From the Howard Hughes Medical Institute, Department
of Biochemistry and Molecular Biology, The Pennsylvania State
University, University Park, Pennsylvania 16802-4500, the ** Department
of Microbiology and Immunology, Baylor College of Medicine, Houston,
Texas 77030, and the
Chromatin and Gene
Expression Group, Anatomy Department, University of Birmingham Medical
School, Birmingham B15 2TT, United Kingdom
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ABSTRACT |
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The coactivator/adaptor protein Gcn5 is a
conserved histone acetyltransferase, which functions as the catalytic
subunit in multiple yeast transcriptional regulatory complexes. The
ability of Gcn5 to acetylate nucleosomal histones is significantly
reduced relative to its activity on free histones, where it
predominantly modifies histone H3 at lysine 14. However, the
association of Gcn5 in multisubunit complexes potentiates its
nucleosomal histone acetyltransferase activity. Here, we show that the
association of Gcn5 with other proteins in two native yeast complexes,
Ada and SAGA (Spt-Ada-Gcn5-acetyltransferase), directly confers upon Gcn5 the ability to acetylate an expanded set of lysines on H3. Furthermore Ada and SAGA have overlapping, yet distinct, patterns of
acetylation, suggesting that the association of specific subunits determines site specificity.
In the eukaryotic nucleus DNA is packaged by histones into
nucleosomes, the repeating subunits of chromatin. The nucleosome core
particle contains about 146 base pairs of DNA and two of each of the
histones H2A, H2B, H3, and H4. Packaging of DNA into chromatin
suppresses transcription, at least in part by occluding the binding of
transcriptional activators and basal transcription machinery to their
cognate DNA sites (1, 2) and by blocking transcriptional elongation
(3). A number of chromatin remodeling activities have been isolated
that assist activators to bind to their sites by promoting a localized
alteration in chromatin structure (4).
In addition to these activities, post-translational modifications of
the core histones within the nucleosome have also been linked to the
transcriptional capacity of chromatin (5, 6). The acetylation of the
highly conserved lysine residues within the amino-terminal domains of
all four core histones has been extensively studied (7). Histone
acetylation has been associated with the interaction of non-histone
proteins with histones (8, 9), histone deposition and nucleosome
assembly (10), higher order packing of chromatin (11, 12), and the
transcriptional activity of cellular chromatin (5, 13). Correlations
between transcription and acetylation are reinforced by studies
demonstrating that at least some active chromosomal domains are
hyperacetylated (14), while inactive or heterochromatin domains are
associated with hypoacetylated histones (15-18). However these general
correlations do not hold true when specific acetylated H4 isoforms are
monitored indicating that particular patterns of lysine acetylation
rather than bulk acetylation is of functional importance (15, 19, 20).
Therefore, an important goal in studying this phenomenon has been the
isolation of native activities that generate such specific patterns of acetylation.
The steady state level of acetylation of histones is a balance between
the action of histone acetyltransferases
(HATs)1 and histone
deacetylases. These activities are often found to be associated with
large multisubunit protein complexes and contain known regulators of
transcription (21). A number of previously characterized
transcriptional coactivators have also been shown to possess HAT
activity, providing a direct molecular link between histone acetylation
and gene activation (22, 23). In the budding yeast Saccharomyces
cerevisiae, the coactivator/adaptor protein Gcn5 is involved in
the regulation of various genes (24-28). Gcn5 requires
acetyltransferase function for both promoter-directed histone
acetylation and Gcn5-mediated transcriptional activation in
vivo (29, 30). In vitro, recombinant Gcn5 has been
shown to efficiently acetylate free histones and not nucleosomal
histone substrates (31), but see also Ref. 32. However, the association of Gcn5 with other proteins in large multisubunit complexes in yeast
potentiates its nucleosomal acetyltransferase activity (33-36).
