuming masses of 5 kDa for nH2A, nH2B, and nH4 and 6 kDa for nH3.
Chromatin Assembly and Characterization--
FLAG-tagged
Drosophila ATP-dependent chromatin assembly factor
(ACF) subunits were expressed in Sf9 cells using a
baculovirus expression system and purified as described previously
(34). FLAG-tagged mouse nucleosome assembly protein 1 (NAP1) was
expressed in bacteria and purified as described previously (35).
Chromatin was assembled from recombinant histones and a 5.4-kb circular DNA template and characterized as described previously (31, 34). A
histone/DNA weight ratio of 1.1 gave optimal assembly under our conditions.
Histone Acetyltransferase and in Vitro Transcription
Assays--
FLAG-tagged human p300 and Gal4-VP16 proteins were
expressed and purified on M2-agarose (Sigma Chemical) according to
standard procedures. HAT and transcription assays were performed as
reported previously (31). For inhibition assays with free histone
tails, p300 was initially recruited to the promoter template by
Gal4-VP16. Histone tails were then added together with acetyl-CoA as
described in the figure legends.
Histone Tail and p300 Interaction Assays--
For binding
assays, recombinant p300 (2 µg) was incubated with the indicated
GST-tail (~1 µg) immobilized on glutathione-Sepharose beads in 200 µl of binding buffer (150 mM NaCl, 20 mM
HEPES, pH 7.5, 0.25 mM EDTA, 10% glycerol, 0.05% Nonidet
P-40, 1 mM phenylmethylsulfonyl fluoride, 2 mM
dithiothreitol, 5 µg/ml leupeptin, and 5 µg/ml aprotinin) in the
presence or absence of acetyl-CoA for 40 min at 30 °C. After
incubation, the beads were gently rotated for 3 h at 4 °C and
washed four times with binding buffer. Equal amounts of beads were
directly suspended in SDS sample buffer, and bound proteins were
resolved by 6% SDSPAGE and detected by Western blot with p300
antibody (Santa Cruz Biotechnology).
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RESULTS |
Substrate-dependent, Differential Modes of Histone
Acetylation--
To investigate the underlying mechanism for
p300-mediated histone acetylation, we first prepared recombinant
wild-type and mutant X. laevis histones (Fig.
1B) that either lacked the
N-terminal histone tails (indicated by prefix g) or
contained lysine-to-arginine (K/R) substitutions (indicated by prefix
m) at the major in vivo acetylation sites (in Fig
1A, prefix Ac). These individual histones were
then used to reconstitute different histone octamers: intact, H2A-tailless (nH2A
), H2B-tailless
(nH2B
), H2A- and H2B-tailless
(nH2A
+ H2B
),
H3-tailless (nH3
), H4-tailless
(nH4
), H3- and H4-tailless
(nH3
+ H4
), totally
tailless (All Tails
), H2A-mutant
(mH2A), H2B-mutant (mH2B), H2A- and H2B-mutant
(mH2A + mH2B), H3-mutant (mH3), H4-mutant
(mH4), H3- and H4-mutant (mH3 + mH4), and totally
mutant (All mTails). The purity of the assembled histone
octamers is shown in Fig. 1C.

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Fig. 1.
Preparation and characterization of diverse
histone substrates. A, schematic summary of histone tail
sequences. Arrows indicate sites of N-terminal deletions for tailless
histones. Ac indicates sites of acetylation. The amino acid
sequences indicate the tail regions fused to GST. B,
analysis of individual recombinant (lanes 1-12) core
histones and purified HeLa histones (lane 13) by 15%
SDS-PAGE and Coomassie Blue staining. Histone globular domains are
indicated by the prefix g and mutant (K/R) full-length
histones with the prefix m. C, analysis of
natural HeLa histone (lane 1) and reconstituted histone
octamers (lanes 2-16) by 15% SDS-PAGE and Coomassie Blue
staining. Reconstituted octamers contained all intact histones
(lane 2), the indicated tailless (lanes 3-9) or
mutant (lanes 10-16) histone(s) plus the complementary
intact histone(s) (lanes 3-16). D, micrococcal
nuclease (MNase) analysis of assembled chromatin. Chromatins
assembled with HeLa (lanes 2 and 3), intact
(lanes 4 and 5), totally tailless (lanes
6 and 7), totally mutant (lanes 8 and
9) histone octamers were treated with 0.5 mU (lanes
2, 4, 6, and 8) and 2 mU
MNase (lanes 3, 5, 7, and
9) for 7 min at 22 °C and analyzed by 1.2% agarose gel
electrophoresis and ethidium bromide staining. A 123-bp DNA ladder
(lane 1) was used as size marker (M).
