Tài liệu Báo cáo khóa học: Trichostatin A reduces hormone-induced transcription of the MMTV promoter and has pleiotropic effects on its chromatin structure pptx

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

and remodeling events in the promoter region. Specifically, a
repositioning of initially randomly positioned nucleosomes
along the distal MMTV long terminal repeat is seen. This
nucleosome rearrangement is similar to the translational
nucleosome positioning that occurs upon hormone activa-
tion. We also note a reduced hormone response in the
presence of TSA. TSA effects have for a long time been
associated with transcriptional activation and chromatin
opening through inhibition of the deacetylation of histones.
However, our results and those of others show that TSA-
induced changes in expression and chromatin structure can
be quite different in different promoter contexts and, thus,
the effects of TSA are more complex than previously
believed.
Keywords: MMTV promoter; chromatin structure; tran-
scription; Xenopus oocytes; TSA.
The role of the nucleosome as the fundamental unit of DNA
packaging has long been accepted, but its purely structural
role has been challenged by an increasing body of experi-
mental data [1]. Recent evidence suggests that the organ-
ization of promoters into nucleosome arrays provides
an additional mechanism of gene regulation [2]. In this
study, we have used a promoter from the 5¢-long terminal
repeat (LTR) region of the mouse mammary tumor virus
(MMTV) to correlate chromatin structure and gene activity.
The MMTV-LTR contains potential regulatory elements
which mediate transcription in the presence of glucocorti-
coid ligands and in the presence of androgen, progesterone
and their respective nuclear receptors (Fig. 1A) [3]. Six
translationally positioned nucleosomes (A–F) cover this

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

chemical modifications of specific amino acids in histones
act as a code by serving as binding sites for various effector
complexes. These complexes can modify the chromatin
structure and hence the expression of a gene [14].
The relationship between the histone acetylation status
of chromatin and transcription has been studied in many
systems using a variety of promoter constructs and native
Correspondence to S. Belikov, Department of Cell and Molecular
Biology, Medical Nobel Institute, Box 285 Karolinska Institute,
SE-171 77 Stockholm, Sweden. Fax: + 46 8 31 35 29,
Tel.: + 46 8 52 48 73, E-mail:
Abbreviations: ChIP, chromatin immunoprecipitation; DMS,
dimethylsulphate methylation; GR, glucocorticoid receptor; GRE,
glucocorticoid response element; HAT, histone acetyltransferase;
HDAC, histone deacetylase; LTR, long terminal repeat; MNase,
micrococcal nuclease; MPE, methidiumpropyl-EDTA–Fe(II); NaBu,
sodium butyrate; TSA, trichostatin A; TA, triamcinolone acetonide.
*Note: Both these authors contributed equally to this work.
(Received 26 August 2003, revised 26 January 2004,
accepted 30 January 2004)
Eur. J. Biochem. 271, 1153–1162 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04019.x
genes. The studies have revealed that both histone acetyl-
transferases (HATs) and histone deacetylases (HDACs)
play a vital role in gene regulation by either allowing
transcription or establishing correct repression. Blocking
of HDACs using inhibitors such as trichoststin A (TSA),
sodium butyrate (NaBu) and trapoxin [15], has revealed
a complicated picture of the exact role of HDACs in
promoter function. TSA has been shown to relieve repres-
sion by non ligand-bound TR/RXR protein bound to

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

oocytes
Xenopus laevis oocytes were prepared and injected as
described previously [20].
Transcription analysis
Quantification of MMTV transcription by S1-nuclease and
DNA analysis was performed as described previously [21],
with one difference. A synthetic oligonucleotide identical
to the lower strand of the )8/+64DNAsegmentofthe
MMTV-LTR was labeled using [
32
P]ATP[cP] (Amersham
Biosciences) and T4-polynucleotide kinase, and used as
probe.
MMTV transcription was also quantified by primer
extension using the following procedure. Homogenate
equivalent to eight oocytes was first treated with
0.5 mgÆmL
)1
proteinase K for 2 h at 37 °Candthen
RNA extracted with Trizol (GibcoBRL) and chloroform
according to the manufacturer’s instructions, precipitated
with 0.7 vol. of isopropanol. One oocyte equivalent was
used for primer extension. The primers were
32
P end-labeled
oligonucleotides with the following sequences: 5¢-GC
GGGAGTTTCACGCCACCAAGATCC-3¢ (MMTV, 10
pmol) and 5¢-GGCTTGGTGATGCCCTGGATGTTAT
CC-3¢ (H4, loading control, 20 pmol). Primer extension was
performed according to a protocol modified from [22]: dry

