Persistent Interactions of Core Histone Tails with Nucleosomal DNA following Acetylation and Transcription Factor Binding

Abstract

In this study, we examined the effect of acetylation of the NH
2
tails of core histones on their binding to nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the NH
2
tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to nucleosomes occupying the central 90 bp occurred without displacement of the histone NH
2
tails from DNA. GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees. However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound transcription factors.

Full text

MOLECULAR AND CELLULAR BIOLOGY,
0270-7306/98/$04.0010
Nov. 1998, p. 6293–6304 Vol. 18, No. 11
Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Persistent Interactions of Core Histone Tails with Nucleosomal
DNA following Acetylation and Transcription Factor Binding
VESCO MUTSKOV,
1
† DELPHINE GERBER,
2
DIMITRI ANGELOV,
3
† JUAN AUSIO,
4
JERRY WORKMAN,
5
AND STEFAN DIMITROV
2
*
Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia,
1
and Institute of Solid State Physics,
Bulgarian Academy of Sciences, 1784 Sofia,
3
Bulgaria; Laboratoire d’Etudes de la Diffe´renciation et l’Adhe´rence
Cellulaires, UMR CNRS/UJF 5538, Institut Albert Bonniot, 38706 La Tronche Cedex, France
2
; Department of
Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6, Canada
4
; and
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The Pennsylvania
State University, University Park, Pennsylvania 16802
5
Received 18 March 1998/Returned for modification 24 April 1998/Accepted 31 July 1998
In this study, we examined the effect of acetylation of the NH
2
tails of core histones on their binding to
nucleosomal DNA in the absence or presence of bound transcription factors. To do this, we used a novel UV
laser-induced protein-DNA cross-linking technique, combined with immunochemical and molecular biology
approaches. Nucleosomes containing one or five GAL4 binding sites were reconstituted with hypoacetylated or
hyperacetylated core histones. Within these reconstituted particles, UV laser-induced histone-DNA cross-
linking was found to occur only via the nonstructured histone tails and thus presented a unique tool for
studying histone tail interactions with nucleosomal DNA. Importantly, these studies demonstrated that the
NH
2
tails were not released from nucleosomal DNA upon histone acetylation, although some weakening of their
interactions was observed at elevated ionic strengths. Moreover, the binding of up to five GAL4-AH dimers to
nucleosomes occupying the central 90 bp occurred without displacement of the histone NH
2
tails from DNA.
GAL4-AH binding perturbed the interaction of each histone tail with nucleosomal DNA to different degrees.
However, in all cases, greater than 50% of the interactions between the histone tails and DNA was retained
upon GAL4-AH binding, even if the tails were highly acetylated. These data illustrate an interaction of
acetylated or nonacetylated histone tails with DNA that persists in the presence of simultaneously bound
transcription factors.
DNA in the cell nucleus exists in the form of chromatin.
Chromatin structure is quite complex, and several different
levels of chromatin packaging have to be perturbed in order for
transcription factors to gain access to their binding sites within
regulatory DNA sequences (12). In this study, we examined the
binding of transcription factors to the first level of chromatin
organization, the nucleosome. The nucleosome is the basic
chromatin subunit; it consists of a DNA fragment about 180 to
200 bp long wrapped around an octamer of core histones, two
each of H2B, H2A, H3, and H4. As demonstrated earlier, the
histone octamer represents a tripartite assembly with an over-
all shape of a cylindrical wedge and a centrally located tet-
ramer, (H3-H4)
2
, flanked by two H2A-H2B dimers (6). The
surface of the histone octamer has 12 periodically located,
binary structural motifs that permit the docking of DNA on the
octamer; discovery of this structure yielded a model for the
nucleosome in which the NH
2
termini emerge at alternating
sides of the DNA (7). This model is in excellent agreement
with the recently resolved crystal structure of the nucleosome
core particle at 2.8 Å (45). The histone tails are located exter-
nal to the core particle and are subject to acetylation, a post-
translational modification which is believed to be involved in
transcriptional regulation (29, 30, 43). Until the late 1980s,
only indirect circumstantial evidence existed to support the
originally proposed hypothesis of Allfrey et al. (3) that acety-
lation remodels chromatin structure and facilitates transcrip-
tion (2, 35). The first direct link between histone acetylation
and transcriptionally active chromatin became apparent with
the development of an immunochemical procedure for the
fractionation of chromatin by use of an antibody which specif-
ically recognizes hyperacetylated histones (29, 30). The use of
the same fractionation scheme with an antibody specific for H4
showed that both transcriptional silencing of the yeast mating
type cassette and telomere silencing are accompanied by a
strong decrease in H4 acetylation levels (13). Thus, all of the
above studies strongly suggest a close relationship between
histone acetylation and transcription.
How can histone acetylation, a posttranscriptional modifica-
tion of the most abundant proteins within the cell nucleus, be
important in transcriptional regulation? At least two different
scenarios can be envisaged. In the first model, the reduction of
the lysine-positive charges within the histone NH
2
tails could
perturb or even abolish their interaction with DNA, hence
loosening the nucleosome and higher-order chromatin struc-
ture (26, 43). This scenario would allow easier transcription
factor binding and thus facilitate transcription (43, 75). This
hypothesis has become very popular, with recent discoveries
suggesting “targeted histone acetylation”: it was found that
components of the basal transcriptional machinery, transcrip-
tion coactivators (11, 14, 15, 47, 51, 77) and transcription core-
pressors (1, 32, 39, 40), possess intrinsic histone acetyltrans-
ferase or histone deacetylase activities. Thus, recruitment of
such coactivators or corepressors by transcription factors to
* Corresponding author. Mailing address: Laboratoire d’Etudes de
la Diffe´renciation et de l’Adhe´rence Cellulaires, UMR CNRS/UJF
5538, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche
Cedex, France. Phone: (33) 4 76 54 94 73. Fax: (33) 4 76 54 94 25.
E-mail: Stefan.Dimitrov@ujf-grenoble.fr.
† Present address: Laboratoire d’Etudes de la Diffe´renciation et
l’Adhe´rence Cellulaires, UMR CNRS/UJF 5538, Institut Albert Bon-
niot, 38706 La Tronche Cedex, France.
6293