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We first analyzed the ability of recombinant p300 to acetylate
individual free histones (Fig.
2A) under identical
conditions. Intact H2A, H2B, H3, and H4 histones each showed a
comparable level of acetylation (Fig. 2A, lanes
1-4). Interestingly, K/R H2A (positions 5 and
9), H2B (positions 2, 9,
12, and 17), and H3 (positions 9,
14, 18, and 23) mutants showed
acetylation levels comparable with those of wild-type free histones,
whereas the K/R H4 (positions 5, 8,
12, and 16) mutant showed a significantly lower
level of acetylation than wild-type H4 (Fig. 2A, lanes
5-8). This persistent acetylation of K/R mutant histones
apparently reflects compensatory acetylation by p300 of minor lysine
substrates in free histones. By contrast, free tailless gH2A, gH2B, and
gH3 mutants were acetylated to a very limited extent, whereas gH4 was
acetylated to a level comparable to that of intact histones (Fig.
2A, lanes 9-12); although interesting, the
possible significance of the latter result is unclear.

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Fig. 2.
Substrate-dependent specificity
of p300-mediated acetylation. A, HAT assay with individual
core histones. Each core histone (200 ng) was subjected to HAT assay
with p300 (20 ng), [3H]acetyl-CoA (2.7 µM)
as described under "Experimental Procedures." Reaction products
were analyzed by 15% SDS-PAGE. The indicated histones are identical to
those described in Fig. 1B. B, HAT assay with
free histone octamers. Assays were identical to Fig. 2A
except that free histone octamers were used. The indicated histones are
identical to those described in Fig. 1C. C, HAT
assay with recombinant chromatin templates. In each case the indicated
chromatin template was incubated with p300,
[H3]acetyl-CoA (2.7 µM), and Gal4-VP16 (20 ng). The histone octamers present in the reconstituted chromatins are
as described in Fig. 2B.
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In agreement with previous studies (22), a similar analysis of histone
acetylation in reconstituted histone octamers confirmed that intact H3
and H4 were highly acetylated, whereas H2A and H2B were only weakly
acetylated (Fig. 2B, lane 2). However, removal of
H3 and H4 tails enhanced the acetylation of H2A and H2B, whereas the
removal of H2A and H2B tails had no significant effect on H3 and H4
acetylation (Fig. 2B, lanes 3-8). Removal of all
histone tails showed acetylation of globular domains, most notably gH4 (Fig. 2B, lane 9). A similar analysis of octamers
reconstituted with mutant histones containing lysine-to-arginine
substitutions revealed that mH3 was still well acetylated (Fig.
2B, lanes 13, 15, and 16),
whereas mH4 was very weakly acetylated (lanes 14-16). Interestingly, mutation of H3 resulted in a ~3-fold enhancement of
wild-type H4 acetylation in the histone octamer (Fig. 2B,
lane 13 versus lane 2), whereas mutations of
other histones in the octamer showed no significant effect on H3
acetylation (lanes 10, 11, 12 and
14 versus lane 2). In contrast, mH2A and mH2B
showed extremely low levels of acetylation in various octamers
(lanes 10, 11, 12, and 16),
consistent with their poor substrate properties when assayed
independently (Fig. 2A).