The 1.8-MDa SAGA complex is a Gcn5-dependent HAT activity,
which contains at least three distinct groups of gene products (23,
33). The first of these are the Ada proteins Gcn5 (Ada4), Ada1, Ada2,
Ada3, and Ada5 (Spt20), isolated as proteins that interact functionally
with the transcription factor Gcn4 and the activation domain derived
from the herpes simplex virus activator VP16. The second group
comprises all members of the TBP-related set of Spt proteins, Spt3,
Spt7, Spt8, and Spt20 (Ada5), except TBP (Spt15), which were isolated
as suppressers of transcription initiation defects caused by promoter
insertions of the transposable element Ty. The third group within SAGA
includes a subset of TBP-associated factors, TAFIIs,
including TAFII90, TAFII68/61,
TAFII60, TAFII25/23, and TAFII20/17
(37).
A second Gcn5-dependent HAT activity, which we named Ada,
has been isolated as a 0.8-MDa multisubunit complex containing the Ada
proteins Gcn5, Ada2, and Ada3. However, the Ada complex does not
contain the other Ada proteins or the Spt or TAFII proteins found in SAGA (33). Gcn5 also functions in additional multisubunit HAT
complexes in yeast (27, 35). In vitro and in vivo
studies indicate that both the SAGA and Ada complexes can promote
acetyl coenzyme A (acetyl-CoA)-dependent transcriptional
activation from nucleosomal templates (29, 30, 38).
Initial studies using recombinant Gcn5 indicated that it displays a
nonrandom specificity for acetylation of lysines, predominantly acetylating histone H3 at lysine 14 (31). In vivo,
acetylation of H3 can occur at lysines 9, 14, 18, and 23. Since
acetylation of lysine 9 of H3 is associated with histone deposition, it
has been suggested that acetylation of other sites may be associated with transcription (10, 31). In this study we have investigated the
histone H3 lysine acetylation specificity of the native
Gcn5-dependent HAT complexes Ada and SAGA in comparison
with recombinant Gcn5 (rGcn5). We used a combination of Western
blotting, peptide assays, and microsequencing analysis to determine the
sites of acetylation for the Gcn5-dependent Ada and SAGA
complexes. We show that while rGcn5 acetylates only lysine 14, purified
Ada complex acetylates both lysines 14 and 18, and SAGA acetylates to
some extent all four lysines in H3. Using mutant variants of Gcn5, we
show that this acetylation pattern is dependent on Gcn5 function and
not other putative HAT activities within these complexes. These results indicate that the association of Gcn5 with different proteins in
multisubunit complexes endows upon it an expanded substrate specificity.
Yeast Strains and Purification of rGcn5, SAGA, and Ada
Complexes--
Yeast Gcn5 was expressed in bacteria as a fusion
protein with six histidine residues at the amino terminus and purified
under denaturing conditions on Ni2+-NTA-agarose (39). SAGA
and Ada complexes were purified from the yeast strain CY396 (40). The
yeast strain PSY316 and its derivative PSY316
SAGA and Ada complexes were purified using a scheme adapted from (33).
Elution of SAGA and Ada from each column was monitored by HAT assays
and immunoblotting. Briefly, whole cell extracts were prepared from 20 liters of the yeast strain CY396 grown to mid-log phase. The extract
was bound batchwise with 20 ml of Ni2+-NTA-agarose
(Qiagen). The resin was then washed in a column with 20 mM
imidazole, followed by elution of the bound proteins with 300 mM imidazole. The Ni2+-NTA-agarose column
eluate was directly loaded onto a Mono Q HR 5/5 column (Amersham
Pharmacia Biotech). Bound proteins were eluted with a 25-ml linear
gradient from 100 to 500 mM NaCl. Peak SAGA and Ada
fractions from the Mono Q column were individually pooled and diluted
to 100 mM NaCl and loaded onto a Mono S HR 5/5 column (Amersham Pharmacia Biotech). For each complex, bound proteins were
eluted with a 25-ml linear gradient from 100 to 500 mM
NaCl. Peak fractions were diluted to 100 mM NaCl and loaded
directly onto a 0.8-ml histone-agarose column (Sigma). Bound proteins
were eluted with a 8-ml linear gradient from 100 mM to 1 M NaCl. Peak fractions were concentrated down to 0.7 ml
using Centriprep-30 (Millipore). Samples were then loaded on a Superose
6 HR 10/30 column (Amersham Pharmacia Biotech) equilibrated with 250 mM NaCl. The calibration of the Superose 6 column for these
samples was as follows: exclusion volume (blue dextran), fraction 12;
thyroglobulin (669 kDa), fraction 22; ferritin (440 kDa), fraction 26;
adolase (158 kDa), fraction 29; ovalbumin (45 kDa, fraction 32).
