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INTRODUCTION |
The compaction of the eukaryotic genome in nucleosomes and the
higher order folding of nucleosome arrays present barriers to
regulatory proteins and the multisubunit enzymes that process genetic
information (reviewed in Refs. 1-5). In the nucleosome core particle,
winding of 147 bp1 of DNA
over an octamer of histones creates severe distortion of the DNA helix
and obscures roughly half of the helix surface (reviewed in Refs. 6 and
7). To counteract the constraints imposed by chromatin architecture,
cells employ several distinct mechanisms. Strategies to destabilize
chromatin include the use of homopolymeric stretches of DNA that resist
bending; architectural, high mobility group-type proteins that
unfold nucleosome arrays; histone-modifying enzymes that covalently
alter specific residues of the histone tails; and
ATP-dependent chromatin remodeling complexes that
facilitate nucleosome mobility (reviewed in Refs. 8-15). In addition,
the passage of an elongating RNA polymerase is also facilitated by
distinct proteins that alter chromatin structure (16).
ATP-utilizing chromatin remodeling complexes can be classified into two
main groups, containing either the SWI2/SNF2 or the related ISWI
ATPases and their close relatives (reviewed in Refs. 17 and 18).
SWI2/SNF2-containing complexes are large assemblies in the megadalton
size range and composed of 11-15 distinct polypeptides. ISWI-containing complexes are smaller and are composed of 2-5 subunits. Both types of chromatin remodelers use the free energy of ATP
hydrolysis to increase nucleosome mobility by changing nucleosome
conformation (19-22). A large body of evidence implicates the
SWI2/SNF2-containing complexes in transcription regulation. The
swi2/snf2 gene was originally identified genetically
as a transcriptional regulator in yeast (reviewed in Refs. 23 and 24).
Recent genetic studies show that Drosophila iswi
is required for engrailed and Ultrabithorax
expression in vivo (25). In addition, SWI2/SNF2- and
ISWI-containing complexes isolated from yeast, flies, and mammals can
assist the transcriptional activation of model chromatin templates
in vitro (26-32).
The histone acetyltransferases (HATs) and histone deacetylases,
which catalyze the reversible modification of specific lysines on the
N-terminal histone tails, are the most extensively studied of the
histone-modifying enzymes. The similarity of Tetrahymena p55
HAT and mammalian HDAC1 to genetically defined transcriptional regulators provided the first link between these histone-modifying enzymes and transcription (33, 34). Subsequent analysis of the growing
family of HATs and HDACs has shown that they can form large, multimeric
complexes that can be recruited to gene promoters (reviewed in Refs.
35-44). Experiments in vitro on reconstituted chromatin
templates have shown that histone acetylation can facilitate transcription (45-54).
Although the separate contributions of histone hyperacetylation and
ATP-driven chromatin remodeling to transcription are well known, the
interrelationships between these two major types of chromatin
modifications and their relative contributions to the activation
process are only beginning to be explored. Genetic studies indicate
that the yeast SWI2/SNF2 and GCN5 genes perform independent but overlapping functions during transcriptional activation (55-61). To date, however, no biochemical studies have examined the
mechanistic relationship between histone hyperacetylation and
ATP-dependent chromatin remodeling. To address this
question, we analyzed the relative contributions of
Drosophila nucleosome remodeling factor (NURF) and histone
acetylation to the activation of a model chromatin template in
vitro.
Drosophila NURF was identified as a four-subunit,
ISWI-containing complex that has been implicated in transcriptional
activation of chromatin (62-67). Other ISWI-containing complexes
related to NURF have subsequently been characterized from
Drosophila (ACF (68) and CHRAC (21)) and human cells
(RSF (30) and WCRF/hACF (69, 70)). In a previous report, we
demonstrated that purified NURF enables activation of a chromatin
template by GAL4-HSF at an early step in the process of transcription
(27). Here, we demonstrate synergism between the hyperacetylation of
histones and nucleosome remodeling by NURF. Histone hyperacetylation
alone did not stimulate transcription from our model chromatin
template. However, in combination, the addition of NURF to
hyperacetylated chromatin leads to synergistic activation of
transcription. We suggest that a hierarchy exists in which
NURF-dependent chromatin remodeling is an obligatory step
during promoter activation. By the use of single and multiple round
transcription experiments we demonstrate that the two types of
chromatin modification stimulate transcription of chromatin both at the
preinitiation and reinitiation stages.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of Recombinant p300 and Human P/CAF
Complexes--
FLAG epitope-tagged human p300 was expressed in
Sf9 cells after infection with recombinant baculovirus, and
protein was purified as described elsewhere (71). Human P/CAF complex
was purified from nuclear extracts of a HeLa cell line expressing FLAG
epitope-tagged P/CAF as described (72).