Next, we extended our assay to chromatin substrates. Partial
micrococcal nuclease digestion of reconstituted chromatin templates generated regularly spaced DNA ladders (Fig. 1D), confirming
recent results (31, 36) that the histone N termini and their
premodifications are dispensable for ACF-dependent
nucleosomal array formation. Consistent with recent observations (31,
35, 37), p300-mediated acetylation of histones within reconstituted
chromatin substrates was completely dependent upon the presence of a
transcriptional activator (Gal4-VP16) that is known to interact
directly with p300 (Fig. 2C; data not shown). These results
also indicate not only high levels of H3 and H4 acetylation but also
highly enhanced levels of H2A and H2B acetylation in chromatin compared
with isolated histone octamers (Fig. 2C, lanes 1 and 2 versus Fig. 2B, lanes 1 and 2). Moreover, in confirmation of previous results
(31), an analysis of chromatins with histone tail deletions showed that H2A and H2B acetylation was strongly dependent upon H3 and H4 tails,
whereas removal of H2A and H2B tails had no effect on H3 and H4
acetylation (lanes 3-9); in addition, an analysis of
chromatins with lysine-to-arginine substitution mutations in the
histone tails failed to show any significant interdependency
(lanes 10-16). Hence, whereas the H3 and H4 tails are
required for optimal acetylation of H2A and H2B, their ability to be
acetylated at major lysine substrate sites is not. Concomitant
lysine-to-arginine substitutions in all four core histone tails
abolished acetylation as effectively as did removal of the tails
(lane 16 versus lane 9); these results confirm
the integrity of the assembled chromatin, because histones lacking
tails or containing the lysine-to-arginine substitutions are readily
acetylated when assayed as free histones or as DNA-free octamers (Fig.
2, A and B). Collectively, these data demonstrate distinct patterns and context-dependent interdependencies
of histone acetylation by p300, with an intrinsic preference for
histone H3 and H4 N termini.
Differential Repressive Effect of Free Histone Tails on
P300-dependent Acetylation--
A possible interpretation
of the histone acetylation results is that the H3 and H4 tails play the
major role in determining p300 function during and/or after its
recruitment by an activator. This possibility was investigated by
analyzing whether free histone tails, especially those of H3 and H4,
are efficient inhibitors of p300 function in solution. To this end, we
expressed and purified intact and K/R mutant (m) core
histone N termini (Fig. 3, prefix n) as GST fusion proteins (Fig. 3A, lanes
1-4 and 11-14), and then purified the tail components
after removal of GST with thrombin digestion. Fig. 3A shows
the Coomassie-stained patterns of electrophoretically resolved
wild-type and mutant H2A, H2B, H3, and H4 tails (lanes 6-9
and 16-19, respectively).

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Fig. 3.
Differential repressive effects of free
histone tails on histone acetylation by p300. A, SDS-PAGE
and Coomassie staining analysis of core histone tails. Intact histone
tails fused to GST (lanes 1-4), free intact histone tails
(lanes 6-9), mutant histone tails fused to GST (lanes
11-14), free mutant histone tails (lanes 16-19), and
GST only (lanes 5 and 15) as indicated. Histone
tails were released from GST-fusion proteins by thrombin digestion as
described under "Experimental Procedures." Molecular mass
marker sizes (lanes 10 and 20) are indicated in
kilodaltons. In A and B, intact and K/R mutant
histone tails are indicated by prefixes n and mn,
respectively. B, HAT assay with free histone tails. Each histone tail (80 ng) was subjected to HAT
assay and analyzed as described in the legend for Fig. 2A.
