Histone deacetylase inhibition redistributes topoisomerase IIb from heterochromatin to euchromatin

Article (PDF Available)inNucleus (Austin, Texas) 2(1):61-71 · January 2011with62 Reads
DOI: 10.4161/nucl.2.1.14194 · Source: PubMed
The genome is organized into large scale structures in the interphase nucleus. Pericentromeric heterochromatin represents one such compartment characterized by histones H3 and H4 tri-methylated at K9 and K20 respectively and with a correspondingly low level of histone acetylation. HP1 proteins are concentrated in pericentric heterochromatin and histone deacetylase inhibitors such as trichostatin A (TSA) promote hyperacetylation of heterochromatic nucleosomes and the dispersal of HP1 proteins. We observed that in mouse cells, which contain prominent heterochromatin, DNA topoisomerase IIβ (topoIIβ) is also concentrated in heterochromatic regions. Similarly, a detergent-resistant fraction of topoIIβ is associated with heterochromatin in human cell lines. Treatment with TSA displaced topoIIβ from the heterochromatin with similar kinetics to the displacement of HP1β. Topoisomerase II is the cellular target for a number of clinically important cytotoxic anti-cancer agents known collectively as topoisomerase poisons, and it has been previously reported that histone deacetylase inhibitors can sensitize cells to these drugs. While topoIIα appears to be the major target for most topoisomerase poisons, histone deacetylase-mediated potentiation of these drugs is dependent on topoIIβ. We find that while prior treatment with TSA did not increase the quantity of etoposide-mediated topoIIβ-DNA covalent complexes, it did result in a shift in their distribution from a largely heterochromatin-associated to a pannuclear pattern. We suggest that this redistribution of topoIIβ converts this isoform of topoII to a effective relevant target for topoisomerase poisons.
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Nucleus 2:1, 61-71; January/February 2011; © 2011 Landes Bioscience
*Correspondence to: Ian G. Cowell; Email: i.g.cowell@ncl.ac.uk
Submitted: 08/05/10; Revised: 11/08/10; Accepted: 11/11/10
DOI: 10.4161/nucl.2.1.14194
Post-translational modifications of histone amino terminal
tails are important for chromatin dynamics and organization.
Methylation and acetylation of specific lysine residues, together
with phosphorylation, polyADP-ribosylation and ubiquitylation
modulate processes such as transcription, cell cycle progression,
DNA damage responses, apoptosis and differentiation.
acetylation is associated with transcription. In contrast, constitu-
tive heterochromatin, which is generally transcriptionally inert,
contains low levels of histone acetylation and is characterized by
trimethylated H3 lysine 9 and H4 lysine 20.
Histone acetylation is governed by the opposing actions of
histone acetyl transferases (HATs) and histone deacetylases
(HDACs). Alterations in the structure or expression of HATs
and HDACs are frequently reported in cancers and are linked
to tumour development. Consequently, HDAC inhibitors
(HDACIs) are undergoing trials as anticancer agents.
alone exert an antiproliferative effect and promote apoptosis
in cancer cell-line models, but in addition HDACIs have been
shown to sensitize cells to the cytotoxic effects of other anticancer
The genome is organized into large scale structures in the interphase nucleus. Pericentromeric heterochromatin
represents one such compartment characterized by histones H3 and H4 tri-methylated at K9 and K20 respectively and
with a correspondingly low level of histone acetylation. HP1 proteins are concentrated in pericentric heterochromatin and
histone deacetylase inhibitors such as trichostatin A (TSA) promote hyperacetylation of heterochromatic nucleosomes
and the dispersal of HP1 proteins. We observed that in mouse cells, which contain prominent heterochromatin, DNA
topoisomerase IIβ (topoIIβ) is also concentrated in heterochromatic regions. Similarly, a detergent-resistant fraction
of topoIIβ is associated with heterochromatin in human cell lines. Treatment with TSA displaced topoIIβ from the
heterochromatin with similar kinetics to the displacement of HP1β. Topoisomerase II is the cellular target for a number
of clinically important cytotoxic anti-cancer agents known collectively as topoisomerase poisons, and it has been
previously reported that histone deacetylase inhibitors can sensitize cells to these drugs. While topoIIα appears to be the
major target for most topoisomerase poisons, histone deacetylase-mediated potentiation of these drugs is dependent
on topoIIβ. We nd that while prior treatment with TSA did not increase the quantity of etoposide-mediated topoIIβ-
DNA covalent complexes, it did result in a shift in their distribution from a largely heterochromatin-associated to a pan-
nuclear pattern. We suggest that this redistribution of topoIIβ converts this isoform of topoII to a eective relevant target
for topoisomerase poisons.