SAGA and Ada complexes were also partially purified from 6 liters of
wild type, gcn5 HAT Assays and Western Blotting--
HAT assays were performed
as described previously (33). In Fig. 1, HAT assays were scaled up
3-fold and performed with HeLa free core histones or nucleosomes in the
presence or absence of [3H]acetyl-CoA. Following
incubation, each reaction was divided into three and the histones
electrophoresed through 15% SDS-polyacrylamide gels. One gel was
processed for fluorography and the other two processed for
immunoblotting, as described (41). For all other immunoblots,
equivalent volumes of Superose 6 fractions of purified SAGA or Ada
complexes were subjected to SDS-PAGE on a 10% gel, transferred to
nitrocellulose, and processed for blotting.
Peptide Synthesis and Assays--
A wild type peptide spanning
the NH2-terminal amino acids 5-28 of human histone H3 and
derivatives containing either no or single non-acetylated lysines
available at amino acids positions 9, 14, 18, or 23 were generated
(Alta Bioscience, University of Birmingham, United Kingdom). The
sequence of the wild type peptide is as follows:
QTARKSTGGKAPRKQLATKAARRSC (the positions of lysine residues are indicated in boldface letters). The mutant versions of this peptide, depicted in Fig. 3, were generated
by substituting acetyl-lysine at certain positions. Assays were
performed using 300 ng of peptides and roughly equivalent activities of
purified rGcn5, Ada, or SAGA complexes in a typical liquid HAT
reaction. Scintillation counts were derived from three individual reactions.
Microsequencing--
HAT assays were performed in the presence
of 5 µg of HeLa nucleosomes, 1 µCi of [3H]acetyl-CoA
and equivalent activities of purified Ada and SAGA complexes. Reactions
were incubated for 40 min at 30 °C and were then subjected to
SDS-PAGE on a 15% gel and transferred to Immobilin PSQ
membrane (Millipore), according to Vettese-Dadey et al.
(41). Histone H3 was excised from the membrane and sequenced as
described (42).
The Gcn5-dependent SAGA and Ada HAT Complexes Show
Expanded H3 Lysine Specificity--
It has been reported previously
that rGcn5 predominantly acetylates histone H3 at lysine 14 (Lys-14),
when free histones are presented as a substrate (31). We investigated
the lysine specificity of purified native Gcn5-dependent
Ada and SAGA HAT complexes in comparison with that of rGcn5. Either
free histones or nucleosomes were incubated, in vitro,
together with rGcn5, Ada, or SAGA complexes in the presence or absence
of acetyl-CoA. These reactions were assayed by SDS-PAGE and
fluorography and by Western blotting, using antisera which recognize
specific acetylated forms of histone H3 (43, 44). Consistent with
previous experiments (33) we found that all three activities acetylated
free histones, but that only native complexes were able to efficiently
acetylate nucleosomal histones under the same conditions (Fig.
1, A and B). Using
an antiserum specific for histone H3 acetylated at Lys-14, we found
that rGcn5 and both native HAT complexes efficiently acetylated this
site on free histone substrates, in an acetyl-CoA-dependent fashion. However using an antiserum that recognizes H3 acetylated at
either lysine 9 or 18, we found that unlike rGcn5, the native Ada and
SAGA complexes are able to acetylate either or both of these sites
(Fig. 1A). When using nucleosomal histones as a substrate, we found the same pattern of site specificity in that H3 modified by
the Ada and SAGA activities is recognized by both the anti-H3.Ac14 and
H3.Ac9/18 antiserum (Fig. 1B).