specific DNA sequences may determine the acetylation status
of core histones and consequently “open” (upon histone acet-
ylation and subsequent removal of histone tails from their
interaction with DNA) or return (upon histone deacetylation)
the nucleosomes to their repressive state (72). In this way,
transcription factor binding and transcription itself will, respec-
tively, be promoted or inhibited by targeted histone acetyla-
tion.
In the alternative model, the acetylation of histones was
viewed as a signal for the binding (elimination) of other factors
(65). This model is essentially based on the use of antibodies
which recognize specific acetylated lysine residues of histone
H4 (37). For example, antibodies against specific H4 lysines
gave a characteristic distribution pattern in polytene chromo-
somes from larval salivary glands of some chironomid insects
(63, 64). Interestingly, the female inactive X chromosome was
not immunolabeled with the different antibodies used (64).
Immunolabeling of human metaphase chromosomes with an-
tibodies against the most highly acetylated forms of H4 also
showed a specific labeling pattern (38). In summary, the im-
munofluorescence studies carried out with these antibodies
demonstrated that constitutive, centric heterochromatin and
facultative heterochromatin in mammalian cells contained un-
deracetylated forms of H4, while acetylated H4 was preferen-
tially located in regions enriched in coding DNA (38, 65). This
specificity of localization of differentially acetylated H4 forms
was suggested to act as a signal for other factors (65).
In this study, we focused on the fate of the histone NH
2
tails
in nucleosome particles reconstituted with hyperacetylated his-
tones and on the binding of the chimeric GAL4-AH transcrip-
tion factor. To this end, we used a unique combination of
UV-induced laser cross-linking together with immunochemical
and molecular biology techniques. We found that the associa-
tion of the histone tails with nucleosomal DNA is both dy-
namic and persistent, surviving both histone acetylation and
GAL4-AH binding.
MATERIALS AND METHODS
Preparation of DNA probes. The 180- and 150-bp DNA fragments containing
the five centered GAL4 binding sites were generated by PCR amplification from
plasmid pG5H as previously described (76). The 154-bp probe with a single
GAL4 binding site at 32 bp from the ends was prepared from plasmid
pBEND401G1 by digestion with SalI and MluI (17).
32
P labeling of the 154-bp probe was carried out with T4 polynucleotide kinase.
The 180- and 150-bp probes were labeled either by T4 polynucleotide kinase
treatment or by PCR, when a higher specific activity was needed. The PCR
mixture contained dATP, dGTP, and dTTP each at a concentration of 200 mM,
dCTP at 100 mM, and 5 mlof[a-
32
P]dCTP (3,000 Ci/mmol; ICN). The fragments
were amplified by numerous cycles and, after separation on a native 8% poly-
acrylamide (acrylamide/bisacrylamide, 29:1)–13 Tris-borate-EDTA (TBE) gel,
were excised from the gel and electroeluted (71). The quantity of the probe was
determined either fluorimetrically or by comparison with a DNA mass ladder
(Gibco-BRL) on ethidium bromide-stained agarose gels.
Oligonucleosome and histone isolation. Linker histone-depleted oligonucleo-
somes were prepared from chicken erythrocyte nuclei. Chicken erythrocyte nu-
clei isolated as described previously (46) were digested with micrococcal nuclease
(5 U per 50 mg of DNA) in 10 mM Tris-HCl [pH 7.8]–50 mM NaCl–1 mM
CaCl
2
–0.5 mM phenylmethylsulfonyl fluoride for 45 min at 37°C. The digestion
was stopped by the addition of EDTA to a final concentration of 5 mM, the
digested material was dialyzed for 6 to 8 h against 0.25 mM EDTA, and oligo-
nucleosomes were recovered in the supernatant after centrifugation of the lysed
nuclei on a bench-top centrifuge for 10 min. Oligonucleosomes were depleted
from H1 and H5 linker histones and from nonhistone proteins by fractionation
in a 10 to 30% sucrose gradient containing 0.65 M NaCl (19). The peak fraction,
containing essentially mononucleosomes and small amounts of dinucleosomes,
was dialyzed against 100 mM NaCl, divided into aliquots, and frozen at 280°C.
Chicken core histones were isolated from the oligonucleosomes by overnight
extraction with HCl (21). Highly hyperacetylated histone octamers were isolated
from HeLa cells grown in butyrate as described by Ausio and van Holde (8).
Briefly, butyrate-grown HeLa cell chromatin was fractionated in the presence of
divalent cations to obtain fractions enriched in hyperacetylated histones. Six
milligrams of the hyperacetylated chromatin fraction (fraction a) was dialyzed
against 0.633 M NaCl–0.1 M potassium phosphate–1 mM dithiothreitol–5 mM
sodium butyrate (pH 6.7). The dialyzed chromatin was loaded onto a hydroxy-
lapatite column (1.5 by 15 cm), and linker histones were eluted with 100 to 120
ml of the above-described buffer. Elution of hyperacetylated core histones was
performed with the same buffer but containing 1 M NaCl. The eluted histones
were concentrated and kept frozen at 280°C until use.
The extent of histone acetylation was assessed on acid-urea-Triton gels (18).
The acetylated histones obtained in this way contained an average of 17 acetyl
groups per histone octamer.
Digestion with trypsin. Tailless nucleosomes were obtained by trypsin diges-
tion. Briefly, 150 ml of nucleosomes (150 mg/ml) in 50 mM Tris-HCl (pH 7.5)–100
mM NaCl was incubated at 37°C with trypsin (Sigma) at a ratio of 1 mgof
trypsin/25 mg nucleosomes. At various times after the beginning of the digestion,
aliquots were removed and transferred to separate tubes, and the digestion was
stopped by the addition of diisopropylfluorophosphate (Sigma) at a final con-
centration of 0.01%. The extent of trypsin digestion was checked by sodium
dodecyl sulfate (SDS)–18% polyacrylamide gel electrophoresis (42).
Transcription factor GAL4-AH purification and nucleosome reconstitution.
The chimeric transcription factor GAL4-AH, containing the DNA binding and
dimerization domains of GAL4 linked to an artificial 15-amino-acid putative
amphipathic helix, was purified as described previously (44).
Reconstitution of nucleosomes containing the
32
P-labeled fragment with one
or five GAL4 binding sites was carried out by either the histone octamer transfer
method (74) or salt dialysis as described by Vettese-Dadey et al. (70). For
octamer transfer, 3 mg of donor nucleosomes was mixed with 30 ng of
32
P-labeled
probe in 1 M NaCl–10 mM Tris-HCl (pH 8.0)–1 mM EDTA in a final volume of
50 ml and incubated for 20 min at 37°C. The reaction mixtures were serially
diluted to 0.9, 0.7, 0.5, and 0.3 M NaCl with dilution buffer (50 mM HEPES [pH
7.5], 1 mM EDTA [pH 8.0]) and incubated at each dilution step for 20 min at
30°C. Finally, the reaction mixtures were brought to 0.1 M NaCl with 10 mM
Tris-HCl (pH 7.5)–1 mM EDTA (pH 8.0)–20% glycerol and incubated for 30
min at 30°C.
For salt dialysis nucleosome reconstitution, 2 to 3 mg of core histones was
mixed with 2.1 mg of carrier thymus DNA and 50 to 100 ng of
32
P-labeled DNA
probe in 2 M NaCl–10 mM Tris-HCl (pH 8.0)–1 mM EDTA (pH 8.0)–10 mM
b-mercaptoethanol–1 mg of bovine serum albumin (BSA) per ml in a total
volume of 100 ml. The reaction mixtures were incubated for 15 to 30 min at room
temperature, transferred to dialysis tubing, and dialyzed at 4°C against 10 mM
Tris-HCl (pH 8.0)–1 mM EDTA (pH 8.0)–10 mM b-mercaptoethanol containing
1.2, 1.0, 0.8, and 0.6 M NaCl. Each dialysis step was carried out for 2 h. Finally,
the reconstituted material was dialyzed overnight against 10 mM Tris-HCl (pH
8.0)–1 mM EDTA (TE). The reconstituted nucleosomes were analyzed on a 4%
native polyacrylamide (acrylamide/bisacrylamide, 19:1)–0.53 TBE gel. Under
optimal conditions, more than 85–90% of the
32
P-labeled fragment was usually
nucleosome reconstituted.
Binding reactions. Nucleosomes reconstituted by octamer transfer or by salt
dialysis were incubated with increasing concentrations of diluted GAL4-AH
(stock solution, 2 mg/ml, diluted in 10 mM HEPES [pH 7.5]–100 mM KCl–10
mM ZnCl
2
–5 mM dithiothreitol–1 mg of BSA per ml). Final reaction mixtures
were brought to 20 ml with binding buffer (20 mM HEPES [pH 7.5], 50 mM KCl,
5% glycerol, 2 mM dithiothreitol, 1 mM ZnCl
2
, 1 mg of BSA per ml) and
incubated for 30 min at 30°C. The binding of GAL4-AH was analyzed on a 4%
polyacrylamide–0.53 TBE gel at 4°C and a constant amperage of 8 mA. The
binding reactions were quantified by use of a PhosphorImager and Image Quant
Software (Molecular Dynamics). UV laser irradiation of the GAL4-AH-bound
nucleosomes was performed immediately after completion of the binding reac-
tions.
Preparation of antibodies. Antibodies against core histones H2A, H2B, and
H4 were prepared by injecting rabbits with histone-RNA complexes essentially as
described previously (4). All antibodies were immunospecifically purified from
sera by use of respective antigens conjugated to CNBr-Sepharose 4B (Pharmacia
Biotech, Inc.).
Immunoblotting. Histones were separated by electrophoresis in SDS–18%
polyacrylamide gels (42). The proteins were transferred to nitrocellulose filters
(Amersham) by electroblotting in 12.5 mM Tris-HCl (pH 8.3)–125 mM glycine–
0.05% SDS–20% methanol for1hataconstant amperage of 200 mA. The
electroblotted proteins were stained with 0.2% India ink in phosphate-buffered
saline (PBS) supplemented with 0.2% Tween 20. After protein visualization, the
filters were rinsed with PBS and blocked for1hin10%nonfat dry milk–0.3%
Tween 20–PBS. The filters were rinsed with PBS and overlaid with affinity-
purified antibodies in PBS–10% fetal calf serum–0.2% Tween 20. After incuba-
tion for 1 h with gentle shaking at room temperature, the filters were washed
three times with PBS–0.5 M NaCl–0.5% Triton X-100 and twice with PBS–0.5 M
NaCl, each washing step lasting 10 min. The filters were incubated for 1 h with
peroxidase-conjugated secondary antibody and, after extensive washing as de-
scribed above, developed by use of an ECL kit (Amersham).
UV laser irradiation. UV laser irradiation was carried out with a single 5-ns
pulse from the fourth harmonics (266 nm) of a Surelite II (Continuum) Nd:YAG
laser. The pulse energy was measured with a calibrated pyroelectrical detector
(Ophir Optronics Ltd.) by use of an 8% deviation beam splitter. The electrical
signal from the detector was transmitted to a computer for further processing.
The sample (usually 20 ml) was irradiated in a 0.65-ml siliconized Eppendorf
6294 MUTSKOV ET AL. MOL.CELL.BIOL.