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

oocytes
Nuclear proteins were isolated by physical separation of the
germinal vesicle from the cytoplasm of injected oocytes
using fine forceps in a pool of isolation buffer (20 m
M
Tris/
HCl, pH 7.5, 0.5 m
M
MgSO
4
,140m
M
KCl). Both fractions
were homogenized in the same buffer with 1% SDS, boiled
for 5 min, and samples run on 10% or 15% SDS/PAGE
gels (for GR and histone analysis, respectively) in Tris/
glycine buffer with 0.1% SDS [24]. Proteins were transferred
onto poly(vinylidene difluoride) (PVDF) membranes (Mil-
lipore) in Tris/glycine buffer containing 20% (v/v) methanol
and 0.037% (w/v) SDS [24] at 20 V for 1 h. Filters were
probed with antibodies to acetylated histones H3 and H4
(Upstate Biotechnology) and acetylated H3 (Upstate Bio-
technology), and antibodies against specific modifications
such as acetylated H3-K9 (Cell Signaling Technology) and
H3-K14 (Abcam) and anti-H3 C-terminal (Abcam). Ana-
lysis of GR was performed at 1 : 1000 dilution of primary
antibody in Tris-buffered saline with 0.05% (v/v) Tween 20
(TTBS) and 5% (w/v) dried milk powder. Secondary
antibody HRP conjugates were used at 1 : 1000 dilution in
TTBS. Protein bands were visualized by chemiluminescence

(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)
1154 C. A
˚
strand et al. (Eur. J. Biochem. 271) Ó FEBS 2004
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

M
dithiothre-
itol and 1· protease inhibitor cocktail (Sigma), 50 lLper
nucleus). After sonication on ice (4· 20 s), samples were
diluted with 1 vol. of buffer I (0.1% sodium deoxycholate,
1% Triton X-100, 2 m
M
EDTA, 50 m
M
Hepes pH 7.2,
150 m
M
NaCl, 1 m
M
dithithreitol and 1· protease inhibitor
cocktail (Sigma) and centrifuged at 13 000 g,4°C, for
10 min to remove insoluble debris. Supernatant, equival-
ent to one nucleus, was used for immunoprecipitation.
Acetylated histones were immunoprecipitated for 4 h with
anti-AcH3 (Upstate Biotechnology), H3 C-terminal and
AcH3-K14 (Abcam) on protein A sepharose beads (Amer-
sham) precoated with calf thymus DNA in 5% (w/v) dry
milk. Complexes were washed (15 min wash) with buffers:
buffer I described above; buffer II [0.1% (w/v) sodium
deoxycholate, 1% (v/v) Triton X-100, 2 m
M
EDTA, 50 m
M
Hepes, pH 7.2, 500 m
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

performed using a Fuji Bio-Imaging analyzer BAS-2500
using
IMAGE GAUGE
V3.3 software.
Results
Trichostatin A has pleiotropic effects on MMTV
transcription
Pools of oocytes were injected with 5 ng GR mRNA into the
cytoplasm, followed by intranuclear injection of 1 ng of
sspMMTV:M13 DNA. TSA (16 n
M
) was added to some of
the pools immediately after DNA injection to assemble
chromatin in the presence of TSA, this is referred to as early
TSA (E). Hormone induction of half of these pools was
performed 18 h later by addition of the synthetic glucocor-
ticoid hormone, triamcinolone acetonide (TA) at a concen-
tration of 10
)6
M
. TSA was added, at the same time, to a
pool of injected oocytes, these are referred to as late TSA
(L). Transcription was allowed to continue for 6 h. Oocytes
were harvested and total RNA was extracted from all pools
(Fig. 1B). S1 nuclease or primer extension analysis of the
injected MMTV reporter transcripts was performed; the
latter using a primer for endogenous histone H4 RNA as an
internal control, followed by quantification on a phosphor-
imager (eight experiments). Both assays generated identical
results.

