Trichostatin A reduces hormone-induced transcription of the
MMTV
promoter and has pleiotropic effects on its chromatin structure
Carolina A
˚
strand
1,
*, Tomas Klenka
1,
*, O
¨
rjan Wrange
1
and Sergey Belikov
1,2
1
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden;
2
D. I. Ivanovsky
Institute of Virology, Moscow, Russia
The deacetylase inhibitor trichostatin A (TSA) has long
been used to study the relationship between gene transcrip-
tion and the acetylation status of chromatin. We have
used Xenopus laevis oocytes to study the effects of TSA on
glucocorticoid receptor (GR)-dependent transcription and
we have related these effects to changes in the chromatin
structure of a reporter mouse mammary tumor virus
(MMTV) promoter. We show that TSA induces a low level
of constitutive transcription. This correlates with a change of
acetylation pattern and a more open chromatin structure
over the MMTV chromatin, and with specific acetylation

region [4], one of which, nucleosome B, covers the DNA
segment around position )60 to )240. This segment
contains four glucocorticoid response elements (GREs)
[4–6]. This whole DNA segment shows increased hyper-
sensitivity to DNase I upon binding of glucocorticoid
receptor (GR) homodimers [4,7,8].
We have used the Xenopus oocyte system to reconstitute
chromatin in vivo using single stranded DNA containing
the MMTV promoter as a template. Single-stranded DNA
reconstitutes chromatin more effectively than double-stran-
ded DNA as the second-strand synthesis is coupled to
chromatin assembly, and thus, seems to mimic the replica-
tion coupled chromatin assembly occurring during S phase
of the cell cycle [9].
While the ordered helical domains in the globular body
of the core histones provide a structure for DNA to wrap
around [10], the N-terminal histone tails have been shown to
protrude through and around the DNA helix in a far less
ordered manner [11]. They harbor positively charged lysine
residues at conserved positions. These lysine residues have
been shown to act as targets for post-translational modifi-
cation [12]. Deletion of H3 and H4 N-terminal tails is a
lethal event in yeast that significantly alters gene regulation,
nucleosome assembly and spacing [13]. It is believed that
reversible modifications of charged residues can alter
chromatin structure by causing changes in the overall
charge of the N-terminal tails, and hence their interactions
with the negatively charged sugar–phosphate DNA back-
bone, or with negatively charged regions located on adjacent
nucleosomes [11]. An alternative view is that the various

exogenous TRbA promoter constructs in Xenopus oocytes
[16], as well as p53/mSin3A-repressed genes in the mam-
malian cell lines [17]. Such effects are due to the inhibition
of HDACs, targeted by specific DNA binding factors to
transcriptionally silent regions as a part of large corepressor
complexes [18]. The endogenous Xenopus H1 gene can be
activated in cell lines by TSA, but only after the mid blastula
transition, when histones become hyperacetylated in the
presence of TSA and NaBu [9,19].
In this study, we analyze TSA-treated chromatin in
Xenopus oocytes, and relate its structure to the function
of an MMTV-LTR reporter construct. We show that TSA
increases the acetylation of bulk histone H3 as well as
H3 acetylation over the MMTV–LTR. Furthermore, TSA
treatment causes a generally more open chromatin struc-
ture, and increases DNA-accessibility to micrococcal nuc-
lease (MNase) in the MMTV promoter. It also triggers a
nucleosome repositioning in the distal part of the MMTV
LTR, similar to the nucleosome rearrangement that occurs
during hormone activation [6]. Our results, and the
results of others, highlight the pleiotropic effects that TSA
administration has on chromatin structure and on gene
expression.
Materials and methods
DNA and plasmids
Construction of the MMTV reporter and the plasmid for
in vitro transcription of rat GR mRNA has been described
previously [6].
Culture and injection of
Xenopus