tube. The size of the laser beam was adjusted by means of a set of circular
diaphragms to perfectly fit the surface area of the sample. Special care was taken
to avoid air bubbles in the sample solution.
Quantitative estimation of protein covalently linked to DNA. The total amount
of protein cross-linked to DNA was determined by repeated phenol extractions.
After irradiation, 20 ml of sample was mixed with 130 ml of TE and 100 mlof
TE-saturated phenol. The solution was then vortexed and centrifuged for 3 min
in a bench-top centrifuge, the aqueous phase was carefully recovered, and the
phenol phase was extracted three more times with 200 ml of TE. The aqueous
phases were pooled, and 100 ml of phenol was added. After the addition of 730
ml of TE to the phenol-phase fraction, the quantities of labeled DNA in both the
phenol and the aqueous phases were measured by Cerenkov counting. The
cross-linking yield was calculated as the ratio of phenol counts to phenol plus
aqueous counts after subtraction of the background counts (irradiated DNA in
the absence of protein). The quantum efficiency was calculated by dividing the
cross-linking yield by the number of photons absorbed by a nucleotide base.
Immunoslot assay. The cross-linking of individual histones was estimated by a
slot immunoassay (48). The covalent histone-DNA complexes in the reconsti-
tuted nucleosomes were separated from the non-cross-linked proteins through
preformed CsCl gradients. The gradients were fractionated, and the fractions
containing the peak of DNA and covalent histone-DNA complexes were pooled.
Five micrograms of the cross-linked material (measured as the amount of DNA)
was dotted onto nitrocellulose filters, and the presence of individual histones was
detected by the protocol described in the Immunoblotting section (see above).
Immunoprecipitation. The immunoprecipitation of individual covalent his-
tone-DNA complexes was performed essentially as described by Moss et al. (48).
Fifty microliters of IgGsorb (The Enzyme Center, Malden, Mass.) was resus-
pended in 0.5 ml of 1% BSA–0.25 mg of laser-irradiated Escherichia coli DNA
per ml in PBS and shaken for1hatroom temperature to block sites of
nonspecific absorption. The pellet obtained after centrifugation for 30 s in a
bench-top centrifuge was resuspended in 0.5 ml of a mixture consisting of the
specific antibody, the irradiated
32
P-labeled reconstituted particles (correspond-
ing to about 3 mg of DNA), and 200 mg of carrier-irradiated nucleosomes. The
ratio of antibody to
32
P-labeled core particles plus carrier-irradiated nucleo-
somes was 1:2.5 in antibody buffer (50 mM HEPES [pH 7.5], 2 M NaCl, 0.1%
SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, 0.1% BSA). The sus-
pension was shaken for 2 to3hatroom temperature and washed five times with
antibody buffer and three times with rinse buffer (50 mM HEPES [pH 7.5], 0.15
M NaCl, 5 mM EDTA). The suspension was centrifuged for 30 s between each
wash. The amounts of immunoprecipitated individual histone-DNA complexes
were measured by Cerenkov counting.
RESULTS
Histones within reconstituted nucleosomes are efficiently
cross-linked to DNA upon UV laser irradiation. UV irradia-
tion with conventional sources does not induce detectable
amounts of core histone-DNA cross-linking (53). However,
high-intensity UV laser irradiation of nuclei and chromatin
leads to efficient histone-DNA cross-linking, and individual
histones within the covalent protein-DNA complexes can be
visualized with the help of specific antibodies (4, 20, 49). His-
tone-DNA cross-linking is essentially determined by the bipho-
tonic mechanism of protein-DNA cross-linking operating in
the presence of high-intensity laser irradiation (4, 33, 53). We
sought to determine the efficiency of histone-DNA cross-link-
ing after nucleosome reconstitution procedures.
Nucleosomes were efficiently reconstituted on a
32
P-labeled
DNA probe by the octamer transfer method, as shown in Fig.
1A. As shown in Fig. 1B, under these conditions, irradiation of
the reconstituted nucleosomes with a single 5-ns laser pulse led
to significant cross-linking of the histones with the labeled
DNA. Moreover, the dependence of the cross-linking yield on
the laser intensity (the dose-response curve) fit perfectly with a
theoretical curve for a biphotonic reaction (see also Fig. 4A
and B). These results confirmed our previous data on the
biphotonic mechanism of protein-DNA cross-linking induced
by high-intensity laser irradiation (4, 5).
UV laser-induced cross-linking of histones to DNA within
reconstituted nucleosomes occurs via their NH
2
tails only. The
structure of the histone core octamer has been determined by
X-ray crystallography to a resolution of 3.1 Å (6, 7). All histone
chains contain a folded motif, the histone fold, and an exter-
nally located NH
2
-terminal tail region which possesses a large
number of positively charged residues. The histone folds con-
tain many sites of interactions with nucleosomal DNA (45). In
addition, the positively charged histone tails also interact with
DNA; however, these domains seem to be devoid of any reg-
ular structure when not bound to nucleosomal DNA (10). Our
earlier immunochemical data indicated that in native chroma-
tin, laser-induced histone-DNA cross-linking was achieved es-
sentially via the NH
2
tails (59). In order to determine whether
this is also the case for reconstituted nucleosomes, we carried
out two types of experiments.
In the first set of experiments, we removed the NH
2
tails of
donor nucleosomes by trypsin digestion and used the truncated
nucleosomes for octamer transfer reconstitution. The recon-
stituted particles, containing trypsin-truncated histones, were
irradiated, and the total amount of cross-linked histones was
compared to that of nucleosomes with native histones. The
kinetics of trypsin digestion of donor nucleosomes are shown
in Fig. 2A. Under these conditions, 1 min of digestion partially
removed the NH
2
tails, while 3 min was sufficient for their
complete elimination. At the same time, the histone fold do-
mains (peptides P
1–5
in Fig. 2A; see also reference 66) re-
FIG. 1. Dose-response curve for reconstituted nucleosomes. (A) Nucleo-
some particles are efficiently reconstituted by octamer transfer. Thirty nano-
grams of the
32
P-labeled 180-bp probe DNA containing five centered GAL4
binding sites was reconstituted into nucleosome cores by the octamer transfer
method. The mobilities of the nucleosome core (Nuc) and the naked DNA are
indicated. The concentrations of donor nucleosomes were 0.1 mg (lane 1), 0.5 mg
(lane 2), 1 mg (lane 3), and 3 mg (lane 4). (B) UV laser-induced histone-DNA
cross-linking proceeds via a biphotonic mechanism. Reconstituted nucleosomes
were irradiated with a single 266-nm laser pulse at different intensities. The
amount of cross-linked
32
P-labeled DNA was measured by the phenol extraction
procedure and plotted against the laser intensity. The experimental points were
computer fitted to reflect two-quantum processes (4, 5).
VOL. 18, 1998 HISTONE ACETYLATION AND TRANSCRIPTION FACTOR BINDING 6295

mained intact. The reconstitution of particles with truncated
nucleosomes was as efficient as that of non-trypsin-digested
native nucleosomes (compare Fig. 1A and the inset of Fig. 2B;
see also references 9 and 69). However, the efficiency of his-
tone cross-linking within reconstituted nucleosomes containing
trypsin-truncated (i.e., without histone NH
2
tails) core histones
was decreased to insignificant levels (Fig. 2B). These data
strongly suggest that the cross-linking of histones to DNA in
reconstituted particles occurred via the histone NH
2
tails.
This conclusion was further supported by the dependence of
histone cross-linking on the concentration of NaCl (Fig. 2C).
In the cross-linking reactions, increasing NaCl concentrations
above 0.3 M led to a pronounced decrease in the efficiency of
cross-linking, which became insignificant at 0.5 to 0.6 M NaCl.
Increasing NaCl concentrations above 0.3 M NaCl released the
NH
2
tails from their interactions with DNA (16, 73). More-
over, direct contact between proteins and DNA is necessary in
order for UV laser-induced cross-linking to occur (UV light is
a “zero-length” cross-linking agent). Thus, the lack of cross-
linking above 0.5 to 0.6 M NaCl was most likely due to the
release of the NH
2
tails from DNA at these salt concentrations.
These data are in agreement with our earlier conclusion that
cross-linking is achieved via histone NH
2
tails. This conclusion
is also supported by the lack of cross-linking for tailless nu-
cleosomes within the range of 0.1 to 0.7 M NaCl (Fig. 2C).
In the second experimental approach, nucleosomes were
reconstituted from purified native histones. These reconsti-
tuted particles were digested with trypsin and irradiated with
the laser, and the covalent histone-DNA complexes were pu-
rified from the non-cross-linked proteins on CsCl gradients.
The amount of individual histones cross-linked to DNA within
the purified complexes was estimated by an immunoslot assay
with highly specific antibodies. The specificity of the antibodies
is illustrated in Fig. 3A. If the NH
2
tails were responsible for
histone-DNA cross-linking, we expected to observe a disap-
pearance of the cross-linked histones within the CsCl-purified
protein-DNA complexes isolated from irradiated and trypsin-
digested (tailless) nucleosomes. This is because of the fact that
the non-cross-linked histone fold domains are dissociated from
DNA during centrifugation in CsCl gradients. Figure 3B illus-
trates that antibody detection of histones H2A, H2B, and H4
in the CsCl fractions indeed was dependent on the presence of
the NH
2
tails of each histone (i.e., lost by trypsin digestion).
Since each antibody reacted with the histone fold domains in
the absence of the histone tails and the presence of histones in
the CsCl fractions was dependent on UV laser-induced cross-
linking (Fig. 3B), these results clearly demonstrate that the
histone-DNA cross-linking was achieved via the nonstructured
tails.
Hyperacetylated NH
2
tails of core histones interact at an
efficiency similar to that of hypoacetylated tails with nucleo-
somal DNA at nearly physiological ionic strengths. Histone
acetylation is a posttranslational modification that correlates
strongly with the transcriptional regulation of numerous genes
(for recent reviews, see references 27, 54, 57, and 72). How-
tuted particles (nuc) determined by using as donors nucleosomes digested with
trypsin for 3 min. (C) NaCl concentration dependence of the yield of cross-linked
DNA on nucleosome particles reconstituted with native histones or with histones
from donor nucleosomes that had been digested with trypsin for 3 min. Both
samples at different NaCl concentrations were irradiated with a single laser pulse
(25 MW/cm
2
), and the dependence of the yield of cross-linked DNA (measured
by phenol extraction) on NaCl concentration was determined. Each experimental
point represents the average of four independent experiments. Because the
errors of measurements at different ionic strengths were found to be essentially
the same, for simplicity error bars are shown for only two experimental points.
FIG. 2. Core histones are cross-linked to DNA via their NH
2
tails. (A)
Electrophoresis (18% polyacrylamide–SDS) of histones isolated from donor
nucleosomes digested with trypsin for the indicated times. P
1–5
, trypsin-resistant
peptides of the core histones, designated as described by van Holde (67). (B)
Dependence of the yield of cross-linked DNA on reconstituted nucleosomes
containing trypsinized histones. Nucleosomes were reconstituted under optimal
conditions (3 mg of donor nucleosomes for 30 ng of
32
P-labeled 180-bp probe
DNA) by using as donors either native nucleosomes or nucleosomes digested
with trypsin for 1 or 3 min. Each sample was irradiated with a single 266-nm laser
pulse at a laser intensity of 25 MW/cm
2
. The amount of cross-linked DNA was
measured by the phenol extraction method, and the yield of cross-linked DNA
was plotted against the time of trypsin digestion of donor nucleosomes. The inset
represents the mobilities of the naked 180-bp DNA fragment and of reconsti-
6296 MUTSKOV ET AL. MOL.CELL.BIOL.