Intact (lanes 1-4) and K/R mutant (lanes 5-8)
histone tails are as indicated in Fig. 3B. C,
schematic representation of the in vitro competition assays
for p300-mediated acetylation and transcription. D,
competitive HAT assays with wild-type histone tails. Chromatin
reconstituted with intact histone octamers was incubated with
Gal4-VP16, p300, and acetyl-CoA at increasing concentrations of intact
N-terminal histone tail(s) as described in Fig. 3C. The
molar ratios of histone within the octamer to free histone tails varied
from 1:3 with 50 ng of tail to 1:20 with 340 ng of tail. Lanes
1-4, intact H2A tail (nH2A); lanes 5-8,
intact H2B tail (nH2B); lanes 9-12, intact H2A
and H2B tails (nH2A+nH2B); lanes 13-16, intact
H3 tail (nH3); lanes 17-20, intact H4 tail
(nH4); lanes 21-24, intact H3 and H4 tails
(nH3+nH4); lanes 25-28, intact H2A, H2B, H3, and
H4 tails (All tails); lanes 29-32,
GST. E, competitive HAT assays with K/R mutant
histone tails. HAT assays were performed as in Fig. 3D but
with K/R mutant (m) N-terminal histone tails.
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Isolated histone tails, normalized on the basis of Coomassie staining,
were then tested as substrates for p300. In contrast to what was
observed with free full-length histones (Fig. 2A), p300
acetylated predominantly wild type H3 and H4 tails with a slight
preference for the H3 tail (Fig. 3B, lanes 1-4).
Interestingly, mutant (lysine-to-arginine substitution) H3 and H4 tails
also were acetylated (Fig. 3B, lanes 7 and
8), although the levels were significantly lower than those
observed for wild type free histone tails. These results, showing that
free H3 and H4 tails are better acetylation substrates for p300 than
are free H2A and H2B tails, indicate that acetylation of the H2A and
H2B tails can be modulated by corresponding globular domains of H2A and H2B.
Next, to check whether free histone tails could act as competitive
inhibitors of p300 function, they were added to a HAT assay in which
chromatin templates assembled with intact core histones served as
substrates (Fig. 3C). As shown in Fig. 3D, a
slight inhibition of Gal4-VP16 dependent p300-mediated acetylation was observed only at the highest tested concentrations of nH2A (lanes 1-4), nH2B (lanes 5-8), or
nH2A+nH2B (lanes 9-12). In contrast, nH3 and nH4
(lanes 13-20) showed a much stronger inhibition of acetylation; their inhibitory concentrations were significantly (~8-fold) lower than those for nH2A and nH2B. Furthermore, an equimolar mixture of nH3+nH4 (lanes 21-24) showed
inhibitory effects comparable to those observed with equivalent masses
of nH3 or nH4, indicating that there was no synergistic effect of nH3
and nH4 tails in inhibition. When an equimolar mixture of all four histone tails (lanes 25-28) was used, the inhibition seemed
slightly less, suggesting that inhibition was proportional mainly to
the concentration of nH3 and nH4. A control assay with GST alone
(lanes 29-32) showed no inhibitory effect. In all the
assays, the final concentration of free tails was kept constant.
Competition assays with point mutated (lysine to arginine) histone
tails (Fig. 3, prefix mn) were also performed. As shown in
Fig. 3E, mnH2A (lanes 1-4), mnH2B (lanes
5-8), and a mixture of mnH2A and mnH2B (lanes 9-12)
all failed to abolish or significantly reduce the acetylation of
chromatin, whereas mnH3 (lanes 13-16), mnH4 (lanes
17-20), and a mixture of mnH3 and mnH4 (lanes 21-24) all diminished the HAT activity of p300 to an extent comparable to that
observed with wild-type histone tails. As expected from these results,
a mixture of all four mutant histone tails (lanes 25-28)
also inhibited p300 activity. Furthermore, identical assays using free
histone octamers as a substrate showed a comparable inhibitory effect
of H3 and H4 tails on p300 HAT activity, although slightly higher
concentrations of the tails were required (data not shown). The results
from these competition assays strongly support the notion of a
preferential association of p300 with H3 and H4 histone
tails in a chromatin context, and in a manner that is independent of
their ability to be acetylated.
Free H3 and H4 Tails Inhibit p300-mediated Transcription--
We
next examined the effect of ectopic free histone tails on p300-mediated
transcription activation. Transcription assays with recombinant
chromatin templates containing Gal4 binding sites upstream of core
promoter sequences were carried out as described previously (31),
except that core histone tails were added together with acetyl-CoA
(Fig. 3C). Transcription from this template is completely
dependent upon an activator (Gal4-VP16), p300, and acetyl-CoA (Fig.