Bacterial Expression and Purification of GAL4-VP16--
Plasmid
pGM2a expressing the GAL4-VP16 fusion protein was constructed by
inserting the XhoI-BamHI fragment of pJL2 (73), which encodes amino acids 95-147 of GAL4 and the carboxyl-terminal 291 amino acids of the VP16 transactivation domain, of plasmid pJL2 into
plasmid pGM1 digested with XhoI and BamHI (27).
Expression of GAL4-VP16 was induced in Escherichia coli BL21
(DE3) pLysE (Novagen) and purified as described (27, 74).
Purification of NURF--
NURF was purified from nuclear
extracts of 0-12 h Drosophila embryos up to the glycerol
gradient step as described (64).
Expression and Purification of Drosophila General Transcription
Factors--
Recombinant Drosophila TFIIA, TFIIB, TFIIE,
and TFIIF were expressed and purified as described (75).
Drosophila TFIID was immunoprecipitated with mAb 2B2 against
Drosophila TAF250 (76) from the MonoQ-TFIID fraction
prepared as described (75). The TFIID complex was washed in 0.1 M KCl-HEMGND buffer (25 mM HEPES-KOH (pH 7.6),
0.1 mM EDTA, 12.5 mM MgCl2, 10%
(v/v) glycerol, 0.1% (v/v) Nonidet P-40, 1 mM
dithiothreitol) and eluted with epitope-containing peptide in 0.1 M KCl-HEMGND containing 0.5 M guanidine
hydrochloride. The eluted TFIID was dialyzed against 0.1 M
KCl-HEMGND and used for in vitro transcription.
Drosophila TFIIH and RNA polymerase II were purified from
Drosophila embryo nuclear extract as described (75, 77).
Recombinant Drosophila TFIIS was synthesized in E. coli BL21 (DE3) (Novagen) and purified as described (78).
Chromatin Assembly--
Chromatin was assembled using a
Drosophila embryo S-190 extract (79) and cosmid pWEGIE-0 as
a template. Cosmid pWEGIE-0 was constructed by insertion of the 1208-bp
AatII-EcoRI fragment of pGIE-0 (see Ref. 80; the DNA
fragment contains five GAL4-binding sites and a TATA box from the
adenovirus type 5 E4 promoter) into the AatII and
EcoRI sites of cosmid pWE15 (Promega) DNA. The S-190 extract
(275 µl; ~7.0 mg of protein) was incubated with purified Drosophila core histones (4.5-5.0 µg) (81, 20) and buffer R (10 mM HEPES-KOH (pH7.6), 0.5 mM EGTA, 1.5 mM MgCl, 10% (v/v) glycerol, 10 mM KCl, 10 mM
-glycerophosphate, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) in a total volume of
630 µl at 4 °C for 30 min. Cosmid pWEGIE-0 DNA (5 µg), ATP
(3 mM), creatine phosphate (30 mM), creatine
phosphokinase (1 µg/ml), and MgCl2 (7 mM)
were added in a final volume of 700 µl, and assembly was carried out
at 26 °C for 6 h.
Purification of Preassembled Chromatin by Sucrose Gradient
Sedimentation--
Sucrose gradient purification of preassembled
chromatin was performed essentially as described (79). Briefly, 2.8 ml
of preassembled chromatin mixture was applied onto a Beckman SW41 tube
and centrifuged in a Beckman SW41 rotor at 26,000 rpm for 16 h at
4 °C. The sucrose gradient buffer was 10 mM HEPES-KOH (pH 7.6), 1 mM EDTA, 35 mM NaCl containing
from 30 to 50% (w/v) sucrose. 1.0-ml gradient fractions were
collected, and DNA concentration was determined after 1% agarose gel
electrophoresis of an aliquot (40 µl) by ethidium bromide staining.
Histone Hyperacetylation of Chromatin by p300 and P/CAF
Complex--
The sucrose gradient fraction was directly subjected to
treatment of the histone acetyltransferases. Typically, 100 µl
(1.4 µg of DNA equivalent) of the gradient fraction was incubated at 26 °C for 30 min with 1 mM dithiothreitol, 1 mM sodium butylate, 1 mM acetyl-CoA (lithium
salt; Amersham Pharmacia Biotech), and ~5 pmol of p300 or P/CAF
complex. Hyperacetylated chromatin was further purified and subjected
to chromatin remodeling and transcription. For conventional
purification of hyperacetylated chromatin, Sepharose CL4B (Amersham
Pharmacia Biotech) was used in a SizeSep-400 spin column (Amersham
Pharmacia Biotech) preequilibrated with elution buffer (10 mM HEPES-KOH (pH 7.6), 0.5 mM EGTA, 5 mM MgCl, 10% (v/v) glycerol, 50 mM KCl, 10 mM
-glycerophosphate, 1 mM
dithiothreitol). Bovine serum albumin (Roche Molecular Biochemicals)
was added to chromatin fractions after purification to a final
concentration of 0.5 mg/ml. DNA content was estimated by agarose gel
electrophoresis and ethidium bromide staining.