Histone deacetylase inhibition redistributes
topoisomerase IIβ from heterochromatin
to euchromatin
Ian G. Cowell,
* Nikolaos Papageorgiou,
Kay Padget,
Gary P. Watters
and Caroline A. Austin
Institute for Cell and Molecular Biosciences; Newcastle University
School of Applied Sciences; Northumbria University; Newcastle upon Tyne, UK
Key words: topoisomerase, histone deacetylase, histone acetyl transferase, chromatin remodelling, heterochromatin, euchromatin,
etoposide, nucleolus, DNA damage
drugs, particularly topoisomerase II poisons.
poisons such as etoposide and epirubicin are of great clinical
importance and are widely used in cancer therapy.
Topoisomerase II is an essential enzyme that allows the pas-
sage of one DNA duplex through a second. During its reaction
cycle the dimeric enzyme introduces an enzyme-bridged DNA
double-strand break (DSB) in one double helix through which
a second duplex can be passed. At this stage each topoisomerase
monomer is coupled to one free end of the first duplex through
a 5'-phosphotyrosyl linkage. Topoisomerase poisons stabilize the
enzyme-linked DSB, which is otherwise transient and rapidly re-
The resultant topoisomerase II-DNA covalent com-
plexes account for the cytotoxic properties of the drugs and are
presumed to be converted to “frank” DSBs by repair processes
and/or result in a damage response following collisions with
transcriptional or replication machinery. Mammals possess two
topoisomerase II isoforms, α and β, encoded by the TOP2A and
TOP2B genes respectively.
Although the isoforms have very
similar enzymatic characteristics in vitro and share 70% amino
acid identity, topoisomerase IIα and -β have different though
possibly overlapping roles in mammalian cells. Topoisomerase
62 Nucleus Volume 2 Issue 1
be specifically nucleolar.
The reasons for these differences are
not clear, but probably reflect, at least in part, species differences
and differences in specimen preparation. Nucleoli of mouse cells
for example are associated at their periphery with AT-rich peri-
centromeric heterochromatin, which clusters in so-called chro-
mocentres around centromeres. These chromocentres can be
readily visualized in fluorescence microscopy of Hoechst or DAPI
stained cells or with antibodies against HP1 proteins (Fig. 1A
and Sup. Fig. 1). Heterochromatin in human cells by contrast, is
less easily visualized. We show here that in mouse epithelial cells,
under standard paraformaldehyde fixation conditions and using
a series of characterized antibodies, that topoisomerase IIβ has a
nucleoplasmic distribution, with an increased concentration in
chromocentres as identified by DAPI and anti-HP1β staining
(Fig. 1A). Although not excluded from nucleoli, topoisomerase
IIβ did not appear to be concentrated in these regions (see Figs.
1A and 2). By contrast, topoisomerase IIα was distributed more
evenly throughout interphase nuclei (see Fig. 1B). Since chro-
mocenters are largely clustered around nucleoli in mouse cells
it was important to unequivocally distinguish between nucleo-
lar and heterochromatic topoisomerase IIβ staining. This was
achieved by co-staining cells with anti-topoisomerase IIβ and
either anti-HP1β to identify heterochromatin or anti-fibrillarin
to identify nucleoli. As shown in Figure 2A, regions of intense
topoisomerase IIβ staining were largely non-overlapping with the
fibrillarin signal, but almost coincident with the HP1 and DAPI
signals, which largely clustered around the outside of nucleoli.
Consistently, centromers, as visualized by CENP-B staining,
were contained within the regions of intense topoisomerase IIβ
(and HP1β and DAPI) staining (Fig. 2B).