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

hormone-inducible transcription.
TSA affects acetylation levels of endogenous histone
pools as well as histones in MMTV containing
minichromosomes
Treatment of oocytes with deacetylase inhibitors such as
TSA may change the bulk acetylation pattern of histones,
and in this way alter the structure of chromatin incorpor-
ating them. To see whether the increased transcription
leakage observed at early addition of TSA can be explained
by an accumulation of acetylated forms of histones in
a time-dependent manner, we looked at the pattern of
TSA-induced histone acetylation.
Using antibodies specific to acetylated forms of H3 and
H4, we endeavored to look at the level of histone hyper-
acetylation 12 h (E) and 18 h (L) before the harvest of
noninjected oocytes. Nuclei isolated from nontreated oocytes
showed significant levels of nuclear AcH4, which did not
increase upon late addition nor on early addition of TSA
(Fig. 2A). A striking response to TSA was seen in the levels of
AcH3: almost no AcH3 was found in nontreated oocytes
while oocytes treated with late TSA showed a significant
increase in AcH3. This increase was even more pronounced
when TSA was added early (Fig. 2A). We conclude that TSA
induces a time-dependent increase in the level of AcH3. We
also looked for specific histone modifications (Fig. 2B) and
noted an increased acetylation of histone H3 lysine residues
14 and 9 upon early addition of TSA.
To monitor the acetylation status of the MMTV
promoter subjected to TSA treatment, we used a chromatin
immunoprecipitation assay (ChIP) and evaluated the

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

TSA on SacIorHinfI accessibility (data not shown). We
therefore used other approaches that were more sensitive to
the small changes in the chromatin structure over a wide
area of the nucleosome B: MNase digestion assay, topology
assay and MPE chemical cleavage together with indirect
end-labeling.
Previous MNase experiments revealed changes in the
canonical nucleosomal ladder over the nucleosome B region
in response to hormone-dependent GR binding [6,20]. As
seen in Fig. 3, increasing amounts of MNase reduced the
nucleosome B region of the hormone-activated promoter
to predominantly mono- and subnucleosomal fragments.
Compare lanes 4–6 (nucleosome B probe) with lanes 1–3
(nucleosome B probe) and also Fig. 3B (left panel). This
hormone-dependent appearance of a subnucleosome is
specific for the nucleosome B area as it is not seen while
reprobing the filter with the vector probe. Compare lanes
4–6 (nucleosome B probe) with lanes 4–6 (M13 vector
probe). The nucleosome B is further affected by the early
addition of TSA. In this case, the nucleosome B area is
distinctly hypersensitive to MNase action, especially at high
concentrations. Late addition of TSA, on the other hand,
had virtually no effect (compare lanes 4–6 with 10–12 and
16–18, nucleosome B probe). Thus, the observed reduction
in hormone response of the system in the presence of TSA
correlates with detectable changes in the local chromatin
architecture of the MMTV promoter. The TSA-induced
leaky transcription also seems to correlate with a loss in
chromatin structure regularity of the bulk chromatin; at
lower MNase concentrations this is seen as increased

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).
1156 C. A
˚
strand et al. (Eur. J. Biochem. 271) Ó FEBS 2004
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

alterations in the chromatin structure that can be detected in
a DNA topology assay as the loss of negative superhelical
turns [20]. These alterations in DNA topology take place
even if histones are not physically disrupted from the
chromatin template [29].
Oocytes were injected with sspBSLSwt, a construct
containing the same MMTV-TK fusion used in other
experiments, but cloned into a Bluescript vector. The size of
the injected DNA was smaller in these experiments, and the
resolution of the topoisomers was thus improved. Following
treatment, oocyte pools were extracted and the DNA
resolved on an agarose gel containing 60 lgÆmL
)1
chloro-
quine to visualize any changes in superhelical density
arising from TSA/hormone treatment. Treatment with
TSA decreased the negative superhelicity by 1.5 superhelical
turns, equal to 1.5 nucleosomes (Fig. 4 lanes/scans 1, 3
and 5). This indicates a more open conformation of the
chromatin, which correlates with a loss of chromatin
regularity and is consistent with increased smearing
observed in the MNase-digested DNA. Interestingly, this
phenomenon was as evident in the presence of ÔearlyÕ TSA
as it was with ÔlateÕ TSA, suggesting that changes in the
topology may occur quickly. The overall change in the
topology caused by GR binding and the MMTV induction
results in an overall loss of about 7 negative supercoils, an
effect which was decreased by TSA treatment by two and
one superhelical turns for ÔearlyÕ and ÔlateÕ TSA, respectively
(Fig. 4 lanes/scans 2, 4 and 6). This indicates that in contrast