Histone Acetyltransferase Assay--
For analysis of histone
acetylation by SDS-PAGE and fluorography, 100 µl (1.4 µg of DNA
equivalent) of the sucrose gradient purified chromatin was processed as
described above, except that 1 nmol of [3H]acetyl-CoA was
introduced in place of 1 mM acetyl-CoA (lithium salt). The
reaction was quenched by the addition of SDS-PAGE sample buffer and
analyzed on 15% SDS-PAGE. The gels were then stained by Coomassie
Brilliant Blue and exposed for fluorography. For Triton-acid-urea gel
analysis, samples were processed as described above, using 1 mM acetyl-CoA (lithium salt). The acetylated chromatin was
then precipitated with trichloroacetic acid and analyzed on a TAU gel,
essentially as described (82).
In Vitro Transcription of Chromatin Template--
In
vitro transcription and primer extension analysis were performed
as described previously (27, 83, 84). For the purified transcription
system, typically 40 ng of chromatin (40 µl of spin column-purified
chromatin) was preincubated in 80 µl (final volume) containing 25 ng
of TFIIA, 15 ng of TFIIB, ~120 ng of immunopurified TFIID, 50 ng of
TFIIE, 50 ng of TFIIF, ~40 ng of purified TFIIH, 10 ng of TFIIS,
~30 ng of RNA polymerase II, 20 mM HEPES-KOH (pH 7.6), 5 mM MgCl, 40 mM KCl, 2.6% (v/v) polyethylene
glycol 8000 (final concentration), 3.75 mM
(NH4)2SO4, 1 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride for 30 min at 26 °C to form preinitiation complexes and then transcribed
for 10 min at 26 °C with the addition of ribonucleotide
triphosphates (0.55 mM final concentration). Transcripts
were detected by primer extension using a 32P-labeled AdE4
primer corresponding to positions 72-99 of the transcribed strand of
pWEGIE-0. cDNA products were analyzed on a 6% denaturing
polyacrylamide gel. Quantitation of transcripts was performed on a Fuji
Bio-Image Analyzer.
For transcription in a crude system, soluble nuclear fraction (nuclear
extract) was prepared from 0-12-h Drosophila embryos as
described (85, 86). Reactions were performed as described above, except
that 70 µg of the soluble nuclear fraction was used instead of
purified general transcription factors and RNA polymerase II.
For the heparin challenge protocol, reactions were performed
essentially as described for transcription with the purified system. A
synthetic RNA polymerase II pause at +5 site was created by the
addition of 0.55 mM ATP, 0.55 mM CTP, and 0.55 mM UTP. Following incubation at 26 °C for 5 min, 400 ng
of heparin and 0.55 mM GTP were added to block reinitiation
of RNA polymerase II, and the reactions were incubated for a further 30 min at 30 °C (87, 88). Transcribed RNAs were analyzed by primer
extension as above.
Micrococcal Nuclease and Restriction Enzyme Digestion of
Chromatin--
Micrococcal nuclease (MNase) digestion analysis and
sequential Southern blot hybridization were performed as described (62, 80, 89). A restriction enzyme accessibility assay was performed essentially as described (90). Typically, 50 µl (~50 ng of DNA equivalent) of spin column-purified chromatin, 0.5 µl of NURF fraction, ATP (0.5 mM final concentration), and 0.5 pmol of
GAL4 derivative (GAL4-VP16) were mixed and adjusted to 65 µl with
elution buffer. The reaction was incubated at 26 °C for 30 min and
subjected to micrococcal nuclease or restriction enzyme digestion. For
MNase digestion, the reaction mixture was directly treated with enzyme and analyzed as described (27). For restriction enzyme digestion, the
reaction mixture was incubated with either 0.5 unit of BamHI (digested at position
46) or 0.5 unit of HaeII (digested
at position +98), at 26 °C for 30 min. Digested DNA samples were
deproteinized, precipitated with ethanol, and redigested with
PstI and ClaI. Samples were loaded on a 1.3%
agarose gel and analyzed by Southern blot hybridization as for the
MNase assay.
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RESULTS |
In Vitro Transcription of Hyperacetylated Chromatin--
To
analyze the relative contributions of histone acetylation and
ATP-dependent chromatin remodeling to transcription, we
used a 9-kilobase pair cosmid (pWEGIE-0) containing five tandemly
repeated GAL4 binding sites upstream of the adenovirus E4 core promoter (80). The experimental procedure for reconstitution and acetylation of
pWEGIE-0 chromatin is outlined in Fig.