TSA mobilizes topoisomerase IIβ from heterochromatin. As
alluded to above, prolonged exposure of mouse cells to the HDAC
inhibitor TSA results in histone hyperacetylation and movement
of heterochromatin clusters from a largely perinucleolar to a
more peripheral distribution and dissociation of heterochromatin
protein HP1. Since like HP1, topoisomerase IIβ is concentrated
in heterochromatin in C127I cells, we hypothesized that TSA
treatment would also lead to its redistribution. C127I cells were
treated with TSA concentrations from 2 to 80 nM for 2 or 5
days and then examined for topoisomerase IIβ and HP1β distri-
bution by immunofluorescence. After 5 days exposure to TSA,
topoisomerase IIβ and HP1β both showed a progressive dissocia-
tion from heterochromatic regions (as visualized by DAPI stain-
ing) with dose of TSA (Fig. 3). After 5 day’s exposure to 50 nM
TSA, little or no heterochromatic concentration of either protein
was apparent. After 2 days exposure to 50 nM TSA most topoi-
somerase IIβ and HP1β was displaced from heterochromatin.
After 5 day’s exposure to 50 or 80 nM TSA, most cells appeared
larger and flatter then untreated cells. Median topoisomerase IIβ
immunofluorescence per cell was increased almost two fold by
TSA (Fig. 4A). This may reflect increased antigen accessibility,
but notably median DNA content per nucleus was also increased,
as assessed by quantitative DAPI fluorescence (Fig. 4C), consis-
tent with accumulation of cells in G
or with aberrant chromo-
some numbers resulting from TSA-induced mitotic defects and
in addition, the nuclei of TSA treated cells are larger than those of
IIα is essential for cell viability and is required for chromosome
decatenation after S-phase,
while top2β null mice develop to
term but die perinatally with neural developmental defects.
Recently, specific roles in transcription regulation and sperm
chromatin remodelling have been uncovered for topoisomerase
The topoisomerase IIα isoform is the major target for
most clinically-relevant topoisomerase poisons including eto-
poside and epirubicin
although the -β isoform appears to
contribute more for some topoisomerase poisons including mito-
xantrone, mAMSA and XK469.
Several mechanisms could explain the sensitization of cells to
topoisomerase poisons by HDAC inhibitors
but notably, it
was recently reported that this potentiation is mediated speci-
cally through topoisomerase IIβ.
This was shown for etoposide
and epirubicin as well as mitoxantrone. HDACIs cause chroma-
tin decondensation, which may affect access of topoisomerase
to DNA or access of topoisomerase poisons to the enzyme,
resulting in elevated DNA damage and cytotoxicity. HDACI
treatment leads to global changes in histone acetylation levels,
including hyperacetylation of heterochromatin and redistribu-
tion of the non-histone heterochromatin protein HP1.
It fol-
lows that this may affect the distribution of topoisomerase II in
the interphase nucleus. Indeed, enzymatically active topoisomer-
ase IIα is concentrated in heterochromatin in mid-late S-phase
in HeLa cells and is delocalized by the HDACI TSA.
We have
studied the nuclear distribution of topoisomerase IIβ and show
here that TSA leads to its redistribution in the nucleus and a
change in the pattern of topoisomerase IIβ-DNA complexes
induced by etoposide in favour of a more euchromatic pattern.
We suggest that altering the distribution of topoisomerase IIβ in
the nucleus and thus topoisomerase IIβ-DNA adducts across the
genome converts topoisomerase IIβ into a more clinically useful
target for topoisomerase poisons including etoposide.
A fraction of topoisomerase IIβ is concentrated in heterochro-
matin. Exposure of cultured cells to HDAC inhibitors results in
cell cycle arrest and apoptosis. Prolonged exposure to low doses
leads to chromatin decondensation associated with hyperacety-
lation of histones H3 and H4. In mouse cells under these condi-
tions, pericentromeric heterochromatin relocates from a largely
perinucleolar to a more peripheral distribution and in mouse
and human cells, HP1 proteins become dissociated from het-
erochromatic regions.