ever, the precise role of this modification remains unclear (28,
65, 72). Acetylation occurs on specific lysine residues within
the core histone NH
2
tails (41, 67). A widely accepted hypoth-
esis is that histone acetylation, by reducing the positive charge
of histone tails, releases them from their interaction with
DNA. This is thought to result in the observed enhanced tran-
scription factor binding to nucleosomes containing acetylated
histones (43, 70, 72). Since we have shown that UV laser
irradiation induces histone-DNA cross-linking via the core hi-
stone NH
2
tails only, this method appears to be an ideal tool
for directly studying the interaction of hyperacetylated histone
tails with DNA. To this end, we reconstituted nucleosomes by
salt dialysis using highly hyperacetylated core histones (17
acetyl groups per histone octamer; Fig. 4C) isolated by a spe-
cial fractionation procedure as described previously (8, 26).
Nucleosome reconstitution with the hyperacetylated core his-
tones was as efficient as reconstitution with the hypoacetylated
core histones (data not shown).
Hyperacetylated and control (hypoacetylated) reconstituted
nucleosomes were UV laser irradiated at different intensities,
and the yield of cross-linking was calculated. The dose-re-
sponse curves for both preparations at 0.1 M NaCl are shown
in Fig. 4A and B. Both dependencies were essentially identical.
Thus, at 0.1 M NaCl, hyperacetylated NH
2
tails interacted as
closely as hypoacetylated tails with nucleosomal DNA. This
conclusion was further confirmed by the immunochemical data
presented in Fig. 4D. The reactions of specific antibodies
against individual core histones with covalent histone-DNA
complexes isolated from irradiated nucleosomes containing hy-
poacetylated and hyperacetylated histones showed the same
intensities.
The efficiencies of histone cross-linking of nucleosomes con-
taining hyperacetylated versus hypoacetylated histones differed
at a higher ionic strength (Fig. 5). Increasing the NaCl con-
centration resulted in decreased cross-linking efficiency, reach-
ing a plateau of insignificant cross-linking for both prepara-
tions. However, for hyperacetylated particles, this effect was
observed at lower NaCl concentrations. The binding of histone
NH
2
tails to DNA is essentially electrostatic in nature (16, 67),
and increasing NaCl concentrations affect electrostatic inter-
actions. Thus, these data indicate a weaker interaction of hy-
peracetylated histone NH
2
tails with nucleosomal DNA at a
higher ionic strength.
The NH
2
tails of core histones and GAL4-AH transcription
factors coexist on the same nucleosomal DNA. We demon-
strated that at nearly physiological ionic strengths, the hyper-
acetylation of histone NH
2
tails only slightly perturbs their
interaction with DNA. Next we asked whether transcription
factor binding affects the interaction of the histone NH
2
tails
with DNA. To answer this question, we carried out immuno-
precipitation experiments (Fig. 6). As a model system, we used
nucleosomal templates containing 180 and 150 bp of DNA
with five centered GAL4 binding sites, because a perturbation,
if induced, should be more apparent in a particle containing
multiple bound transcription factors. The 180-bp particle con-
tains almost 40 bp of linker DNA and, since the sites of DNA
interaction with the histone NH
2
tails are not well known (7,
45), it is possible that some of the histone tails might interact
with the linker DNA. If this is the case, the binding of
GAL4-AH should not affect the histone tail-DNA interactions,
since the GAL4 binding sites are located within the nucleoso-
mal DNA and not the linker DNA. The use of the linkerless
150-bp particle overcomes this problem, since it allows for
studying the effect of an interaction of the histone NH
2
tails
with nucleosomal DNA only.
Briefly,
32
P-labeled particles, with or without five bound
GAL4-AH molecules, were UV laser irradiated with identical
doses, and the covalent histone-DNA complexes were immu-
noprecipitated with highly specific antibodies against individ-
ual histones (Fig. 3A). The precipitation was carried out under
conditions in which the non-cross-linked histones were com-
pletely removed from the DNA (for details, see Materials and
Methods). A comparison of the amounts (measured as
32
P
counts) of precipitated individual histone-DNA complexes for
the different samples allowed us to judge the efficiency of
histone NH
2
tail-DNA cross-linking in the presence or absence
of bound GAL4-AH dimers. This comparison provided a mea-
sure of the effect of GAL4-AH binding on histone tail inter-
actions with DNA.
FIG. 3. Immunochemical evidence for selective histone NH
2
tail-DNA cross-
linking induced by UV laser irradiation. (A) Specificity of the histone antibodies
used. Hen erythrocyte histones were separated by 18% polyacrylamide–SDS gel
electrophoresis, electroblotted, and stained with India ink (lane 1) or reacted
with immunopurified antibodies against H4 (lane 2), H2B (lane 3), and H2A
(lane 4). (B) Immunoslot assay for the presence of core histones in cross-linked
protein-DNA complexes obtained upon irradiation of nucleosomes. Nucleo-
somes containing 180 bp of DNA were reconstituted by histone octamer transfer
by using as donors either native nucleosomes or nucleosomes digested with
trypsin for 1 or 3 min. The samples were irradiated with identical doses, and the
cross-linked histone-DNA complexes were separated from the free histones on
CsCl gradients. The CsCl gradients were fractionated, and the fractions contain-
ing the DNA peak were pooled. Five micrograms (measured as DNA) from each
pooled sample was dotted on a nitrocellulose filter and reacted with antibodies
against H2A, H2B, and H4 and preimmune IgG (0). a, Nonirradiated particles;
b, irradiated particles; c and d, irradiated particles containing 1- and 3-min
trypsin-digested histones, respectively; e and f, control slots showing the reaction
of the antibodies with nucleosomes containing native or 3-min trypsin-digested
histones.
VOL. 18, 1998 HISTONE ACETYLATION AND TRANSCRIPTION FACTOR BINDING 6297

Initially, we performed experiments to determine the opti-
mal conditions for the saturation of GAL4-AH on all five sites
on the reconstituted particles (Fig. 7). Once these conditions
were determined, we irradiated the samples and carried out
the immunoprecipitation experiments. The immunoprecipita-
tion data are presented in Fig. 8. As shown in Fig. 8A, the
cross-linking yield for individual histones in hypoacetylated
180- and 150-bp particles containing five GAL4-AH dimers
was decreased compared to that in samples lacking bound
GAL4-AH, suggesting that some perturbation in histone tail-
DNA interactions occurred. However, this perturbation was
found to be relatively small, the highest level being observed
for the 180-bp particle H2A (33% cross-linking yield decrease).
Histones H2A and H4 of the hyperacetylated 180-bp particle
containing 10 bound GAL4-AH molecules were cross-linked
with essentially the same efficiency as those of the GAL-AH-
bound hypoacetylated particle, while H2B cross-linking was
affected (about a 50% decrease in the cross-linking yield, com-
pared to 10% in the hypoacetylated particle; Fig. 8B). Thus,
histone hyperacetylation may be responsible for the selective
perturbation of H2B NH
2
tail-DNA interactions within hyper-
acetylated nucleosome particles bound to GAL4-AH.
FIG. 4. Hypoacetylated and hyperacetylated nucleosomes are cross-linked with the same efficiency in 100 mM NaCl.
32
P-labeled 180-bp probe DNA was
reconstituted in nucleosomes with control, hypoacetylated, or hyperacetylated histones (17 acetyl groups per histone octamer). Samples, in a solution of 100 mM NaCl,
were irradiated with single 266-nm laser pulses at different intensities, and the yield of cross-linked DNA was measured by the phenol extraction procedure. (A and
B) Dose-response curves for hypoacetylated (A) and hyperacetylated (B) nucleosomes. (C) Acid-urea-Triton gel electrophoresis of hypoacetylated (lane 1) and
hyperacetylated (lane 2) histones used for reconstitution. The number of acetylated groups is indicated by numbers 0 to 4. (D) Immunoslot assay of the reaction of
antibodies to H2A, H2B, and H4 and preimmune IgG (0) with covalent histone-DNA complexes isolated after centrifugation in CsCl gradients of irradiated
nucleosomes containing hypoacetylated (a) and hyperacetylated (b) histones; c, immunoslot assay of the material from control (hypoacetylated), nonirradiated particles
after centrifugation in CsCl. The experiment was carried out as described in the legend to Fig. 3B.
6298 MUTSKOV ET AL. MOL.CELL.BIOL.