1A. After assembly with the
Drosophila embryo S-190 extract and purified
Drosophila core histones, chromatin is purified by sucrose
gradient sedimentation. This procedure removes the bulk of nonhistone
proteins, leaving histones as the predominant proteins in the chromatin
preparation, as judged by SDS-PAGE and silver staining (79). The
purified chromatin is then modified with a HAT and acetyl-CoA, followed by repurification on a Sepharose CL4B spin column, to preclude acetylation of remodeling proteins and transcription factors that are
introduced subsequently. Chromatin is next incubated with saturating
amounts of the transcription activator GAL4-VP16 and saturating amounts
of purified NURF and ATP to allow the mobilization of nucleosomes. This
is followed by the assembly of preinitiation complexes with either a
Drosophila soluble nuclear fraction that has little
ATP-dependent chromatin remodeling activity (27, 80) or a
purified transcription system consisting of native and recombinant
Drosophila general transcription factors and RNA polymerase
II. Transcription is then initiated upon the addition of ribonucleotide
triphosphates (NTPs), and the RNA products after 10 min of
transcription are analyzed by primer extension. We note that
Drosophila embryo extracts contain histone
acetyltransferases and deacetylases that can modify histones during
chromatin assembly (45, 47, 82). However, we and others (47, 82) have
found the chromatin product after in vitro assembly yields
histones whose final levels of acetylation are extremely low, thus
providing an appropriate substrate for histone hyperacetylation.

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Fig. 1.
Histone hyperacetylation facilitates
transcription of chromatin. A, experimental protocol.
B, histone hyperacetylation by human p300. Top,
15% SDS-PAGE and Coomassie Blue staining. Middle,
[3H]acetyl-labeled histones were detected by
fluorography. Bottom, Triton-acid-urea (TAU) gel
electrophoresis and silver staining showing monoacetylated (1 Ac), diacetylated (2 Ac), and triacetylated (3 Ac) forms of H4. C, primer extension analysis of RNAs
transcribed using Drosophila embryo nuclear extract under
conditions as indicated. After histone hyperacetylation by p300 and
further purification by CL4B gel filtration, a portion (40 ng) of the
chromatin was incubated with 0.2 pmol of GAL4-VP16, 0.2 µl of NURF
(2.5 ng/µl glycerol gradient fraction), and 0.5 mM ATP
for 30 min to allow chromatin remodeling. The sample was then incubated
with nuclear extract, followed by the addition of NTPs for
transcription. D, in vitro transcription using
purified components. Primer extension analysis of transcription
reactions was performed as above, except for the use of purified
Drosophila general transcription factors (GTFs)
and RNA polymerase II (Pol II). In C and
D, the -fold activation is the average of three independent
experiments. Hence, the numerical values do not fully correlate with
visual inspection of the presented autoradiograph.
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NURF and Histone Hyperacetylation Synergistically Facilitate
Transcription of Chromatin--
We utilized recombinant human p300
as a potent HAT enzyme to hyperacetylate nucleosomal histones (71,
91); p300 preferentially acetylates nucleosome core histones in
vitro at the same sites that are acetylated in vivo
(92). We confirmed that histone hyperacetylation by p300 occurs in our
reactions by the incorporation of [H3]acetyl-CoA into all
four core histones (Fig. 1B, middle
panel). In addition, Triton-acid-urea gel electrophoresis
showed that p300 quantitatively modifies, in an acetyl
CoA-dependent fashion, the bulk of histone H4 in
reconstituted chromatin, leading to a conversion from a predominantly
unacetylated form (for purposes of discussion, we refer to this state
as "unacetylated") to the mono-, di-, and triacetylated states
(Fig. 1B, bottom panel). The
sequential operations of histone hyperacetylation and
ATP-dependent chromatin remodeling by NURF resulted in very
high level activation of the E4 promoter in a crude transcription
system (139-fold; Fig. 1C, lane 16).
(In concurrence with previous studies in vivo and in
vitro, initiation from the E4 promoter occurs at
A+1 and T
6 positions (103, 104);
unless otherwise noted, we use the sum of the transcription signals
from both positions as a measure of -fold activation. In addition, the
numerical values given are averages of three experiments.) This
139-fold level of activation was ~5-fold higher than the activation
conferred by NURF in the absence of histone hyperacetylation (30-fold;
Fig. 1C, lane 4). The ~5-fold
increase was dependent on both p300 and acetyl CoA, since it was not
observed upon the omission of either reagent (Fig. 1C,
lanes 8 and 12). Given that p300 is
known to harbor other activities (51, 71, 91), the requirement for
acetyl-CoA suggests that it is the HAT activity of p300 that is
responsible for the further increase of transcription. As previously
noted (e.g. Refs. 27 and 80), transcription of chromatin was
also highly dependent on the presence of the VP16 activation domain (data not shown; Fig. 1C).
We next investigated whether histone hyperacetylation could substitute
for the ATP-dependent nucleosome remodeling step by omitting NURF from the reaction protocol. Interestingly, there was
little or no activation of the E4 promoter in the absence of remodeling
by NURF, despite bulk histone hyperacetylation by p300 (1.1-fold; Fig.