Ada and SAGA Are Bona Fide Gcn5-dependent HAT
Activities--
These results indicate that when Gcn5 is associated
with other proteins in a HAT complex, it acquires an expanded substrate specificity. We have shown, using a combination of purified activities from gcn5
Ada and SAGA activities from a Superose 6 gel filtration column were
incubated in HAT reactions or assayed by Western blotting with
antiserum against the Gcn5 or Ada2 components of these complexes. Both
the wild type Ada (Fig. 2A)
and SAGA (Fig. 2B) complexes efficiently acetylate
nucleosomal histones; however, this activity is lost using activities
purified from gcn5 The SAGA and Ada HAT Complexes Show Distinct Patterns of H3 Lysine
Acetylation--
The data presented in Fig. 1 indicate that both the
Ada and SAGA complexes acetylate lysines other than Lys-14 on histone H3. We wanted to further investigate the specificity of these complexes. Therefore, we generated a set of peptides that spanned the
H3 amino terminus. The wild type peptide carries non-acetylated lysines
at all four positions (Lys-9, Lys-14, Lys-18, Lys-23), while
derivatives of this peptide were either acetylated at all four lysines
or retained only single non-acetylated lysines at positions Lys-9,
Lys-14, Lys-18, or Lys-23 (Fig.
3A). These peptides were used
as substrates in HAT reactions to determine the lysine specificity of
the distinct purified HAT complexes. We found that rGcn5 was able to
efficiently acetylate only the wild type peptide or the peptide with a
non-acetylated Lys-14 (Fig. 3B), consistent with the results
of Kuo et al. (31). However, the Ada and SAGA complexes were
able also to acetylate both the Lys-14 and the Lys-18 peptide,
indicating an expanded lysine specificity over rGcn5. In addition SAGA
was able to acetylate the Lys-9 and Lys-23 peptides, albeit to a lesser
extent (Fig. 3B). This suggests that the histone acetylation
specificity of Gcn5 is dependent on the context of associated factors
in a given complex.
Native HAT Complexes Generate Distinct Patterns of Nucleosomal H3
Acetylation--
Collectively the data presented above indicate that
the two Gcn5-dependent HAT complexes, Ada and SAGA, and
rGcn5 show partially overlapping yet distinct patterns of lysine
acetylation in the H3 amino terminus. Both of these native HAT
complexes most likely modify nucleosomal histones in the nucleus.
Consistent with this notion rGcn5 is able to efficiently acetylate only
free histones, while the Ada and SAGA complexes can also acetylate
nucleosomal histones (Fig. 1B). To rigorously identify the
lysine(s) acetylated by each of these native complexes we subjected
in vitro labeled nucleosomal histone H3 to microsequence
analysis followed by direct determination of radioactivity at each
position in the H3 sequence. The results demonstrate that the Ada
complex acetylates both Lys-14 and Lys-18 (Fig.
4A), while SAGA acetylates all
four lysines to varying extents (Fig. 4B). These data
obtained from the microsequence analysis are in good agreement with the
results obtained from the Western blotting (Fig. 1) and peptide assays
(Fig. 3).
The transcriptional adaptor Gcn5 was originally demonstrated to be
a histone acetyltransferase with a nonrandom pattern of acetylation,
predominantly modifying H3 at Lys-14 in vitro (31). Very
recently, rGcn5 has also been shown to monoacetylate nucleosomal H3 at
lysine 14, albeit less efficiently than free histones, using optimum
NaCl or MgCl2 concentrations (32). However, the association of Gcn5 in high molecular weight multiprotein complexes potentiates its
nucleosomal histone acetyltransferase activity (33-36). In addition,
we have shown in this study that this association also directly endows
upon Gcn5 an expanded lysine specificity. The Gcn5-dependent complex Ada preferentially acetylates
Lys-14 > Lys-18, while SAGA acetylates Lys-14 > Lys-18 > Lys-9 = Lys-23 on synthetic H3 peptide substrates and on
nucleosomal H3. These results indicate that Gcn5 generates a
context-dependent acetylation pattern as a consequence of
its association with other factors. The Ada and SAGA complexes share at
least a few common components, namely Gcn5, Ada2, and Ada3. However the
SAGA complex is distinguished from the Ada complex by the fact that it
additionally contains Spt proteins (33) and a subset of
TAFII proteins (37). Gcn5 is also associated in other
apparently distinct HAT complexes (27, 34, 35). These findings have
suggested that these multiple Gcn5-dependent HAT complexes
may serve distinct roles in yeast transcriptional regulation (23). It
is therefore possible that individual Gcn5-containing complexes may
generate overlapping, yet distinct patterns of nucleosomal acetylation,
which may signal a specific function. In this scenario, the components
unique to each complex are likely to determine the target lysines
acetylated by Gcn5.