The binding of five GAL4-AH dimers to both hypo- and
hyperacetylated nucleosomes did not completely perturb the
interaction of histone NH
2
tails with nucleosomal DNA. In all
cases, the reduction in cross-linking efficiency was twofold or
less with GAL4-AH binding. This finding raises the possibility
that the hyperacetylation of histones may have a similar mag-
nitude of effect on GAL4-AH binding to nucleosomal DNA.
To test this idea, we reconstituted nucleosome particles with
hypoacetylated and hyperacetylated histones containing one or
five GAL4 binding sites and studied quantitatively the binding
of GAL4-AH to both types of particles. The results of these
studies are presented in Fig. 9. The binding of GAL4-AH was
enhanced 2 to 2.5 times in hyperacetylated nucleosomes. This
effect was found to be more apparent for the template with one
GAL4 binding site. Thus, the hyperacetylation of histones had
only a modest effect on GAL4-AH binding to nucleosomal
DNA, as expected from the cross-linking data given above.
These results are in excellent agreement with the previously
published data of Vettese-Dadey et al. (70), who reported that
only the most highly acetylated histone, H4, substantially af-
fected binding, which was more apparent for the basic helix-
loop-helix (bHLH) protein USF than for GAL4-AH.
DISCUSSION
Transcription regulation in eukaryotes requires the coordi-
nated binding of numerous basal and specific transcription
factors (60). This binding, however, is impeded by nucleosomes
in the presence of transcription factor cognate DNA sequences
(22, 52). The histone core octamer binds to nucleosomal DNA
with a high affinity; thus, the resulting nucleosomal complex is
very stable (6, 7). Thus, in many instances transcription factors
have to overcome the nucleosome barrier in order to gain
access to their DNA binding sequences. It should be noted that
different transcription factors bind to nucleosomal DNA with
different levels of affinity relative to naked DNA (12, 52). It has
been proposed that core histone NH
2
tails play an important
role in hindering the interaction of transcription factors with
nucleosomal DNA (43, 72). Accordingly, the hyperacetylation
of lysine residues within core histone tails may substantially
weaken histone NH
2
tail-DNA interactions by displacing the
tails away from the DNA. This situation in turn may facilitate
the binding of transcription factors to DNA (43, 70). To test
this model, in this work we examined the effect of both histone
hyperacetylation and transcription factor binding on the inter-
action of histone NH
2
tails with nucleosomal DNA by a com-
bination of UV laser-induced cross-linking and molecular bi-
ology techniques.
UV laser-induced histone-DNA cross-linking within recon-
stituted nucleosomes. We showed that histone-DNA cross-
linking within reconstituted nucleosomes is achieved via core
histone NH
2
tails in two different sets of experiments. In the
first set, we demonstrated that no histone-DNA cross-links
were induced upon laser irradiation of nucleosomes reconsti-
tuted with trypsin-truncated (tailless) histones (Fig. 2B). In
agreement with this interpretation, the reduction of histone
tail-DNA interactions by rising ionic strength (16) led to a
manifold decrease in the efficiency of cross-linking (Fig. 2C).
In the alternative set of experiments, we first cross-linked the
histones in reconstituted particles and then digested the his-
tone NH
2
tails with trypsin. The digestion of the tails released
the histones from the DNA upon centrifugation in CsCl gra-
dients (Fig. 3B), demonstrating again that the UV laser-in-
duced cross-linking was accomplished solely via the N-terminal
region of the histone molecule. This conclusion is in agreement
FIG. 5. UV laser-induced cross-linking detects some perturbations in the
histone NH
2
tail-DNA interactions in nucleosomes containing hyperacetylated
histones. Particles containing 180 bp of DNA and reconstituted with hypoacety-
lated (h) or hyperacetylated (}) histones were irradiated with a single laser
pulse (25 MW/cm
2
) at different ionic strengths (50 to 700 mM NaCl), and the
yield of cross-linked DNA was measured. Data derived from three independent
experiments are presented as a graph of the percentage of cross-linking DNA
yield versus NaCl concentration. For simplicity, the error bars at one NaCl
concentration only are shown.
FIG. 6. Schematic presentation of the experimental strategy for studying the
effect of five nucleosome-bound GAL4-AH molecules on histone NH
2
tail-DNA
cross-linking efficiency. For experimental details, see Materials and Methods.
VOL. 18, 1998 HISTONE ACETYLATION AND TRANSCRIPTION FACTOR BINDING 6299

with our previous data on laser-induced histone-DNA cross-
linking in native chromatin (59). It should be noted that the
efficiencies of histone NH
2
tail cross-linking and the respective
interactions of the histone NH
2
tails with the DNA, unlike
those for the C-terminal domain of H2A (66), were essentially
the same for mononucleosomes and for high-molecular-weight
chromatin (3a, 59). Thus, our experimental model, a nucleo-
some containing 180 bp of DNA, is a representative one for
studying histone NH
2
tail interactions with DNA.
Why is histone cross-linking achieved via the NH
2
tails only?
One possible explanation is the following. In order for the
photochemical reaction resulting in cross-linking to occur, two
events have to take place at the same time: (i) very close
protein-DNA contact and (ii) a favorable orientation of both
chromophores, the DNA base and the amino acid residue.
Some of the amino acid residues from the histone fold-domain
are in close contact with DNA (45). However, the histone folds
are organized in a relatively rigid structure in the histone
octamer, in contrast to their NH
2
tails, which seem to be
flexible (16). Thus, flexibility of the histone tails may present
favorably oriented amino acids for efficient protein-DNA
cross-linking.
Interaction of histone NH
2
tails with DNA within reconsti-
tuted nucleosomes containing hyperacetylated histones. The
fact that UV histone-DNA cross-linking occurs exclusively via
the NH
2
tails provides an approach to examine the effect of
histone acetylation on the interaction of NH
2
tails with DNA.
Since histone acetylation is restricted to the NH
2
tails and since
direct contact between the tails and nucleosomal DNA is
needed for cross-linking to occur (53), the cross-linking yield is
a direct measure of the extent of the histone tail-DNA inter-
actions. To this end, we reconstituted nucleosomes by the salt
dialysis method using highly hyperacetylated histone octamers
(17 acetyl groups per histone octamer) (Fig. 4C) and compared
their efficiency for cross-linking with that of particles contain-
ing hypoacetylated histones (Fig. 4 A and B and Fig. 5). The
cross-linking yields for both samples at 100 to 150 mM NaCl
were very similar, demonstrating that at these nearly physio-
logical ionic strengths, the NH
2
tails were not released from
nucleosomal DNA upon histone acetylation. However, raising
the ionic strength led to an earlier decrease in the cross-linking
yield for hyperacetylated nucleosomes (Fig. 5). Thus, bearing
in mind that the histone NH
2
tail interaction is essentially
electrostatic (16), these data reflect weakening in the interac-
tion between the hyperacetylated histone tails and DNA with-
out their release from the DNA at physiological ionic
strengths, in contrast to previous models (for a review, see
reference 72). This conclusion is further enhanced by recent in
vivo data showing that the hyperacetylated histones of actively
transcribed ribosomal genes can be cross-linked to DNA by use
of UV laser irradiation (49). The above finding is also sup-
ported by data on chemically induced histone-DNA cross-link-
ing (performed partially via the NH
2
tails) which indicated
changes in hyperacetylated histone NH
2
tail-DNA interactions
but not their total displacement from the DNA (23).
Reconstituted oligonucleosome complexes with the same
highly hyperacetylated histones as those used in the present
work were studied in the past with the help of sedimentation
and electron microscopy techniques (26). Both types of anal-
ysis showed that at nearly physiological ionic strengths (100 to
150 mM NaCl), the hyperacetylated oligonucleosomes re-
mained in an extended conformation, in contrast to their hy-
poacetylated counterparts. Thus, the weakening of hyperacety-
lated histone NH
2
tail-DNA interactions detected in this study
obviously affects the compaction of the nucleosomal filament.
Binding of GAL4-AH to reconstituted nucleosomes contain-
ing hypoacetylated and hyperacetylated histones. We also ad-
dressed the question of whether the weakened hyperacetylated
histone-DNA interactions affected transcription factor binding
and if these histone-DNA interactions were in turn affected by
transcription factor binding. To this end, we studied the inter-
actions of the chimeric transcription factor GAL4-AH with
hypoacetylated and hyperacetylated nucleosomes, since
GAL4-AH can invade nucleosomes with relatively small
changes in affinity relative to naked DNA (12, 52). For both
FIG. 7. Titration of reconstituted nucleosomes with GAL4-AH. (A) Recon-
stituted particles containing the 180-bp DNA fragment and bearing five central
GAL4-AH binding sites were incubated without (lane 2) or with increasing
amounts of GAL4-AH (lanes 3 to 7). The GAL4-AH-bound nucleosomes were
separated from the unbound nucleosomes on a 4% polyacrylamide gel. An
autoradiogram of the gel is shown. The concentrations of GAL4-AH used were
as follows: 0 (lane 2), 25 nM (lane 3), 51 nM (lane 4), 154 nM (lane 5), 309 nM
(lane 6), and 515 nM (lane 7). The arrows Lane 1 consists of the loaded DNA
fragment used in the reconstitution. The arrows indicate the positions of the
different nucleosome complexes. (B) Same as panel A but for particles contain-
ing the 150-bp DNA fragment.
6300 MUTSKOV ET AL. MOL.CELL.BIOL.