4A) (31). As shown in Fig.
4B, nH2A (lanes 1-4), nH2B (lanes
5-8), and a mixture of nH2A and nH2B (lanes 9-12)
showed only a slight inhibitory effect on transcription at the highest
concentrations tested. In contrast, a significant inhibitory effect on
transcription was observed in the presence of comparable concentrations
of nH3 (lanes 13-16) or nH4 (lanes 17-20). In
addition, the experiments with a mixture of nH3 and nH4 (lanes
21-24) tails or of all four histone tails (lanes
25-28) failed to reveal any synergistic or cooperative effects of
the tails on this inhibition.

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Fig. 4.
Selective repressive effect of free H3 and H4
tails on p300- and Gal4-VP16-dependent transcription.
A, chromatin templates reconstituted with intact recombinant
histone octamers were transcribed according to the protocol of Fig.
3C and as detailed in An et al. (2002).
B, transcription was performed as in A but with
increasing concentrations of the indicated free histone tails. The
molar ratio of total free histone tails to total histones in chromatin
was 4:1, 20:1, and 40:1 in each set of dose-response assays.
C, transcription was performed as in Fig. 4B but
with increasing concentrations of mutant free histone tails.
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We also examined whether free mutant histone tails with
lysine-to-arginine substitutions could block the transcription
activation by p300. Consistent with the results of the HAT
competition assays, mutant H3 and H4 tails, alone or together, showed
strong inhibitory effects on the p300-dependent
transcription (Fig. 4C, lanes 13-24). By
contrast, mutant H2A and H2B tails, alone or together, minimally affected transcription at the highest concentrations tested (Fig. 4C, lanes 1-12). A control analysis showed that
comparable concentrations of GST alone did not affect transcription
(Fig. 4C, lanes 29-32). Taken together, these
results suggest that association of p300 with N-terminal tails of H3
and H4 can regulate p300-mediated transcription in a manner that is
largely independent of their acetylation at natural acetylation sites.
Preferential Interaction of p300 with H3 and H4 Tails in
Vitro--
To examine how the histone tails influence p300 function
more directly, we analyzed the ability of intact and mutated (Fig. 3A, K/R substitutions) histone tails to interact with a
fixed concentration of p300. Equimolar amounts of individual GST-tail fusion proteins (Fig 3A, lanes 1-4 and 11-14)
were prebound to GST beads and incubated with p300. After extensive
washing of the beads, bound proteins were eluted and analyzed by
Western blot with p300 antibody. To investigate the possible
involvement of histone tail acetylation in p300 interactions, the
assays were conducted in the absence or presence of acetyl-CoA. As
shown in Fig. 5A, p300 binding
was highly selective for intact H3 and H4 tails (lanes 5 and
6) relative to intact H2A and H2B tails (lanes 3 and 4). GST alone showed no binding (lanes 2 and
12). Interestingly, the binding of intact H3 and H4 tails,
which lack any acetylated residues because of expression in bacteria,
was independent of the presence of any accompanying acetylation.

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Fig. 5.
In vitro interactions of histone
H3 and H4 tails with p300. A, purified recombinant p300 was
incubated with GST-histone tail fusion proteins in the presence and
absence of acetyl-CoA. Intact and K/R mutant histone tail fusion
proteins are as described in Fig. 3A. The input lane
contained 1/10 of the p300 used in each interaction assay.
B, quantitation and summary of GST-tail pull-down assays.
Averaged values from three experiments, including that in A,
are presented.