RNA pellets were resuspended in 4 lLofeachprimer
dilution and 2 lL5· First Strand buffer (GibcoBRL),
primers were then annealed at 95 °C for 10 min, 55 °Cfor
25 min, 45 °C for 10 min. Extension was performed in
20 lLat45°Cwith1lL Superscript II (–RNaseH) RT
(GibcoBRL), in 10 m
M
dithiothreitol, 0.5 m
M
dNTP for a
further 40 min. Samples were diluted 1 : 1 (v/v) with
denaturing loading buffer and run on 6% polyacrylamide
sequencing gels: extension products were analyzed and
quantified on a Fuji Bio-Imaging analyzer BAS-2500 using
IMAGE GAUGE
V3.3 software.
Chromatin and protein–DNA analysis
Micrococcal nuclease (MNase) digestion and in situ cleavage
by methidiumpropyl-EDTA–Fe(II) (MPE) was performed
as described previously [6] as was the supercoiling assay [23]
that used a chloroquine concentration of 60 lgÆmL
)1
.
Radioactivity scans and quantifications were performed
using a Fuji Bio-Imaging analyzer BAS-2500 with
IMAGE
GAUGE
V3.3 software.
Analysis of proteins extracted from
Xenopus

(GibcoBRL). Quantification was via
IMAGE GAUGE
V3.3
software. For an internal standard and loading control, the
oocytes were incubated in oocyte medium also containing
[
35
S]methionine (Amersham Biosciences) for 5 h. After
Western blotting, the filters were analyzed for radioactivity
using a Fuji Bio-Imaging analyzer BAS-2500 as above.
Chromatin immunoprecipitation
Pools of oocytes were injected with 4.5 ng sspMMTV [6] and
treated with or without TSA prior to fixation with 1% (v/v)
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formaldehyde for 10 min at ambient temperature. Chro-
matin immunoprecipitation (ChIP) was performed according
to a protocol described previously with some modifications
[25]. Cells were washed and 12 nuclei per pool were dissected
and collected in sonication buffer (20 m
M
Tris/HCl, pH 7.2,
60 m
M
KCl, 15 m
M
NaCl, 1 m
M
EDTA, 1 m

NaCl, 1 m
M
dithiothreitol and
1· protease inhibitor cocktail]; buffer III [0.25
M
LiCl, 0.5%
(v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 1 m
M
EDTA, 10 m
M
Tris/HCl, pH 8.0, 1 m
M
dithiothreitol and
1· protease inhibitor cocktail] and TE, pH 8.0 (1 m
M
dithiothreitol, 1· protease inhibitor cocktail). Bound mater-
ial was eluted in elution buffer [0.5% (w/v) SDS, 0.1
M
NaHCO
3
,0.5lgÆlL
)1
proteinase K]. Crosslinking was
reversed at 65 °C overnight and DNA was purified by
extraction with phenol/chloroform and isopropanol preci-
pitation. PCR was performed in 21 cycles with primers
covering the nucleosome B ()291/+42), the nucleo-
some F ()1044/)732) and the M13 vector (2699/2990), and
products were analyzed on a 6% (w/v) polyacrylamide
sequencing gel. Radioactivity scans and quantifications were

Hormone induction of the MMTV promoter caused a
dramatic increase in transcription over the basal level. This
basal level was  0.5% of full induction (Fig. 1C, compare
lanes 1, 2 and 3, 4). However, TSA alone tended to induce
transcription at a weak level, also in the absence of hormone
(Fig. 1C, compare lanes 1, 2 and 5, 6 and 9, 10). A further
effect of TSA treatment was a significant reduction in
hormone-induced transcription ( 50% of full induction
when added early, compare lanes 3, 4 and 11, 12). Neither of
these effects depended on the TSA concentration over the
range used in these experiments, i.e. 16 n
M
and 64 n
M
(data
not shown). This agrees with similar studies that used TSA
to affect gene transcription [9,16,26]. A TSA concentration
of 16 n
M
was used for subsequent experiments as this was
enough to elicit a reproducible response.
The TSA-induced, hormone-independent, or leaky,
transcription was clearly seen only in the case of early
TSA-treated oocyte pools (Fig. 1D), [1.74 ± 0.4% (E) vs.
0.60 ± 0.5% (L); n ¼ 8]. Similarly, the TSA-mediated
effect of reducing the response to hormone was less evident
when added late [52.5 ± 17.1% (E) of full hormone
induction compared to 76.2 ± 21.6% (L) (n ¼ 8)]. We
conclude that TSA causes a weak hormone-independent
transcription of the MMTV promoter and partly inhibits