We have recently demonstrated that purified native yeast HAT complexes,
including Ada, and SAGA can promote transcription in an
acetyl-CoA-dependent fashion in vitro (38, 45).
These results are reinforced by additional in vitro and
in vivo data demonstrating a requirement for Gcn5-directed
HAT activity for promoter acetylation and transcriptional activation
(29, 30). Since Gcn5 is a transcriptional adaptor protein, it was
originally inferred that the modification of Lys-14 by rGcn5 represents
a transcription linked acetylation pattern, distinct from the
predominant mapped deposition-related acetylation site of Lys-9 in
yeast (31, 46). However, our data suggest that the SAGA complex
acetylates all four lysines including Lys-9, thereby linking lysines
other than Lys-14 in transcriptional activation. We demonstrate that Gcn5 contained in native type A HAT complexes can generate acetylation patterns on H3 at least, which overlap with those generated by type B
histone acetyltransferases for histone deposition and chromatin assembly (10). Therefore, transcription-linked acetylation does not
simply involve acetylation of Lys-14 on H3, but may encompass a variety
of lysine modifications. However, a common theme between both native
HAT Gcn5 complexes and rGcn5 used in this study is the acetylation of
Lys-14. This may indicate a pivotal role for this lysine in the
Gcn5-dependent regulation of transcription.
A recent study examined the function of histone acetylation in
vivo by mutation of target lysines in yeast strains with and without a functional GCN5 gene. The authors found that Gcn5 was required for full levels of acetylation at multiple sites in H3 and H4
in vivo (47). In particular Gcn5 is directly or indirectly required for acetylation of H3 Lys-9 and H3 Lys-18 in vivo,
while mutation of Lys-14 in gcn5
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
gcn5 used for
transformation of the GCN5 HAT domain mutants LKN and KQL are described
(29).
, KQL, and LKN mutant strains, which were grown at
30 °C to an absorbance at 600 nM of 2.0. Whole cell extracts were prepared by glass bead disruption of cells, and complexes
were purified using 5 ml of Ni2+-NTA-agarose, Mono Q HR 5/5
column and Superose 6 HR 10/30 columns, as described above.
RESULTS
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Fig. 1.
Native Gcn5-containing complexes show an
expanded lysine acetylation specificity on histone H3.
A, HAT assays and Western blotting using free histones as
substrates. Free core histones were incubated in HAT assays in the
presence (+) or absence ( ) of radiolabeled acetyl-CoA, either alone
(control) or together with rGcn5, purified Ada, or SAGA complexes, as
indicated. Histones were then subjected to SDS-PAGE, Coomassie Blue
staining, and fluorography or immunoblotted with anti-H3.Ac14 or
anti-H3.Ac9/18 antiserum. B, HAT assays and Western blotting
using nucleosomal substrates. The migratory positions of histones are
indicated.
yeast strains in HAT assays or activity gel assays, that
Gcn5 is most likely the only or at least predominant catalytic subunit
of the Ada and SAGA complexes (33). However it remains formally
possible that the expanded repertoire for site specificity of these
complexes is due to an additional unidentified catalytic subunit. To
further address this issue we assayed purified Ada and SAGA complexes
from a wild type or deleted gcn5 strain or yeast strains carrying point
mutations within Gcn5. The KQL mutant Gcn5 has been demonstrated to be
defective in HAT activity and transcriptional activation, while the LKN
mutant maintains the catalytic activity and transcriptional potential
of Gcn5 (29).
strains. Consistent with the results of Wang
et al. (29) the KQL mutant Gcn5 complexes are unable to
acetylate nucleosomal (Fig. 2, A and B) and
free histone substrates (date not shown), while LKN mutants are
unaffected. These data reinforce the evidence that the acetylation
activity of both the Ada and SAGA complexes is solely dependent
on Gcn5 function.