In the light of this HDAC-mediated
chromatin remodelling, and the role of topoisomerase IIβ in
sensitization of cells to topoisomerase poisons by HDAC inhibi-
tors, we hypothesized that HDAC inhibitors such as TSA or VPA
may alter the subnuclear distribution of topoisomerase IIβ. This,
either directly or through increased accessibility of decondensed
chromatin could lead to increased cytotoxicity associated with
topoisomerase poisons. Previous studies have reported differing
subnuclear distributions for topoisomerase IIβ. Topoisomerase
IIβ has variously been reported to be concentrated in the nucleoli
or at the periphery of heterochromatic regions in fixed cells,
and in live cells,
while others have not found the protein to
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small decrease in the median levels of etoposide-induced, extrac-
tion resistant topoisomerase IIβ immunofluorescence when anti-
bodies 18513 or 30400 were used, but did not significantly affect
the signal with antibody 3535 (Fig. 5).
In cells treated with etoposide for 2 hours before DRT extrac-
tion, topoisomerase IIβ as detected by antibody 18513 had a clus-
tered appearance, with bright areas coincident with the bright
DAPI regions (Fig. 6A). This heterochromatic pattern was
apparent in more than 70% of cells, while the remainder dis-
played a more diffuse, speckled pattern. The variation in pattern
untreated cells (see Fig. 3), Topoisomerase IIβ protein
levels were not changed by TSA in western blot analy-
sis where protein loading was standardized by micro-
grams of protein loaded (Fig. 4D). By comparison, it
was previously noted that HDACIs did not significantly
alter topoisomerase IIβ protein level in human MCF7
cell extracts.
The level of HP1β immunofluorescence
remained unchanged by TSA (Fig. 4B), consistent with
previous observations.
When topoisomerase IIβ dis-
sociated from heterochromatin it appeared in a fine
speckled pattern throughout the nucleus and was occa-
sionally excluded from heterochromatic regions (Fig. 3
fourth and fifth rows).
Effect of HDAC inhibition on etoposide-induced
topoisomerase-DNA complexes. The topoisomerase
II reaction mechanism allows the passage of one DNA
duplex through another by transiently cleaving the
first DNA helix to create an enzyme-bridge DNA gate
through which the second duplex is transported. The
break is subsequently ligated and the DNA structure
During the cleavage reaction, a covalent
enzyme-DNA intermediate is formed between a tyro-
sine residue of each topoisomerase II monomer and the
5'-phosphate group of the cleaved DNA. This enzyme-
bridged double-strand break normally exists tran-
siently, but the action of topoisomerase poisons such
as etoposide is to stabilize these intermediates. Both
topoisomerase IIα and β are targets for etoposide, and
etoposide-induced topoisomerase II-DNA complexes
can be visualized and quantified using a fluorescence
microscopy based method (Trapped in AgaRose DNA
ImmunoStaning or TARDIS).
This method involves
ionic detergent and salt extraction of agarose-embed-
ded cells, resulting in removal of proteins not covalently
coupled to the nuclear DNA, which is too bulky to elute
from the agarose. Covalently coupled proteins are then
detected by immunofluorescence. In a variation of this
approach adherent cells are extracted in a similar fash-
ion on glass coverslips under slightly less harsh condi-
tions (DRT extraction) without agarose embedding.
We used this latter technique to examine the effect of
TSA treatment on both the quantity and subnuclear
distribution of topoisomerase IIβ-DNA complexes.
While very low levels of nuclear topoisomerase IIβ fluo-
rescence were observed in DRT-extracted control cells,
in cells treated with etoposide topoisomerase IIβ immu-
nofluorescence was easily detectable (Figs. 5 and 6). The median
immunofluorescent signal in DRT-extracted, etoposide-treated
cells corresponded to approximately 20% of the total topoisom-
erase IIβ complement measured in normally fixed (i.e., non-
extracted) cells (Sup. Fig. 2). From knowledge of the action of
etoposide, and analogous experiments employing the more harsh
extraction conditions of the TARDIS protocol,
we conclude
that the etoposide-induced topoisomerase IIβ signal in DRT-
extracted cells corresponds to covalent topoisomerase-DNA com-
plexes. Notably, prior exposure to TSA (5 days 50 nM) caused a
Figure 1. Topoisomerase IIβ staining co-localizes with DAPI-bright and HP1
staining regions. (A) Mouse C127I cells were xed with paraformaldehyde and
permeabilized (see Materials and Methods) and processed for immunouores-
cence with the anti-topoisomerase IIβ rabbit antisera 3535β, 18513β, 30400β or
anti-topoisomerase IIβ MAB H8, and rat MAB MAC353 and were counterstained
with DAPI. (B) C127I cells were similarly processed using anti-topoisomerase IIα
rabbit antiserum 18511. Z-stacks of uorescence images were captured. Decon-
volved equatorial sections are shown. Scale bar in main image, 10 mm; scale bar in
insert 4 μm.