acetylation status of the so-called B- and nucleosome F [4]
and compared these patterns with the M13 vector (Fig. 2C).
As a control for the potential loss of histone–DNA contacts
during treatment, an antibody against the carboxyterminal
segment of histone H3, which is not subjected to any known
modifications, was also included [27]. The ChIP analysis
Ó FEBS 2004 TSA effects on transcription and chromatin structure (Eur. J. Biochem. 271) 1155
demonstrated TSA-dependent five- to 10-fold increase in
histone H3 acetylation which involved both the MMTV
promoter, the nucleosome B, the distal MMTV LTR, here
presented by the nucleosome F, and the M13 vector DNA
(Fig. 2C).
We conclude that early TSA addition increases the
acetylation status of bulk histones as well as the histones
organizing the minichromosomes.
Structural alterations in nucleosomal organization
caused by TSA treatment
Changes in the acetylation status of histones by TSA
treatment may cause changes in the organization of
chromatin. Such altered chromatin may no longer be able
to repress transcription from inducible promoters and it
may have less capacity to organize effective transcription in
the induced state. We used several methods to look at
chromatin structure and chromatin remodeling within the
MMTV. Chromatin remodeling can be followed by in situ
chromatin digestion with appropriate restriction enzymes
[8,28]. A restriction enzyme accessibility assay utilizing a
SacIorHinfI restriction site revealed a hormone-dependent
remodeling of the chromatin in this region [6]. However,
similar experiments failed to show any significant effect of

Fig. 1. TSA decreases hormone-induced transcription of the MMTV
promoter, and increases basal transcription in the absence of hormone.
(A) The reporter DNA construct, the pMMTV:M13 used for injection
with the primer used for primer extension analysis of DMS methyla-
tion protection (solid black arrow), and the restriction enzyme cleavage
sites that are referred to in the text. White boxes designate GRE
hexanucleotide elements numbers I to IV, the black box shows the
NF1 site, dark gray boxes show the Oct 1 sites, and light gray box
shows the TATA sequence. The nucleosome B probe used in the
MNase experiments is shown below. (B) Time-course of the oocyte
injection experiment. Collagenased oocytes were allowed to recover for
18 h prior to injection of GR mRNA and DNA, TSA addition [ÔearlyÕ
(E) or ÔlateÕ (L)] and hormone induction. RNA and DNA were
extracted from pools of eight oocytes each. (C) Representative dena-
turing acrylamide gel showing analysis in duplicate of the MMTV
transcription in the presence of hormone and TSA. (D) Phosphor-
imager analysis of MMTV transcription assayed by primer extension
normalized to H4. The lower panel shows a smaller scale graph
highlighting the increase in basal transcription. Error bars signify SD
(n ¼ 8).
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smearing of the nucleosomal pattern both in the promoter
and vector sequences (Fig. 3A, both panels, compare lanes
1, 7 and 13 and Fig. 3B, right panel).
Topological changes in chromatin induced by TSA
treatment and GR binding
We have demonstrated previously that hormone-dependent
activation of the MMTV promoter is associated with