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To investigate the possible role of lysine residues at the major
acetylation sites for regulating p300-tail interactions, we conducted
identical binding assays with mutant histone tails bearing
lysine-to-arginine substitutions (Fig. 5, prefix m) fused to
GST. As with unmodified tails, positive interactions were detectable exclusively with H3 and H4 tails (Fig. 5A, lanes
13-16), although the absolute levels of bound p300 were 2-3-fold
lower than those observed with wild-type counterparts (lanes
5 and 6 versus 15 and 16). In
this case, however, there was an apparent effect of acetyl-CoA on
p300-mutant histone tail interactions, as reflected by moderate
1.8-3-fold increases in the binding of p300 to mutant H3 and H4 tails
(lanes 15 and 16 versus lanes 19 and
20). This suggests that, unlike the major lysine substrates,
the acetylation of minor lysine residues may functionally contribute to
p300 recruitment by acetylation when the major lysine substrates are
absent. The binding efficiency with the different histone tails and the
effect of acetylation on the interactions are quantitatively presented in Fig. 5B as average percentages of p300 retention on beads
from three separate experiments. Taken together, these results, in agreement with the tail competition assays, indicate a stable association of p300 with histone H3 and H4 tails that is
acetylation-independent and functionally significant for
p300-mediated transcriptional activation.
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DISCUSSION |
We recently demonstrated a direct link between the presence and
acetylation status of histone H3 and H4 tails and
p300-dependent transcription from chromatin templates (31),
but the underlying mechanisms remain to be unraveled. In the present
work, and through systematic analyses with purified wild type and
mutant recombinant histones, we describe discrete patterns of
p300-mediated acetylation with different histone substrates and a
selective, acetylation-independent interaction of p300 with H3 and H4
tails. Our data clearly underscore an intrinsic preference of p300 for
physical and functional interactions with H3 and H4 tails, and possible
roles of their interaction in transcriptional regulation are discussed below.
Substrate and Context Effects on p300-mediated Histone
Acetylation--
As a first step toward understanding the mechanisms,
we investigated the histone acetylation activity of p300 with various substrates. One important observation, both confirming and extending previous results, is the effect of context on histone acetylation by
p300. Thus, whereas all histones are good substrates when tested individually, H3 and H4 are the preferred substrates when free histone
octamers are assayed at a limiting p300 concentration. By contrast, at
the same limiting p300 concentration, all histones are acetylated in
chromatin in response to a transcriptional activator.
Context effects are also evident from studies with histone tail
deletions. Thus, the joint deletion of H3 and H4 tails results in
increased H2A and H2B acetylation in free histone octamers but, most
significantly, decreased H2A and H2B acetylation in chromatin. In
contrast, joint deletion of H2A and H2B tails has no significant effect
on acetylation of H3 and H4 tails either in free octamers or in
chromatin. Similar studies with histones containing lysine-to-arginine
substitutions at the major acetylation sites indicated some modest
interdependencies of acetylation events in free histone octamers but
almost none in chromatin substrates. The latter results with histone
tails that are largely intact has important implications for
acetylation-independent binding of p300 to chromatin (see below).
In almost all cases, these results are most simply explained by an
intrinsic preference of p300 for H3 and H4 substrates and by
competitive substrate interactions. This conclusion is supported by the
observation that isolated H3 and H4 tails are both preferentially acetylated (by p300) relative to isolated H2A and H2B tails (Fig. 3B). In contrast, the context-dependent
requirement of H3 and H4 tails for maximal H2A and H2B acetylation
within chromatin must have another basis. This could reflect structural
differences between tails within free and nucleosomal histones or the
stabilization and enhanced catalytic activity of p300 on chromatin
through interactions either with Gal4-VP16 (35) or (via its
bromodomain) with unacetylated (27) or acetylated (26, 28-30) H3 and
H4 tails.