1C, lane 14). To confirm the above
transcription results, which were obtained with the
Drosophila soluble nuclear fraction, we prepared a purified
Drosophila polymerase II transcription system using
bacterially expressed and native components (see "Experimental
Procedures") and performed the same set of experiments. With the
purified transcription system, we were able to demonstrate again the
synergy between histone hyperacetylation and nucleosome remodeling
by NURF. Transcription of chromatin with the purified polymerase II
system showed that activation was increased further by ~5-fold when
histone hyperacetylation by p300 is included in the protocol (202-fold;
Fig. 1D, lane 16, versus
33-, 42-, and 37-fold; lanes 4, 8, and
12). Moreover, little or no activation was observed when
NURF was omitted from the protocol (1.1-fold; Fig. 1D,
lane 14). Thus, core histone hyperacetylation can
operate synergistically with NURF but is unable to replace it
functionally in activating transcription of chromatin, suggesting that
ATP-dependent nucleosome remodeling is an obligatory event
for the GAL4-E4 promoter.
We performed additional experiments that underscore a role for
histone hyperacetylation in facilitating transcription of chromatin. Introduction of desulfocoenzyme A (DSA), an
acetyltransferase inhibitor, along with p300 and acetyl CoA blocked the
synergistic activation of chromatin (35-fold; Fig.
2A, lane
22), while synergy was retained when desulfocoenzyme A
was introduced after chromatin was hyperacetylated (163-fold; Fig.
2A, lane 18). We also deliberately reintroduced acetyl-CoA at a late stage of the protocol (after the
removal of p300), to assess whether synergistic activation might be
caused by undetected HAT activities that could be contaminating the
NURF preparation or are dominant in the transcription extract. As shown
in Fig. 2B, continuous exposure of the chromatin template to
acetyl-CoA during ATP-dependent remodeling, preinitiation
complex formation, and transcription did not further increase
activation (32-fold (Fig. 2B, lane 2)
versus 30-, 29-, and 28-fold (lanes 6,
10, and 14)). Taken together, the results
indicate that hyperacetylation of bulk histones can operate
synergistically with NURF in GAL4-VP16-mediated transcription of
chromatin.

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Fig. 2.
Histone acetyltransferase activity of p300
facilitates transcription of chromatin. A, primer
extension showing transcription of chromatin template treated with the
acetyltransferase inhibitor desulfocoenzyme A (DSA).
Experimental protocol is shown at the top. 10 mM
desulfocoenzyme A was introduced simultaneously with p300 and
acetyl-CoA (DSA (i)) or with GAL4 activator and NURF
(DSA (ii)). B, primer extension showing
transcription after reintroduction of 1 mM acetyl-CoA for
the remodeling and subsequent steps.
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Hyperacetylation of Core Histones Fails to Mobilize
Nucleosomes--
To examine how histone hyperacetylation might assist
in transcription, we analyzed the structure of the acetylated chromatin by digestion with micrococcal nuclease (MNase), which cleaves nucleosome linker DNAs. After histone hyperacetylation with p300, chromatin was repurified and incubated with GAL4-VP16, digested with
MNase, and processed for Southern blot hybridization with an E4
promoter-specific oligonucleotide. As previously reported, MNase
digestion of chromatin reconstituted with the Drosophila extract produces a "ladder" of DNA fragments with a repeat length of ~180 bp, corresponding to periodically spaced nucleosomes (Ref. 27; Fig. 3A, panel
1; two digestion points per panel). Previous studies (27, 28) also indicated that, in the absence of NURF, a
saturating amount of GAL4-VP16 is able to bind to the five cognate sites (spanning ~100 bp) without disturbing periodic nucleosome spacing at the E4 promoter. We observed similar results (Fig. 3A, panel 2; data not shown).
(GAL4-VP16 binding to linker DNA protects it from MNase digestion and
increases the yield of fragments corresponding to dinucleosomes).

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Fig. 3.
Histone hyperacetylation by p300 does
not remodel nucleosomes. A, nucleosome organization of
Gal4-E4 promoter after histone hyperacetylation by p300. An aliquot of
chromatin used for in vitro transcription after the
remodeling step was analyzed by MNase digestion (two digestion points)
and Southern blot hybridization, using a radiolabeled oligonucleotide
probe specific for the E4 promoter region ( 57 to 25 bp).
B and C, restriction enzyme accessibility. An
aliquot of chromatin after the remodeling step was digested with
BamHI (B) or HaeII (C) and
analyzed by Southern blot hybridization using a oligonucleotide probe
corresponding to positions 72-99 of the E4 promoter. The uncut
PstI/ClaI fragment and the BamHI- or
HaeII-cut fragments are indicated by arrowheads.
The percentage of digestion is given by the average value from three
sets of experiments. D, locations of restriction enzyme
cleavage sites on the GAL4-E4 promoter are given in the diagram.
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Upon remodeling by NURF, the mobilization of nucleosomes away from GAL4
binding sites to slightly distal locations produces a smeared ladder of
DNA fragments in the MNase digestion assay (Ref. 27; Fig.