samples, saturation of nucleosomes with five GAL4-AH
dimers led to a decrease in core histone cross-linking efficiency,
as judged by immunoprecipitation with specific antibodies
against individual core histones (Fig. 8). However, interactions
of the NH
2
tails with DNA were still detected after GAL4-AH
binding (greater than 50% for hyperacetylated H2B and H4
and 70% for H2A). Since the five GAL4 sites covered 90 bp of
the core particle DNA, this result clearly demonstrates that in
both hypoacetylated and hyperacetylated nucleosomes, the his-
tone NH
2
tails remained associated with DNA, which was
simultaneously bound by GAL4-AH. Interestingly, the effect of
GAL4-AH binding on the interactions of the H2B tail with
DNA was largely acetylation dependent (a fivefold greater
reduction in cross-linking). However, this result was somehow
not surprising, considering the position occupied by the histone
H2B tails in the core particle (7, 45).
It should be noted that we have no data on the fate of the
histone H3 tails within reconstituted nucleosomes containing
bound GAL4-AH transcription factors. Although we raised
antibodies against histone H3, these antibodies were found not
to function at the high ionic strengths (see Materials and Meth-
ods) necessary for specific immunoprecipitation of individual
FIG. 8. Binding of five GAL4-AH molecules does not substantially affect the histone NH
2
tail-DNA interactions in reconstituted particle preparations containing
either hypoacetylated or hyperacetylated histones. (A)
32
P-labeled hypoacetylated 150-bp (I) and 180-bp (II) DNA particles containing or not containing five GAL4-AH
molecules were laser irradiated at identical doses, and the covalent complexes containing individual cross-linked histones were immunoprecipitated with specific
antihistone antibodies (see Materials and Methods for details). The amount of immunoprecipitated DNA was measured by Cerenkov counting. A histogram showing
the percentage of immunoprecipitated individual covalent histone-DNA complexes in the presence of five nucleosome-bound GAL4-AH molecules relative to that in
the absence of bound GAL-AH is shown. 1, presence of five GAL4-AH factors; 2, absence of these factors. The results are averaged over three independent
experiments with each of the antibodies used. a.u., arbitrary units. (B) Same as panel A but with 180-bp DNA particles reconstituted with either hypoacetylated or
hyperacetylated histones. N, particles containing hypoacetylated histones; H, particles containing hyperacetylated histones. The data represent average values from
several experiments. For hypoacetylated nucleosomes, six, four, and five independent immunoprecipitations were carried out with antibodies against H2A, H2B, and
H4, respectively. The results for hyperacetylated nucleosomes are averaged over three independent experiments with each of the antibodies used.
VOL. 18, 1998 HISTONE ACETYLATION AND TRANSCRIPTION FACTOR BINDING 6301

covalent histone-DNA complexes. However, our antibodies
functioned in immunoblotting, and by using them we found the
same efficiencies of cross-linking of hyperacetylated and hy-
poacetylated H3 tails to nucleosomal DNA in an immunoslot
assay (data not shown).
Based on the above data, hyperacetylation of histones
should not be expected to play a dramatic role in the enhance-
ment of GAL4-AH binding. In fact, this was found to be the
case: the binding of GAL4-AH was enhanced 2 to 2.5 times for
hyperacetylated nucleosomes (Fig. 9). The increased affinity of
GAL4-AH for its binding sites on hyperacetylated nucleo-
somes was more apparent for one GAL4 binding site tem-
plate. Recently, Vettese-Dadey et al. (70) demonstrated that
GAL4-AH binding to nucleosomes containing acetylated his-
tones was modestly stimulated. By using a gel retardation assay
coupled to immunochemical techniques, these authors were
able to show that only core particles containing the most highly
acetylated forms of histones had the highest affinity for GAL4-
AH. This affinity was found to be two to three times higher
than that for hypoacetylated particles. These results fully agree
with the data presented here for nucleosomes containing
highly hyperacetylated histones. Interestingly, Vettese-Dadey
et al. (70) observed stronger effects of H4 acetylation on the
binding of the bHLH protein USF (70). It will be interesting to
determine in future studies if USF has a stronger effect on the
DNA binding of histone tails. It has also been recently shown
that for transcription factors which involve a large DNA bind-
ing domain, such as TFIIIA, acetylation does not have any
effect on the efficiency of their binding to nucleosomal DNA
(34).
Histone hyperacetylation and transcription. Previous stud-
ies on the effect of histone acetylation on transcription factor
binding to nucleosomes have proposed that acetylation may
release histone tails from DNA, thereby stimulating transcrip-
tion factor access (reviewed in reference 72). This model was
based in part on the observation that removal of the histone
tails with trypsin (43, 69) similarly stimulated transcription
factor access to nucleosomal DNA. However, the present study
FIG. 9. Binding of GAL4-AH to reconstituted nucleosomes containing hypoacetylated or hyperacetylated core histones. (A)
32
P-labeled 180-bp probe DNA
containing five GAL4 binding sites was reconstituted in nucleosome cores with either hypoacetylated (lanes 1 to 6) or hyperacetylated (lanes 7 to 12) core histones and
allowed to react with increasing amounts of GAL4-AH. The mobilities of the naked DNA and the nucleosome complexes (Nucl.) are indicated. The concentrations
of GAL4-AH used in these experiments were 0 (lanes 1 and 7), 22 nM (lanes 2 and 8), 68 nM (lanes 3 and 9), 90 nM (lanes 4 and 10), 135 nM (lanes 5 and 11), and
180 nM (lanes 6 and 12). (B) Graphic representation of data from representative experiments shown in panel A. The decrease in unbound nucleosome bands versus
GAL4-AH concentrations is shown. Quantification was performed with the help of a PhosphorImager. The percentage of unbound nucleosomes was calculated as the
ratio of the nucleosome band signal to the radioactivity signal of the whole lane h, hypoacetylated histones; }, hyperacetylated histones. (C) An experiment similar
to that in panel A was carried out but with a 154-bp probe containing a single GAL4 binding site. The concentrations of GAL4-AH used were 0 (lanes 1 and 7), 22
nM (lanes 2 and 8), 45 nM (lanes 3 and 9), 110 nM (lanes 4 and 10), 220 nM (lanes 5 and 11), and 440 nM (lanes 6 and 12). (D) Dependence of the percentage of
unbound nucleosomes on GAL4-AH concentrations for the data presented in panel C. The measurements were performed as described for panel B.
6302 MUTSKOV ET AL. MOL.CELL.BIOL.