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

of TA and TSA resulted in a digestion pattern indistin-
guishable from that observed after treatment with TA alone
Fig. 2. TSA treatment causes acetylation of bulk histones and acetyla-
tion of histones in MMTV-containing minichromosomes. Pools of dis-
sected nuclei from noninjected oocytes were analyzed by SDS/PAGE.
(A) Western blot probed with anti-acetylated H3 (upper panel) and
anti-acetylated H4 (lower panel). In vivo [
35
S]methionine-labeled pro-
teins in the nuclear extract were detected on the filter after blotting and
were used as an internal standard. This showed that equal amounts of
protein were loaded in each lane (not shown). (B) Western blot probed
with antibodies against AcH3-K14, AcH3-K9 and H3 C-terminal.
TSA was either not added (-), added early (E) or added late (L),
according to the schedule in Fig. 1B. (C) Effects of TSA treatment on
H3 acetylation at different regions of the MMTV promoter and vector
sequences. The DNA-injected oocytes were treated (early) or not
treated with TSA. The ChIP assay was performed as described in
Materials and methods. Radioactively labeled PCR fragments from
nucleosome B, nucleosome F and vector are shown to the left, and
corresponding bars to the right. Bars represent the intensity of the
bands, normalization was performed according to actual histone
H3-DNA binding (H3 C-term) in TSA-treated and nontreated
oocytes. Dark bars show TSA-treated cells.
Ó FEBS 2004 TSA effects on transcription and chromatin structure (Eur. J. Biochem. 271) 1157
(Fig. 5A and B, compare lane 4 and 8, lower scan). We
conclude that TSA can cause specific nucleosome rear-
rangements in the distal MMTV-LTR similar to the
hormone-induced rearrangements in this DNA segment.
TSA treatment does not affect GR binding

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,

compare lanes 1 and 2 and corresponding radioactivity
scans). Addition of TSA to the oocyte media had virtually
no effect on the DMS digestion pattern (Fig. 6B, compare
lanes 1, 2 and 3, 4). This shows that TSA has no effect on
GR-DNA binding.
Discussion
We have shown that TSA treatment of oocytes alters the
MMTV transcription profile and causes changes in the bulk
chromatin structure, as well as specific changes in the
promoter region. To the best of our knowledge this is the
first report to demonstrate a specific translational reposi-
tioning of nucleosomes induced by TSA or any other
HDAC inhibitor.
The fact that we see effects more clearly when TSA is
present during chromatin assembly (early addition) suggests
that the deacetylation step of chromatin maturation is being
blocked. Pools of newly synthesized histone H4 diacetylated
at the evolutionarily conserved K5 and K12 residues are
known to exist in a variety of organisms [32], and diAcH4 is
also the main form of stored H4 in Xenopus oocytes [19].
Newly synthesized H3 has also been found in a diacetylated
form in Drosophila and Tetrahymena, although this appears
to be more transient, and the pattern of lysine residues
acetylated in this manner is far less well conserved between
species [32]. We were able to detect only tiny amounts of
Fig. 3. Nucleosomal organization of the
MMTV promoter. (A) Autoradiogram of a
Southern blot of chromatin digested with
increasing amounts of MNase, probed with
vector M13 DNA (left) and reprobed with

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.
1158 C. A
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strand et al. (Eur. J. Biochem. 271) Ó FEBS 2004
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