3A, panel 4). Importantly, in the
absence of remodeling by NURF, the hyperacetylated chromatin retained
periodic nucleosome spacing at the E4 promoter, as shown by the
integrity of the DNA ladder (Fig. 3A, panel
14). Hence, histone hyperacetylation by p300 could not
substitute for NURF to mobilize nucleosomes over GAL4 binding sites.
Furthermore, the combination of histone hyperacetylation by p300 and
chromatin remodeling by NURF did not lead to an increase in the
kinetics of MNase digestion (Fig. 3A, panels
4, 8, and 12 versus
panel 16).
As an alternative probe of chromatin accessibility, we used restriction
endonucleases instead of MNase. Chromatin was treated with
BamHI (cleavage position
46, within the region of DNase I
hypersensitivity induced by GAL4-VP16 and NURF; Ref. 27) or with
HaeII (cleavage position +98, in the downstream nucleosome). In the absence of remodeling by NURF, Southern blot analysis showed that BamHI digestion of hyperacetylated chromatin is only
slightly increased over unacetylated controls (18% versus
21, 20, and 22% digestion) (Fig. 3B, lane
14 versus lanes 2,
6, and 10). Similarly, HaeII digestion
was not substantially affected by histone hyperacetylation in the
absence of remodeling by NURF (1.0% versus 1.5, 1.5, and 1.1% digestion) (Fig. 3C, lane 14 versus lanes 2, 6, and
10). In contrast, remodeling by NURF caused a significant
increase of BamHI digestion from ~20 to ~60%. This
level of digestion was found for both unacetylated chromatin (Fig.
3B, lanes 2, 6, and 10 versus lanes 4,
8, and 12) and hyperacetylated templates (Fig. 3B, lane 14 versus
lane 16), confirming that histone
hyperacetylation did not alter nucleosome structure appreciably.
Similarly, remodeling by NURF increased HaeII digestion from
1.0 to 1.5% to 14-19% (Fig. 3C, lanes
2, 6, 10, and 14 versus lanes 4, 8,
12, and 16), independent of the acetylation state
of chromatin. Overall, the results indicate that the structural changes
induced by histone hyperacetylation that increase transcription occur
beyond the level of the nucleosome core particle and most likely
involve changes in higher order chromatin folding. (A study reporting
increased accessibility to restriction nuclease upon histone
hyperacetylation may not be inconsistent with our results, because the
restriction enzyme reaction contained ATP and HeLa nuclear
extract, a potential source of NURF-like nucleosome remodeling
activities; see Ref. 46).
NURF and Histone H3 Hyperacetylation Facilitate Transcription of
Chromatin--
Given that p300 potently acetylates all four core
histones, we used PCAF, a HAT with more restricted substrate
specificity, to analyze the effects of histone hyperacetylation (PCAF
acetylates histone H3 at lysine 14 preferentially; see Ref. 92; Fig.
4B). We found that histone H3
hyperacetylation by the purified PCAF complex leads to a 4.5-fold
increase of transcription over the level detected in its absence
(166-fold (Fig. 4C, lane 16)
versus 37-, 33-, and 36-fold (lanes 4,
8, and 12)). Consistent with the observations for
p300, histone H3 hyperacetylation by PCAF could not substitute for NURF
in remodeling chromatin for activation by GAL4-VP16 (Fig.
4C, lane 14). Moreover, MNase and
restriction enzyme digestion of histone H3 hyperacetylated chromatin
showed a lack of nucleosome mobilization at the E4 promoter when NURF was omitted from the protocol (Fig.
5A, panel
14 versus panel 16; Fig. 5,
B and C (lane 14 versus lane 16)). These findings underscore the
obligatory requirement for nucleosome mobilization by NURF for
transcription of our model chromatin template. The results also suggest
that hyperacetylation of one histone (H3) may be sufficient to create
synergy with NURF in facilitating activation by GAL4-VP16.

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Fig. 4.
P/CAF complex facilitates transcription of
chromatin. A, experimental protocol. B,
histone H3 hyperacetylation by human P/CAF complex. Top,
SDS-PAGE and Coomassie Blue staining showing core histones in the
chromatin sample (0.5 h) as in Fig. 1. Bottom,
[3H]acetylated core histones revealed by fluorography as
in Fig. 1. C, in vitro transcription using a
purified transcription system. Primer extension analysis was performed
as in Fig. 1, with the exception of using human P/CAF complex instead
of human p300.
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Fig. 5.
Histone hyperacetylation by P/CAF complex
does not remodel nucleosomes. A, nucleosome
organization of Gal4-E4 promoter after histone hyperacetylation by
P/CAF complex. An aliquot of chromatin used for in vitro
transcription (1.0 h) was analyzed by MNase digestion (two digestion
points) and processed as in Fig. 3. B and C,
restriction enzyme accessibility. An aliquot of chromatin used for
in vitro transcription (1.0 h) was digested with
BamHI (B) or HaeII (C) and
analyzed as in Fig. 3.