provides a different view of the dynamic interactions of tran-
scription factors and histone tails with nucleosomal DNA,
based upon two crucial observations. First, our data clearly
demonstrate that both hypoacetylated and hyperacetylated hi-
stone NH
2
tails bound to nucleosomal DNA. Hyperacetylation
of histone tails weakened histone tail-DNA binding but did not
abolish the interaction. Second, greater than 50% of the NH
2
tail-DNA interactions persisted during the occupancy of 90 bp
of nucleosomal DNA by GAL4-AH dimers (saturation of the
five GAL4 binding sites) within either hypoacetylated or hy-
peracetylated particles. Thus, our data indicate that the histone
tails remained associated with nucleosomal DNA when acety-
lated and also when the nucleosomal DNA was cooccupied by
DNA binding transcription factors. These data argue against a
simple model in which histone tails are mere inhibitors of
transcription factor access through mutually exclusive binding.
Instead, these data support a more dynamic role of histone
tails and their acetylation in enhancing factor access and in
transcription regulation.
It is becoming increasingly clear that transcription may be
regulated by histone NH
2
tails indirectly, since they can be the
target for repressor proteins (31, 56). For example, Edmonson
et al. (24) showed that the in vitro binding of the NH
2
tails of
histones H4 and H3 to the transcription repressor Ssn6-Tup1
complex is negatively regulated by histone acetylation. Fur-
thermore, mutations within the NH
2
tails of histones H3 and
H4 which abolish Tup1-histone binding led to in vivo enhance-
ment of transcription by more than one order of magnitude.
However, even if enhanced transcription factor binding and
displacement of repressor proteins operate synergistically via
histone acetylation, this activity can account for only a portion
of the several hundredfold enhancement of transcription ob-
served in vivo; consequently, other activities, such as those of
chromatin remodeling factors (36, 55, 61, 62, 68), activator
proteins (60), and so forth, must participate in the activation
process. Recently, an example demonstrating the complexity of
the activation of transcription was the finding that tumor sup-
pressor p53 can be acetylated by its coactivator, p300 (which
until recently was thought to have a specific histone acetyl-
transferase activity only), resulting in a remarkable enhance-
ment of its binding to DNA and activation of its biochemical
function (28).
The omnipresent histones. In this study, we demonstrated
that the binding of five GAL4-AH dimers to DNA (90 bp of
DNA occupancy) in both hypoacetylated and hyperacetylated
nucleosomes results in a weak alteration of the histone NH
2
tail-DNA interaction. At the same time, the saturation of the
five GAL4 binding sites induces a massive disruption of folded
histone domain-DNA interactions (52, 74). These findings sug-
gest that in vivo, during the process of transcription, histones
might not leave DNA but instead might remain anchored to it
(25, 49, 50, 58) through their NH
2
tails (49, 50). Indeed,
Nacheva et al. (50), by using chemical cross-linking with his-
tone NH
2
tails, showed that the actively transcribed hsp70 gene
(this gene does not have nucleosome organization when ac-
tively transcribed) contained histones in amounts comparable
to those of the nonactive gene. However, when a procedure for
cross-linking via the histone folded domains was used, no his-
tones were found on the hsp70 gene, indicating that histone
folded domain-DNA interactions were disrupted. The second
line of evidence came from the study of Mutskov et al. (49).
These authors, by using histone NH
2
tail UV laser-induced
cross-linking, demonstrated the presence of hyperacetylated
histones on the coding sequences of actively transcribed, non-
nucleosomally organized rat ribosomal genes. All of these data
clearly show that actively transcribed genes are covered with
histones and that the histones remain attached to the DNA via
their NH
2
tails.
ACKNOWLEDGMENTS
We thank E. Moudrianakis and S. Khochbin for helpful and stimu-
lating discussions as well as for careful reading of the manuscript.
This work was supported by grants from CNRS, INSERM (contract
4E006B), and Region Rhoˆne-Alpes (project Emergence).
REFERENCES
1. Alland, L., R. Muhle, H. Hou, Jr., J. Potes, L. Chin, N. Schreiber-Agus, and
R. A. DePinho. 1997. Role for N-CoR and histone deacetylase in Sin-3-
mediated transcriptional repression. Nature 387:49–55.
2. Allegra, P., R. Sterner, D. F. Clayton, and V. G. Allfrey. 1987. Affinity
chromatographic purification of nucleosomes containing transcriptionally
active DNA sequences. J. Mol. Biol. 196:379–388.
3. Allfrey, V. G., R. Faulkner, and A. E. Mirsky. 1964. Acetylation and meth-
ylation of histones and their possible role in the regulation of RNA synthesis.
Proc. Natl. Acad. Sci. USA 51:786–794.
3a.Angelov, D., V. Mutskov, and S. Dimitrov. Unpublished data.
4. Angelov, D., V. Stefanovsky, S. I. Dimitrov, V. Russanova, and I. Pashev.
1988. A picosecond UV laser induced protein-DNA crosslinking in recon-
stituted nucleohistones, nuclei and whole cells. Nucleic Acids Res. 16:4525–
4538.
5. Angelov, D., M. Berger, J. Cadet, C. Marion, and A. Spassky. 1994. High-
intensity ultraviolet laser probing of nucleic acids. Trends Photochem. Pho-
tobiol. 3:643–663.
6. Arents, G., R. W. Burlingame, B.-C. Wang, W. E. Love, and E. N. Moudri-
anakis. 1991. The nucleosomal core histone octamer at 3.1A resolution: a
tripartite protein assembly and a left-handed superhelix. Proc. Natl. Acad.
Sci. USA 88:10148–10152.
7. Arents, G., and E. N. Moudrianakis. 1994. DNA protein interactions in
chromatin and the structure of the histone octamer, p. 93–108. In H. Sarma
and M. Sarma (ed.), Structural biology: the state of the art. Proceedings of
the Eighth Conversation. State University of New York, Albany.
8. Ausio, J., and K. van Holde. 1986. Histone hyperacetylation: its effect on
nucleosome conformation and stability. Biochemistry 22:1421–1428.
9. Ausio, J., F. Dong, and K. E. van Holde. 1989. Use of selectively trypsinized
nucleosome core particles to analyze the role of the histone tails in the
stabilization of the nucleosome. J. Mol. Biol. 206:451–463.
10. Bane´res, J.-L., A. Martin, and J. Parello. 1997. The N tails of histones H3
and H4 adopt a highly structured conformation in the nucleosome. J. Mol.
Biol. 273:503–508.
11. Bannister, A. J., and T. Kouzarides. 1996. The CBP coactivator is a histone
acetyltransferase. Nature 384:641–643.
12. Beato, M., and K. Eisfeld. 1997. Transcription factor access to chromatin.
Nucleic Acids Res. 25:3559–3563.
13. Braunstein, M., A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach. 1993.
Transcriptional silencing in yeasts is associated with reduced nucleosome
acetylation. Genes Dev. 7:592–604.
14. Brownell, J., and C. D. Allis. 1995. An activity gel assay detects a single
catalytically active histone acetyltransferase subunit in Tetrahymena macro-
nuclei. Proc. Natl. Acad. Sci. USA 92:6364–6368.
15. Brownell, J., J. Zhou, T. Ranally, R. Kobayashi, D. Edmonson, S. Roth, and
C. D. Allis. 1996. Tetrahymena histone acetyltransferase A: a homolog to
yeast Gcn5p linking histone acetylation to gene activation. Cell 84:843–851.
16. Cary, P. D., T. Moss, and E. M. Bradbury. 1978. High resolution proton-
magnetic-resonance studies of chromatin core particles. Eur. J. Biochem.
89:475–482.
17. Chen, H., B. Li, and J. L. Workman. 1994. A histone DNA-binding protein,
nucleoplasmin, stimulates transcription factor binding to nucleosomes and
factor-induced nucleosome disassembly. EMBO J. 13:380–390.
18. Dimitrov, S., and A. P. Wolffe. 1997. Fine resolution of histones by two-
dimensional polyacrylamide gel electrophoresis: developmental implica-
tions. Methods Companion Methods Enzymol. 12:57–61.
19. Dimitrov, S. I., V. Russanova, and I. Pashev. 1987. The globular domain of
histone H5 is internally located in the 30 nm chromatin fiber: an immuno-
chemical study. EMBO J. 6:2387–2392.
20. Dimitrov, S. I., V. Stefanovsky, L. Karagyozov, D. A. Angelov, and I. G.
Pashev. 1990. The enhancers and promoters of the Xenopus laevis ribosomal
spacer are associated with histones upon active transcription of the ribo-
somal genes. Nucleic Acids Res. 18:6393–6397.
21. Dimitrov, S. I., G. Almouzni, M. Dasso, and A. Wolffe. 1993. Chromatin
transitions during early Xenopus embryogenesis: changes in histone H4
acetylation and in linker histone type. Dev. Biol. 160:214–227.
22. Dimitrov, S. I., and A. P. Wolffe. 1995. Chromatin and nuclear assembly:
experimental approaches toward the reconstitution of transcriptionally ac-
tive and silent states. Biochim. Biophys. Acta 1260:1–13.
23. Ebralidze, K. K., T. R. Hebbes, A. L. Clayton, A. W. Thorne, and C. Crane-
Robinson. 1993. Nucleosomal structure at hyperacetylated loci probed in
VOL. 18, 1998 HISTONE ACETYLATION AND TRANSCRIPTION FACTOR BINDING 6303