USF to nucleosomal DNA in vitro [33–35]. The restriction
enzyme accessibility assay, which is a sensitive method that
is capable of detecting even subtle changes in chromatin
structure, also failed to reveal any difference between
chromatin treated with TSA and untreated chromatin (data
not shown).
Fig. 4. Changes in DNA topology upon hormone induction of the
MMTV promoter and addition of TSA. Southern blot of pBSLSwt
minichromosomes extracted from GR-injected oocyte pools treated
with TSA and/or hormone, followed by separation on an agarose gel
containing 60 lgÆmL
)1
chloroquine to reveal the superhelical density;
probed with radiolabeled DNA fragment )103/+431. One oocyte
equivalent per lane. Phosphorimager profiles of scanned lanes showing
the distributions of superhelical species are shown below. The circle
indicates the most frequent topoisomer(s).
Fig. 5. Nucleosome positioning analysis in situ by MPE digestion.
(A)SouthernblotofMPE-cleavedDNAfromtreatedoocytepools,
probed with radiolabeled 513 bp probe (EcoRV-SacI fragment, +425/
)108). The diagram to the left shows the positions of nucleosomes on
the MMTV promoter [6], the transcriptional start site and the cleavage
sites for SacI(S),HinfI (H) and BamHI (B). (B) Radioactivity scans
from selected lanes.
Ó FEBS 2004 TSA effects on transcription and chromatin structure (Eur. J. Biochem. 271) 1159
The structural changes over the whole minichromosome
upon TSA treatment, as indicated by the increased acces-
sibility of MNase, have been reported previously [16]. Our
MNase digestion results, when coupled with the topology
changes resulting from the loss of 1.5 superhelical turns

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

nucleosome positioning pattern over initially randomly
organized nucleosomes in the MMTV promoter [6]. After
treatment of injected oocytes with TSA alone, an altered
pattern of MPE cleavage over nucleosomes C and D is seen
(Fig. 5), reflecting a partial, hormone-independent chroma-
tin remodeling event. This mimics the situation occurring
during hormone induction, where GR bound to the
nucleosome B region of the MMTV promoter renders an
array of six positioned nucleosomes (A–F). A TSA-induced
chromatin remodeling event occurs in the HIV promoter.
This promoter harbors a nucleosome positioned at the
initiation site that is disrupted upon TNF-a-induced
expression. Treatment with TSA results in a similar
chromatin remodeling of the HIV promoter and in consti-
tutive transcription [40]. However, we are not aware of any
previous report of a specific translational positioning being
induced by TSA. Our observations suggest that nucleosome
repositioning during hormone induction [6] is not solely
explained by GR binding to the GRE sequences within the
nucleosome B region, but also depends on the subsequent
histone acetylation in the surrounding area. Indeed, it was
shown that histone H4 acetylation at Lys8 is responsible
directly for the recruitment of the SWI/SNF complex to
IFN-b gene during activation [41]. Another possibility is
that the TSA-induced remodeling of the MMTV promoter
that we have observed is triggered by acetylation of a
transactive factor(s), which might lead to specific binding to
the distal part of the MMTV-LTR and thereby direct the
translational nucleosome positioning.
The acetylation status of histones H3 and H4 associated

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.
1160 C. A
˚
strand et al. (Eur. J. Biochem. 271) Ó FEBS 2004

signal in ChIP experiments might indicate a loss of histone
in the respective area [27]. Our results and the results of
others [25] show an increase in acetylation status of histone
H3 over the MMTV-LTR upon TSA treatment. This
hyperacetylation was also seen in the vector sequences.
Interestingly, our results show that a small but clearly
detectable increase in the level of basal transcription occurs
upon the TSA treatment. This shows that TSA can only
insignificantly overcome the repressive nature of the chro-
matin. We also discovered a reduction of  50% in the
hormone response of the system in the presence of TSA,
whereas previous studies on MMTV have reported aug-
mentation of hormone induction by TSA [26,45,46].
However, addition of HDAC inhibitors resulted in the
down-regulation of the MMTV transcription to various
extents in several studies [43,47,48]. The inhibitory effect in
our experiments was more evident in the case of early TSA
treatment, suggesting that HDAC activity during chromatin
maturation may not only help to establish sufficiently
repressive chromatin, but may also be necessary for the
formation of transcriptionally competent chromatin [49].
Deacetylase activity has for a long time been associated
with transcriptional repression through the deacetylation of
histones [50]. However, several studies have shown that
HDACs are required for both transcriptional activation and
repression [51]. One recent example of the down-regulation
by HDAC inhibitors is that of the STAT 5 target genes [52],
where transcription involves recruitment of HDAC1 [53].
Chromatin remodeling of these genes is not affected by
TSA, but recruitment of the components of the basal

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

Hermanson for providing the antibody against AcH3-K9. This
work was supported by the Swedish Cancer Foundation (project
2222-BOZ-18XBC) and the Royal Swedish Academy of Sciences
(12682). Project support was also provided by the European Commis-
sion, TMR, to O
¨
. W. (Network Contract ERBFMRXCT-98–0191).
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