64 Nucleus Volume 2 Issue 1
γH2AX is present in a faint speckled pattern in the nucleus, typi-
cally with one or a small number of bright foci, presumably origi-
nating from spontaneous DNA double-strand breaks (DSBs).
However, in cells exposed to TSA for 5 days, γH2AX was con-
centrated almost exclusively adjacent to large chromocentres
(see Fig. 7). These TSA-induced γH2AX foci were not as intense
as those induced by etoposide, which also form predominantly
outside heterochromatin,
but contributed to an overall increase
in background γH2AX (Fig. 7A and B). Notably, it has recently
been demonstrated that HDAC inhibition slows replication and
leads to replication-associated DNA damage and H2AX phos-
and we suspect that this is likely to be the cause of
the TSA-induced peri-heterochromatic gH2AX seen here. Thus
increased background H2AX phosphorylation and the redistri-
bution of topoisomerase IIβ are likely to be independent effects
of the TSA-mediated remodelling of heterochromatin.
between cells may have been due to the short drug exposure in
an asynchronous population of cells. In contrast, little if any het-
erochromatic concentration of extraction-resistant topoisomerase
IIβ was apparent in cells pre-treated with TSA before exposure to
etoposide (Fig. 6A and compare D and F). Results were similar
with antibody 30400 and 3535. Thus, consistent with the TSA-
induced redistribution of total topoisomerase IIβ (see Fig. 2),
the nuclear distribution of etoposide-induced topoisomerase
IIβ-DNA complexes is altered by TSA, to a more euchromatic
Notably, TSA treatment alone did not significantly affect the
quantity of DRT extraction-resistant topoisomerase IIβ (Fig.
S2), nor did it affect the magnitude of etoposide induced histone
H2AX phosphorylation, a marker for DNA double-strand breaks
(not shown). However, we did find that it had an unexpected
effect on the background pattern of γH2AX. In control cells,
Figure 2. Topoisomerase IIβ is concentrated in chromocentres often surrounding nucleoli in mouse epithelial cells. (A) Cells were xed and processed
for immunouorescence with anti-topoisomerase IIβ (green) and anti-brillarin or MAC353 (red) and were counterstained with DAPI. Z-stacks of uo-
rescence images were captured. Deconvolved equatorial sections are shown for the non-merged images. Equatorial xy planes together with xz and yz
sections are shown for the merged images. Scale bar 10 μm. (B) Cells were xed and stained for topoisomerase IIβ or HP1β (green) and centromeres
(CENP-B, red).
www.landesbioscience.com Nucleus 65
The effect of HDACIs on the distribution of
topoisomerase IIβ in human cells. Since HDACIs
have been shown to potentiate topoisomerase poi-
sons in human cancer cell lines, we also exam-
ined the effect of TSA and valproic acid (VPA)
on topoisomerase II distribution in the human
lung cancer cell line A549. Topoisomerase IIβ
was distributed in a nucleoplasmic pattern, with
some concentration in the perinucleolar regions
of most cells (Fig. 8A). The perinucleolar pat-
tern was more pronounced when cells were per-
meabilized prior to fixation (Fig. 8B). Nucleoli of
human cells are typically surrounded by clusters
of heterochromatin identified by late replication
timing, histone methylation and the presence of
HP1 proteins.
In human cells, HP1α gives the
most robust pericentromeric staining pattern of
the three HP1 species.
When we compared
the distribution of HP1α to that of topoisomer-
ase IIβ, HP1 substantially coincided with the
areas of brightest topoisomerase IIβ signal (Fig.