to the uninduced promoter, TSA treatment during hormone
activation leads to a less open chromatin structure. This
observation agrees with the reduced hormone-dependent
transcription from the MMTV promoter in the presence of
TSA (Fig. 1D).
TSA treatment causes nucleosome repositioning
within the MMTV LTR
For mapping of the translational nucleosome positioning
along the MMTV LTR, we have used the chemical nuclease
MPE, which has a strong preference for internucleosomal
regions and shows significantly less sequence bias in cleaving
DNA than MNase [6,30]. The MPE cleavage data suppor-
ted the previous finding [6] that hormone induction causes
a dramatic remodeling event within the MMTV LTR,
resulting in hypercutting over the nucleosome B area,
protection of the nucleosome C area and repositioning of
initially randomly positioned nucleosomes (Fig. 5A and B,
compare lanes 1, 2, and 3, 4 and corresponding scans). Quite
unexpectedly, we observed a hormone-independent
remodeling event within the MMTV-LTR after the addition
of TSA. On early addition of TSA alone, the pattern
of remodeling was seen over the region covered by
nucleosomes C–F, which resembles the pattern obtained
by hormone treatment in the absence of TSA (Fig. 5A and
B, compare lanes 5, 6 to lanes 1, 2 and lanes 3, 4 and
corresponding scans). This effect was detectable but less
evident when TSA was added after chromatin assembly, i.e.
late TSA treatment (compare lanes 5, 6 and 9, 10). No
significant effects of TSA treatment were detected on
nucleosome B. On the other hand, simultaneous addition

to the chromatin template
Graphical calculation of the total GR expressed in an
oocyte following injection of 5 ng GR mRNA, and com-
parison to a standard dilution curve (Fig. 6A) allowed us to
estimate an average of 67 ng of GR protein is present in
each oocyte under the injection conditions used. This is
equivalent to 0.76 pmol per oocyte (relative molecular mass
of GR ¼ 87 500). We also analyzed the nuclear localization
of GR in nuclei microdissected from TSA treated/untreated
oocytes and found no difference in localization patterns
between the oocyte pools (data not shown). To find out
whether the reduced hormone response of the MMTV
promoter after addition of TSA could be a result of
compromised binding of GR to GREs in a hyperacetylated
chromatin context, we analyzed GR–DNA interactions by
dimethylsulphate (DMS) methylation [8], and the cleavage
of DNA by alkali [31]. The method allows easy detection of
DNA–protein interactions via the N7 position of guanines
in the major groove and via the N3 position of adenines in
the minor groove. The DMS cleavage pattern was devel-
oped by primer extension (Fig. 6B). The pattern of the
nonhormone-induced MMTV promoter is virtually identi-
cal to that obtained for naked DNA (data not shown).
Hence, there is no protein binding detected by the DMS
methylation assay in the MMTV promoter in the
noninduced state. Addition of hormone resulted in a drastic
reduction in DMS methylation (protection) over the
glucocorticoid response elements. In agreement with our
previous results [20], we observed  40% DMS methylation
of the corresponding guanines over the GREs 1–4 (Fig. 6B,

nucleosome B probe (right). Positions of
bands corresponding to tri-, di-, mono-
and subnucleosomal bands are indicated.
(B) Phosphorimager profiles of individual
lanes from (A), indicating changes in MNase
digestion on treatment of oocytes with TA
or TSA. Positions of tri-, di-, and the mono-
nucleosomal bands are indicated, as is the
hormone-induced subnucleosomal fragment.
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AcH3 in resting oocytes in our experiments, and the other
researchers have not detected AcH3 in HeLa cells [32]. 2D
PAGE analysis of Xenopus oocytestreatedwithNaBuor
TSA have revealed little change in the overall H4 acetyla-
tion, and inconclusive changes to AcH3 until the
mid-blastula transition [19]. In agreement with this, our
experiments did not show any change in the level of AcH4
following treatment with TSA, but we did see a distinct and
time-dependent increase in the hyperacetylation of histone
H3 as well as an increased acetylation at specific sites, i.e.
lysines 14 and 9 (Fig. 2B).
There are a number of reasons to suspect, apriori,that
changes in the acetylation status of histones result in
alterations in chromatin structure and DNA–protein inter-
actions. We have used several methods to address this issue.
We have not found any differences in GR binding to GREs
with or without TSA (Fig. 6). At the same time, histone
acetylation facilitates the binding of TFIIIA, GAL4 and