Functional Implications of H3 and H4 Tail-dependent
Association of p300 with Chromatin--
Competition studies (Figs. 3
and 4) allowed an analysis of the effect of each histone tail on p300
activity and showed a dosage-dependent repression of p300
function that was specific for H3 and H4 tails. Similar inhibitory
effects were also observed with free histone H3 and H4 tails containing
lysine-to-arginine substitutions in major acetylation sites. This
suggests that p300 may recognize distinct features of the H3 and H4
tails other than acetylated lysines, as recently reported for GCN5
bromodomain and histone H4 tail interaction (27). Therefore, the
observed inhibitory effects could reflect masking of substrate
recognition sites (e.g. bromodomains) in p300
and/or the induction of conformational changes that decrease p300
acetylation activity. The in vitro binding assays (Fig. 5)
further confirm dominant interactions of p300, not only with wild type
H3 and H4 tails but also with mutant tails containing
lysine-to-arginine substitutions, again suggesting the selective
recognition of H3 and H4 tails by p300.
Although structural studies of bromodomain-acetylated peptide complexes
support the hypothesis that acetylated tails may directly recruit
factors that are functionally associated with chromatin (26, 28-30),
our results suggest that p300 alone may associate with chromatin
through interactions with unmodified H3 and H4 tails. Indeed, our
results are in good agreement with a recent report (38) showing that
histone acetylation plays a minor role (at least quantitatively) in the
stable bromodomain-dependent association of p300 with
chromatin or isolated histones. However, our finding that H3 and H4
interact equally well with p300 differs slightly from the finding (38)
that p300 interacts preferentially with H3. These divergent results may
reflect differences in the assays employed
namely GST-p300 bromodomain
binding with a mixture of all four core histones versus
binding of individual histone tail-fused proteins with full-length p300.
It is worth noting that incubation with acetyl-CoA promotes
interactions between p300 and mutant H3 and H4 tails containing lysine-to-arginine substitution at the major acetylation sites (Fig.
5A, lanes 15 and 16 versus
19 and 20). Because these mutations do not abrogate
acetylation of the free histones (Fig. 2A), secondary acetylation may compensate for reduced interactions that result from
the lysine-to-arginine mutations at the major acetylation sites but may
involve mainly other residues. In this regard, previous studies have
indicated that other residues or regions of the histone tails may
provide major sites for acetylation-independent recognition of histone
tails by bromodomains (27, 30).
The Role of Histone Modifications in Gene Regulation--
The
finding that free H3 and H4 tails interact strongly with p300, together
with the observation that acetylation of major lysine substrates of
histone tails seems not to be necessary for this association, raises
the possibility that the acetylation of specific lysine residues may
mainly be involved in recruiting other regulatory factor(s). Such
factors could be associated with subsequent chromatin reorganization
events that lead to an active state of transcription. In this respect,
our observations support the view that the recognition of modified
histone tails by various factors or protein complexes (10, 11, 19, 39,
40) may account for the indispensable features of histone H3 and H4
tails in our studies. In the context of this model, the identification and characterization of potential factors/complexes that associate with
modified histone tails will facilitate the elucidation of the
mechanistic role of histone modifications. Indeed, recent studies have
documented modified histone tails as recognition motifs for highly
conserved domains in chromatin-associated factors (10, 11, 19, 39, 40).
Moreover, specific tail modifications seem to be primarily, if not
solely, responsible for the recruitment and anchoring of various
transcriptional regulatory factors in transcription (26, 28-30, 41,
42). In this regard, a recent study (43) has demonstrated that the
histone H3 N terminus can bind to the NuRD complex when lysine 9 is
methylated, but that methylation at lysine 4 effectively disrupts this
interaction. These results suggest specific roles for different tail
modifications in regulating the association or recruitment of various factors.
Our findings suggest a novel role(s) for H3 and H4 tails in p300
recruitment and retention that is correlated with essential roles of
histone H3 and H4 tails in p300-mediated transcription (31). However,
it is not yet clear at which stage(s) the described p300-tail
interactions play a role. They could be required before or during
activator-targeted recruitment of p300 to promoters or for continuous
and more stable association of p300 with chromatin after recruitment
and in fulfillment of the p300 coactivator function. In any case, our
results suggest that direct contact between p300 and histone H3 and H4
tails is involved, at some stage, in p300 function. Further
characterization of the timing and function of histone acetylation
events during the course of the overall transcription reaction will
facilitate the elucidation of the functional role of histone tails and
their modifications.