nuclei by DNA-histone crosslinking. Nucleic Acids Res. 21:4734–4738.
24. Edmonson, D. G., M. M. Smith, and S. Y. Roth. 1996. Repression domain of
the yeast global repressor Tup1 interacts directly with histones H3 and H4.
Genes Dev. 10:1247–1259.
25. Ericsson, C., U. Grossbach, B. Bjorkroth, and B. Daneholt. 1990. Presence
of histone H1 on an active Balbiani ring gene. Cell 60:73–83.
26. Garcia-Ramirez, M., C. Rocchini, and J. Ausio. 1995. Modulation of chro-
matin folding by histone acetylation. J. Biol. Chem. 270:17923–17928.
27. Grunstein, M. 1997. Histone acetylation in chromatin structure and tran-
scription. Nature 389:349–352.
28. Gu, W., and R. G. Roeder. 1997. Activation of p53 sequence-specific DNA
binding by acetylation of the p53 C-terminal domain. Cell 90:595–606.
29. Hebbes, T. R., A. W. Thorne, and C. Crane-Robinson. 1989. A direct link
between core histone acetylation and transcriptionally active chromatin.
EMBO J. 7:1395–1402.
30. Hebbes, T. R., A. L. Clayton, A. W. Thorne, and C. Crane-Robinson. 1994.
Core histone acetylation co-maps with generalized DNase I sensitivity in the
chicken b-blobin chromosomal domain. EMBO J. 13:1823–1830.
31. Hecht, A., T. Laroche, S. Strahl-Bolsinger, S. M. Gasser, and M. Grunstein.
1995. Histone H3 and H4 termini interact with SIR3 and SIR4 proteins: a
molecular model for the formation of heterochromatin in yeast. Cell 80:583–
592.
32. Heinzel, T., R. M. Lavinsky, T.-M. Mullen, M. So¨derstro¨m, C. D. Laherty, J.
Torchia, W.-M. Yang, G. Brard, S. D. Ngo, J. R. Davie, E. Seto, R. N.
Eisenman, D. W. Rose, C. K. Glass, and M. G. Rosenfeld. 1997. A complex
containing N-CoR, mSin3 and histone deacetylase mediates transcriptional
repression. Nature 387:43–48.
33. Hockensmith, J. W., W. L. Kubasek, W. R. Worachek, E. M. Evertsz, and
P. H. von Hippel. 1991. DNA-protein interactions. Methods Enzymol. 208:
211–236.
34. Howe, L., and J. Ausio. 1998. Nucleosome translational position and not
histone acetylation determines TFIIA binding to nucleosomal Xenopus laevis
5S rRNA genes. Mol. Cell. Biol. 18:1156–1162.
35. Ip, Y. T., V. Jackson, J. Meyer, and R. Chalkley. 1988. The separation of
transcriptionally engaged genes. J. Biol. Chem. 263:14044–14052.
36. Ito, T., M. Bulger, M. J. Pazin, R. Kobayashi, and J. T. Kadonaga. 1997.
ACF, an ISWI-containing and ATP-utilizing chromatin assembly and re-
modeling factor. Cell 90:145–155.
37. Jeppesen, P., A. Mitchell, B. M. Turner, and P. Perry. 1992. Antibodies to
defined histone epitopes reveal variations in chromatin conformation and
underacetylation of centric heterochromatin in human metaphase chromo-
somes. Chromosoma 101:322–332.
38. Jeppesen, P., and B. M. Turner. 1993. The inactive X chromosome in female
mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic
marker for gene expression. Cell 74:281–289.
39. Kadosh, D., and K. Struhl. 1997. Repression by Ume6 involves recruitment
of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to
target promoters. Cell 89:365–371.
40. Khochbin, S., and A. P. Wolffe. 1997. The origin and utility of histone
deacetylases. FEBS Lett. 419:157–160.
41. Kuo, M.-H., J. A. Brownell, R. E. Sobel, T. Ranalli, R. G. Cook, D. G.
Edmonson, S. Y. Roth, and C. D. Allis. 1996. Transcription-linked acetyla-
tion by Gcn5p of histones H3 and H4 at specific lysines. Nature 383:269–272.
42. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
43. Lee, D. Y., J. J. Hayes, D. Pruss, and A. P. Wolffe. 1993. A positive role for
histone acetylation in transcription factor access to nucleosomal DNA. Cell
72:73–84.
44. Lin, Y. S., M. Carey, M. Ptachne, and M. R. Green. 1988. How different
transcription activators can cooperate. Cell 54:659–664.
45. Luger, K., A. W. Ma¨der, R. K. Richmond, D. F. Sargent, and T. J. Richmond.
1997. Crystal structure of the nucleosome core particle at 2.8 A resolution.
Nature 389:251–260.
46. Mirzabekov, A. D., D. V. Pruss, and K. K. Ebralidze. 1989. Chromatin
superstructure-dependent crosslinking with DNA of the histone H5 residues
Thr1, His25 and His62. J. Mol. Biol. 211:479–491.
47. Mizzen, C. A., X. J. Yang, T. Kokubo, J. E. Brownell, A. J. Bannister, T.
Owen-Hughes, J. Workman, L. Wang, S. L. Berger, T. Kouzarides, I. Naka-
tani, and C. D. Allis. 1996. The TAFII250 subunit of TFIID has histone
acetyltransferase activity. Cell 86:1261–1270.
48. Moss, T., S. I. Dimitrov, and D. Houde. 1997. UV-laser crosslinking of
proteins to DNA. Methods Companion Methods Enzymol. 11:225–234.
49. Mutskov, V. J., V. Russanova, S. I. Dimitrov, and I. Pashev. 1996. Histones
associated with non-nucleosomal rat-ribosomal genes are acetylated, while
those bound to nucleosome organized gene copies are not. J. Biol. Chem.
271:11852–11857.
50. Nacheva, G. A., D. Y. Guschin, O. V. Preobrazhenskaya, V. L. Karpov, K. K.
Ebralidze, and A. D. Mirzabekov. 1989. Changes in the pattern of histone
binding to DNA upon transcription activation. Cell 58:27–36.
51. Ogryzko, V. V., R. L. Schiltz, V. Russanova, B. H. Howard, and Y. Nakatani.
1996. The transcriptional coactivators p300 and CBP are histone acetytrans-
ferases. Cell 87:953–959.
52. Owen-Hughes, T., and J. L. Workman. 1994. Experimental analysis of chro-
matin function in transcriptional control. Crit. Rev. Euk. Gene Expression
4:403–441.
53. Pashev, I. G., S. I. Dimitrov, and D. Angelov. 1991. Laser-induced protein-
DNA crosslinking. Trends Biochem. Sci. 16:323–326.
54. Pazin, M. J., and J. T. Kadonaga. 1997. What’s up and down with histone
deacetylation and transcription? Cell 89:325–328.
55. Peterson, C. L., and J. W. Tamkun. 1995. The SWI-SNF complex: a chro-
matin remodeling machine? Trends Biochem. Sci. 20:143–146.
56. Roth, S. Y., M. Shimuzu, L. Johnson, M. Grunstein, and T. Simpson. 1992.
Stable nucleosome positioning and complete repression by the yeast a2
repressor are disrupted by amino-terminal mutations in histone H4. Genes
Dev. 6:411–425.
57. Roth, S. Y., and C. D. Allis. 1996. Histone acetylation and chromatin assem-
bly: a single escort, multiple dances? Cell 87:5–8.
58. Solomon, M. J., P. L. Larsen, and A. Varshavsky. 1988. Mapping protein-
DNA interactions in vivo with formaldehyde: evidence that histone H4 is
retained on a highly transcribed gene. Cell 53:937–947.
59. Stefanovsky, V., S. I. Dimitrov, V. R. Russanova, D. Angelov, and I. G.
Pashev. 1989. Laser-induced crosslinking of histones to DNA in chromatin
and core particles: implications in studying histone-DNA interactions. Nu-
cleic Acids Res. 23:10069–10081.
60. Tjian, R., and T. Maniatis. 1994. Transcriptional activation: a complex puz-
zle with few easy pieces. Cell 77:5–8.
61. Tsukiyama, T., P. B. Becker, and C. Wu. 1994. ATP-dependent nucleosome
disruption at a heat-shock promoter mediated by the binding of GAGA
transcription factor. Nature 367:525–532.
62. Tsukiyama, T., C. Daniel, G. Tamkun, and C. Wu. 1995. ISWI, a member of
the SWI2/SNF2 ATPase family, encodes the 140 kDa subunit of the nucleo-
some remodeling factor. Cell 83:1021–1026.
63. Turner, B. M., L. Franchi, and H. Wallace. 1990. Islands of acetylated H4 in
polytene chromosomes and their relationship to chromatin packaging and
transcriptional activity. J. Cell Sci. 96:335–346.
64. Turner, B. M., A. J. Birley, and J. Lavender. 1992. Histone H4 isoforms
acetylated at specific lysine residues define individual chromosomes and
chromatin domains in Drosophila polytene nuclei. Cell 69:375–384.
65. Turner, B. M., and L. P. O’Neill. 1995. Histone acetylation in chromatin and
chromosomes. Semin. Cell Biol. 6:229–236.
66. Usachenko, S. I., S. G. Bavykin, I. M. Gavin, and E. M. Bradbury. 1994.
Rearrangement of the histone H2A C-terminal domain in the nucleosome.
Proc. Natl. Acad. Sci. USA 91:6845–6849.
67. van Holde, K. 1988. Chromatin. Springer-Verlag KG, Berlin, Germany.
68. Varga-Weisz, P. D., M. Wilm, E. Bonte, K. Dumas, M. Mann, and P. B.
Becker. 1997. Chromatin-remodelling factor CHRAC contains the ATPases
ISWI and topoisomerase II. Nature 388:598–602.
69. Vettese-Dadey, M., P. Walter, H. Chen, L. J. Juan, and J. L. Workman. 1994.
Role of the histone amino termini in the facilitated binding of a transcription
factor, GAL4-AH, to nucleosome cores. Mol. Cell. Biol. 14:970–981.
70. Vettese-Dadey, M., P. A. Grant, T. R. Hebbes, C. Crane-Robinson, C. D.
Allis, and J. L. Workman. 1996. Acetylation of histone H4 plays a primary
role in enhancing transcription factor binding to nucleosomal DNA in vitro.
EMBO J. 15:2508–2518.
71. Volgelstein, B., and D. Gillespie. 1979. Preparative and analytical purifica-
tion of DNA from agarose. Proc. Natl. Acad. Sci. USA 76:615–619.
72. Wade, P. A., D. Pruss, and A. P. Wolffe. 1997. Histone acetylation: chromatin
in action. Trends Biochem. Sci. 22:128–132.
73. Walker, I. O. 1984. Differential dissociation of histone tails from core chro-
matin. Biochemistry 23:5622–5628.
74. Walter, P. P., T. A. Owen-Hughes, J. Coˆte´, and J. L. Workman. 1995.
Stimulation of transcription factor binding and histone displacement by
nucleosome assembly protein 1 and nucleoplasmin requires disruption of the
histone octamer. Mol. Cell. Biol. 15:6178–6187.
75. Wolffe, A. P., and D. Pruss. 1996. Targeted chromatin disruption: transcrip-
tion regulators that acetylate histones. Cell 84:817–819.
76. Workman, J. L., and R. E. Kingston. 1992. Nucleosome core displacement in
vitro via a metastable transcription factor-nucleosome complex. Science 258:
1780–1784.
77. Yang, H. J., V. Ogryzko, J. Nishikawa, B. Howard, and Y. Nakatani. 1996.
Ap300/CBP-associated factor that competes with the adenoviral oncoprotein
E1a. Nature 382:319–324.
6304 MUTSKOV ET AL. MOL.CELL.BIOL.