of the DNA over the whole construct, suggest that the
chromatin is indeed more relaxed in the absence of
hormone. Interestingly, this phenomenon is as evident in
the presence of ÔearlyÕ TSA as it is with ÔlateÕ, suggesting that
changes in the topology may occur quickly, and that these
changes may be a sensitive readout of histone hyperacety-
lation. The MMTV-LTR specific structural changes in
chromatin, detected by MPE, require a longer exposure to
TSA to develop and they require exposure to TSA during
second-strand synthesis and chromatin assembly. On the
other hand, hormone activation results in an overall loss of
about seven negative supercoils. This effect was decreased
by TSA treatment by one or two superhelical turns
following TSA treatment, which indicates that addition of
TSA leads to formation of less open structure. This is in
striking correlation with a reduced hormone response in
TSA-treated oocytes. Our results are in good agreement
with those previously published from studies in vitro [29]
and in vivo [36] on the effects of TSA on DNA topology.
However, changes in the DNA topology of minichromo-
somes assembled with acetylated/nonacetylated histones
were not significant in other studies [16,37]. Experimental
data are consistent with an idea that in a hyperacetylating
environment, the net charge over positively charged lysine
residues on histone tails will be neutralized, thus altering
histone–DNA [38] and, possibly, histone–histone [11] inter-
actions. This alteration would eventually lead to a decrease
in chromatin compaction [39].
Previously, we have shown that hormone induction in
Xenopus oocytes results in the establishment of a specific

with different parts of the MMTV promoter has been
studied recently using ChIP [42–44]. Rather unexpectedly,
it was shown that upon activation, promoter-proximal
histones (the nucleosome B area) become deacetylated
whereas the acetylation of both H3 and H4 of nuclesome
F was increased [42]. Addition of TSA resulted in only an
insignificant increase of the acetylation level of histone H4 in
the nucleosome B region [43]. These conclusions were made
assuming that the overall amount of histone–DNA cross-
links induced by formaldehyde in the nucleosome B and F
areas are the same. However, this might not be the case,
given the strong remodeling of nucleosome B that occurs
during transcription activation [6,7]. This remodeling might
result in the partial loss of histone–DNA contacts in the
nucleosome B area. Thus, the decrease of the acetylated
Fig. 6. DMS methylation protection over the nucleosome B segment.
(A)SDS/PAGEandWesternblotoftotaloocyteproteinextracts
following injection of 5 ng GR mRNA, probed with GR polyclonal
antibodies. 0.5 and 0.25 oocyte equivalent was compared to a standard
curve of GR protein of known concentration purified from rat liver
[56]. (B) DMS methylation protection over the nucleosome B segment
in the presence/absence of TA and TSA. Oocytes in groups of five were
treated with DMS, see Materials and methods. The methylation pat-
tern was developed by primer (+42/+15) extension. Corresponding
guanidine residues that are protected after hormone induction are
indicated with arrows. Radioactivity scans of corresponding lanes are
shown to the right.
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transcription machinery is blocked [52]. Interestingly, GR is
able to recruit HDAC activity and thereby deacetylate
histone H4, and in this way also repress the expression of
IL-1b -stimulated granulocyte-macrophage colony-stimula-
ting factor [54]. One may speculate that a specific pattern of
modified histone tails is required to recruit the basal
transcription machinery, and that TSA can distort this
pattern and thus reduce the hormone-induced transcrip-
tional response [41,52]. To understand these events, it will be
essential to map the detailed pattern of histone modifica-
tions that occurs in the MMTV promoter during transcrip-
tion activation, as has recently been done in the PHO5
promoter [27]. This work is in progress in our laboratory.
HDAC inhibitors are exciting and promising anticancer
drugs [55], not only for their ability to inhibit histone
deacetylases but also due to their strong potency to induce
growth arrest, to promote differentiation and to induce
apoptosis. It is believed that they exert their effects via
up-regulation of gene expression [55]. However, our results
and the results of others [43,48] suggest that the down-
regulation of viral tumor promoters may be equally
important for the clinical effects HDAC inhibitors, and
thus also for their possible future use as pharmaceuticals.
Acknowledgements
We are grateful to Ulla Bjo
¨
rk for skilful technical assistance and
Dr Birgitta Gelius for skilful nuclear dissections and for performing the
GR localization experiment. We thank Dr Jiemin Wong for kindly
sharing the ChIP protocol for Xenopus oocytes, and Dr Ola

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