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NURF and Histone Hyperacetylation Facilitate Initiation and
Reinitiation of Transcription--
Our transcription reactions usually
proceed for only 10 min. In this way, the total number of rounds of
transcription is restricted. Therefore, our assay predominantly should
reveal effects on the preinitiation or initiation stages of
transcription. Nonetheless, the observed activation of the E4 promoter
could also include a contribution from transcription reinitiation
during the 10-min interval. Accordingly, we modified the protocol to
allow only a single round of transcription on the chromatin template
(Fig. 6A). After
hyperacetylation by p300 or PCAF, remodeling by NURF, and assembly of
preinitiation complexes, initiation is allowed with only three rNTPs
(ATP, CTP, and UTP), creating an artificial pause at +6 (the location
of the first G in the transcript). GTP and heparin are then introduced
to permit read-through of the first round transcript and to block
reinitiation of transcription, respectively (87, 88, 93) (Fig.
6A).

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Fig. 6.
Histone hyperacetylation and NURF facilitate
transcription preinitiation and reinitiation. A,
experimental protocol. B, primer extension analysis showing
multiple round (left) and single round transcription
(right) after histone hyperacetylation by p300. 5 min after
initiation with ATP, CTP, and UTP, heparin was introduced into the
reaction, followed by GTP; transcription was allowed for 10 min.
Transcripts originating from two major start sites at 6 and +1 on the
E4 promoter are indicated. For multiple round transcription, reactions
were performed as in single round transcription, except for the
omission of heparin. C, primer extension analysis showing
multiple round and single round transcription after histone
hyperacetylation by P/CAF complex. The reaction was performed as above,
except for using P/CAF complex instead of p300. -Fold activation in
B and C is the average of three independent
experiments.
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The single round transcription experiments revealed that transcription
from the
6-position of the E4 promoter was stimulated ~20-fold when
chromatin was treated with NURF (Fig. 6B, lanes 10, 12, and 14). There were no single
round transcripts when NURF was omitted from the protocol (data not
shown). When histone hyperacetylation by p300 was combined with the
action of NURF, we observed an additional ~1.9-fold increase in the
single-round transcript from the
6 start site (38-fold (Fig.
6B, lane 14) versus 21-fold
(lane 16)). Hence, as anticipated, NURF, and
histone hyperacetylation combined with NURF, stimulate transcription at
the preinitiation or initiation stage. Interestingly, single-round
transcription occurred only from the
6-position of the E4 promoter.
This unexpected finding raises the possibility that subsequent rounds
of transcription starting from +1 may be dependent on subtle
repositioning of promoter-proximal nucleosome(s), caused by passage of
the first polymerase.
When multiple rounds of transcription were allowed (without heparin),
NURF in the absence of histone hyperacetylation conferred the typical
~36-fold activation, or ~1.8 rounds of transcription in 10 min from
the
6-position (Fig. 6B, lanes 2,
4, and 6). Multiple round transcription of
chromatin hyperacetylated by p300 and remodeled by NURF gave
~183-fold activation, or ~4.8 rounds of transcription in 10 min, a
~2.7-fold increase in reinitiation attributable to histone
hyperacetylation (Fig. 6A, lane 8 versus lane 6). (For calculations of
the rounds of transcription, we have not included transcripts
initiating at +1, although reinitiation from this position is also
increased when histone hyperacetylation is combined with remodeling by
NURF). Our results indicate that both types of chromatin modification
can stimulate reinitiation of transcription. We have obtained similar
findings showing a ~1.8-fold increase of single round transcription
and a ~2.8-fold increase in the rounds of transcription when the
combined effects of NURF and histone H3 hyperacetylation by PCAF are
compared with the effects of NURF alone (Fig. 6C).
 |
DISCUSSION |
An increasing number of in vitro experiments
utilizing chromatin templates provide compelling evidence that
noncovalent and covalent modifications of chromatin can directly
participate in the process of transcriptional control by
sequence-specific activators. To date, however, studies show that
ATP-dependent chromatin remodeling or histone
hyperacetylation can each function to facilitate transcription. But
there is little or no information on how these two major types of
chromatin modification might be mechanistically integrated in the
transcription process. In this report, we have begun to address this
issue by comparing the relative contributions of histone
hyperacetylation and ATP-dependent chromatin remodeling to
transcription of a model chromatin template in vitro. We
found evidence of synergism between histone hyperacetylation and
nucleosome remodeling by NURF in allowing
GAL4-VP16-dependent activation of the adenovirus E4 minimal
promoter. In combination, the two modifications caused dramatic
stimulation of preinitiation or initiation as well as reinitiation
of transcription.