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Fig. 2.
The Ada and SAGA complexes are dependent on
Gcn5 for HAT activity. A, Ada complexes were partially
purified from wild type (WT) and gcn5 yeast strains or
yeast strains carrying a mutation within Gcn5 which do (KQL)
or do not (LKN) affect HAT activity. Fluorograms from
nucleosomal HAT assays and Western blots performed using anti-Gcn5 or
anti-Ada2 antisera, using fractions from the final Superose 6 column,
are shown. The migratory positions of histones are indicated.
B, HAT assays and Western blotting using partially purified
SAGA fractions are shown, as described above.
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Fig. 3.
The Ada and SAGA complexes directly acetylate
lysines other than lysine 14 on H3 peptides. A,
schematic diagram depicting the H3 NH2-terminal peptides
used. The positions of non-acetylated lysines in each peptide are
indicated. B, recombinant yeast Gcn5 (rGcn5) or
purified Ada and SAGA complexes were incubated together with wild type
peptide (WT), peptide without any non-acetylated lysines
(open circles), or peptides with only single non-acetylated
lysines at any given position (9, 14, 18, or 23). Shown are histograms
of scintillation counts from liquid HAT assays. Data are normalized to
the relative activity achieved using the WT peptide.
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Fig. 4.
The Ada and SAGA complexes generate distinct
patterns of acetylation on histone H3 from nucleosomes. HAT assays
were performed using nucleosome substrates incubated together with
purified Ada (A) or SAGA complexes (B).
Radiolabeled histone H3 was subsequently purified and subjected to
microsequence analysis followed by direct determination of
radioactivity at each position in the H3 sequence. The counts/min (cpm)
for each cycle are plotted against the amino acid detected for that
cycle. Potential acetylation sites are indicated.
DISCUSSION
cells conferred a strong synthetic growth defect. Our results provide a direct molecular explanation for
these observations, in that native Gcn5-containing HAT complexes, such
as SAGA and Ada, can acetylate multiple H3 lysines. The
Gcn5-dependent acetylation events are important for normal
progression of the cell cycle and for transcriptional activation (29,
47). Furthermore, a critical level of histone acetylation is essential
for cell viability (47). Interestingly a study by Thompson et
al. (48) has shown that histone H3 is required for full repression
at yeast telomeres and silent mating loci and that the acetylatable
lysines of H3 play an important role in silencing. While single-site
mutations at H3 lysines 9 or 14 had little effect on telomeric
repression, substitution of 3 or 4 lysines simultaneously resulted in
strong telomeric derepression. This result suggested that a number of modified lysines in H3 may be required for derepression (48). Our
results suggest that such a function could be provided by expanded
lysine specificity of Gcn5 contained within native HAT complexes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Shelley Berger for yeast strains and Robert Durso for technical assistance.
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FOOTNOTES |
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* This work was supported in part by Grant GM47867 from the National Institute of General Medical Sciences (awarded to J. L. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by Postdoctoral Fellowship PF-98-017-01-GMC from the American Cancer Society.
¶ Holds a fellowship from the Austrian Science Foundation (FWF).
Postdoctoral associate of the Howard Hughes Medical Institute.
§§ Supported by the Wellcome Trust.
¶¶ Asssociate Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802-4500. Tel.: 814-863-8256; Fax: 814-863-0099; E-mail: jlw10{at}psu.edu.
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ABBREVIATIONS |
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The abbreviations used are: HAT, histone acetyltransferase; SAGA, Spt-Ada-Gcn5-acetyltransferase; TBP, TATA-binding protein; TAFII, TBP-associated factor; PAGE, polyacrylamide gel electrophoresis; H3.Ac14, histone H3 acetylated at lysine 14; NTA, nitrilotriacetic acid.
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REFERENCES |
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