To demonstrate the effects of histone hyperacetylation on
GAL4-VP16-dependent transcription of chromatin, we used two
characterized histone acetyltransferases, p300 and PCAF, as enzymatic
tools. In addition to its potent HAT activity on all four nucleosomal histones (71, 92), p300 has also been shown to acetylate
sequence-specific transcription factors such as p53 (94) and to
function as a multifunctional transcriptional coactivator
(e.g. Refs. 51 and 95). Therefore, to deploy only the HAT
activity of p300, it was necessary first to hyperacetylate chromatin
with p300 and then to remove it by gel filtration prior to introduction
of NURF, GAL4-VP16, and the transcription machinery. (This technical
requirement constrained the order of addition of the chromatin
modifiers in our reaction protocol, which should not be taken
necessarily as the order of chromatin modifications as they occur
in vivo).
The combination of both chromatin modifications led to a very high
level (>100-fold) of transcriptional activation, with a close
correlation between increased activation and bulk hyperacetylation of
histones. Such a state of histone modification on the reconstituted 9-kilobase pair cosmid chromatin may be compared with the "global" status of histone hyperacetylation for an activated locus in
vivo, e.g. as found at the chick
-globin gene
cluster (96). Interestingly, preferential hyperacetylation of histone
H3 on cosmid chromatin by PCAF, a more specific HAT enzyme, gave a
level of synergistic activation similar to that provided by p300,
indicating that general modification of a specific histone could
suffice to assist the transcription process. Histone hyperacetylation
can also be limited to several or more nucleosomes surrounding
promoter regions (59, 60, 97, 98). This aspect of histone
hyperacetylation is not addressed by our study, but it was recently
shown that GAL4-VP16 targeting of p300 can cause histone H3 and H4
hyperacetylation within proximal nucleosomes (54, 102) and
transcriptional activation of chromatin in vitro (54).
The requirement for the VP16 activation domain of GAL4-VP16 in
revealing the effects of chromatin modifications on transcription suggests that histone hyperacetylation and ATP-dependent
nucleosome remodeling help to stimulate recruitment of general
transcription factors and the RNA polymerase II machinery by the
sequence-specific activator. It is of interest that, despite the
ability to synergize with NURF, histone hyperacetylation alone was
unable to stimulate recruitment of the transcription apparatus. This
suggests that ATP-dependent nucleosome repositioning is
obligatory to activate the E4 promoter and that the two modes of
chromatin modification enable GAL4-VP16-dependent
transcription using distinct mechanisms. It should be noted that the
obligatory requirement for NURF in this study is not inconsistent with
studies of other promoters (e.g. human immunodeficiency
virus-1, hsp26, and AdML promoters) demonstrating significant effects
of histone hyperacetylation on transcription, in the apparent absence
of remodeling by NURF (45-47, 51, 52, 54). In those studies,
transcription of hyperacetylated chromatin templates was likely to have
occurred in the presence of NURF-like activities present in the high
salt nuclear extracts employed as a source of the transcriptional
machinery. It is also possible that the E4 promoter may possess a
specific requirement for nucleosome remodeling by NURF.
How might histone hyperacetylation facilitate
GAL4-VP16-dependent recruitment of the transcription
apparatus in the context of ATP-dependent nucleosome
mobility? While it is possible that hyperacetylation of chromatin could
help recruitment by extending the range or efficiency of nucleosome
remodeling by NURF, we did not observe significant enhancement of
nucleosome mobility under saturating NURF conditions, as measured by
MNase and restriction enzyme digestions. Moreover, histone
hyperacetylation of chromatin did not promote the binding of the GAL4
activator, shown in a previous study (99), since effects on activator
binding were bypassed in our experiments by the use of saturating
amounts of GAL4-VP16. Rather, we favor two nonexclusive mechanisms by
which chromatin modification could facilitate recruitment of the
transcription apparatus. It is possible that histone hyperacetylation
could decondense the higher order folding of nucleosome arrays in a manner not readily detectable by probing MNase or restriction endonucleases. Such a view is consistent with studies in which histone
hyperacetylation was found to unfold arrays of 5 S nucleosomes and
dramatically enhance transcription by RNA polymerase III (100). Alternatively, histone H4 hyperacetylation could directly enhance recruitment of the general transcription factor TFIID to promoter chromatin through increased affinity for the bromodomains of the TAFII250 subunit (101).
Thus, the recruitment of the transcription apparatus by GAL-4VP16 could
be assisted by at least three distinct, chromatin-based processes: 1)
the repositioning or sliding of nucleosomes to distal locations,
mediated by the GAL4 DNA binding domain and catalyzed by NURF, 2) the
unfolding of higher order structure caused by histone hyperacetylation,
and 3) the increased binding of TFIID to hyperacetylated nucleosomes.
For the GAL4-UAS-minimal E4 promoter, the disposition of nucleosomes
over promoter sequences may render nucleosome repositioning obligatory
for activation, despite higher order unfolding, thus creating the
observed regulatory hierarchy. While such a scenario may be envisaged
for some gene promoters, it is likely that other gene promoters may
possess intrinsic mechanical properties and a series of DNA
cis-regulatory elements that dictate a different
hierarchical order (and a different set) of chromatin modifications.
Our present findings should provide a useful foundation for future
studies to elucidate the multistep process of activating natural
promoters in the context of chromatin.