8C). When cells were treated with TSA or VPA,
topoisomerase IIβ adopted a more pan-nuclear
distribution, as was observed following HDACI
treatment of mouse cells (Fig. 3). HP1α still pre-
sented a focal distribution under these conditions,
as observed previously,
but these foci were no
longer associated with bright topoisomerase IIβ
staining (Fig. 8C). Thus, as in mouse C127I cells,
topoisomerase IIβ is partially concentrated in het-
erochromatic regions of the nucleus of A549 cells
and is redistributed into a more uniform pattern
upon HDACI treatment. We also investigated the
distribution of topoisomerase IIβ in two human
lymphoblastoid cell lines K562 and Nalm-6.
Topoisomerase IIβ appeared nucleoplasmic in dis-
tribution in these cells, but after mild detergent
extraction (CSK buffer) the remaining topoisom-
erase IIβ was concentrated in the DAPI-bright
heterochromatic regions surrounding nucleoli or
at the nucleolar periphery (Sup. Fig. 3), similar to
the distribution in A549 cells.
Topoisomerase IIβ mediates the potentiat-
ing effect of HDACI. We investigated the abil-
ity of the HDACI valproic acid to potentiate the
growth inhibitory/cytotoxic effects of topoisomer-
ase poisons in A549 cells using a series of topoi-
somerase poisons with different selectivities for
Figure 3. TSA-mediated disassociation of topoisom-
erase IIβ and HP1β from heterochromatin. C127I cells
were cultured in media containing the indicated
concentrations of TSA for two or ve days before x-
ing and processing for immunouorescence with the
antibodies shown. Stacks of images were collected
and deconvolved images were merged into maximum
intensity projections.
66 Nucleus Volume 2 Issue 1
deacetylase inhibitors with topoi-
somerase poisons.
What causes topoisomerase IIβ
to be retained in heterochromatin?
The very similar subnuclear localiza-
tion of topoisomerase IIβ and HP1β
and their parallel dispersal from het-
erochromatin following TSA treat-
ment suggests that the two proteins
might interact in a protein-protein
complex. Another piece of evidence
that suggests this is the very similar
phenotype of mice null for TOP2b
and CBX1, the genes encoding
topoisomerase IIβ and HP1β respec-
tively. Both TOP2b and CBX1 null
mice develop apparently normally in
utero, but die perinatally from respi-
ratory failure due to failure to inner-
vate the diaphragm.
We carried
out a co-immunoprecipitation
experiment to determine whether
HP1β and topoisomerase IIβ exist
in a stable protein complex. While
antibodies 18513 and MAC353 ef-
ciently precipitated their cognate
antigens (topoisomerase IIβ and
HP1β respectively), 18513 immuno-
precipitates did not contain detect-
able HP1β and MAC353 did not
specifically precipitate topoisomerase
IIβ (data not shown). Thus, topoi-
somerase IIβ and HP1β do not exist
in a protein complex, at least not one
that is stable under the conditions
in which the cell extracts were pre-
pared. So the heterochromatic accu-
mulation of topoisomerase IIβ can
probably not be explained by associa-
tion with HP1β. Other candidates
include interaction with another het-
erochromatin protein such as HP1α or KAP1 or with a modi-
fied histone such as H3K9me3 (involved in targeting HP1β)
H4K20me3. Whether any of these features are involved remains
to be elucidated. Notably, topoisomerase II has previously been
shown by photo bleaching studies to be quite mobile within the
thus interactions resulting in the observed heterochro-
matic concentration of topoisomerase IIβ are presumable not of
high affinity.
Several mechanisms have been proposed to account for the sen-
sitization of cells to topoisomerase poisons by HDACIs. These
include modulation of apoptotic pathways
and the general
decondensation of chromatin following histone hyperacetylation,
topoisomerase IIα and -β, see Table 1 and Sup. Table 1. The
most robust potentiation was observed with mitoxantrone and
mAMSA, while marginal potentiation was obtained for etopo-
side and no significant effect was seen for doxorubicin or epirubi-
cin. Cells lacking topoisomerase IIβ have previously been shown
to be resistant to mitoxantrone and mAMSA,
indicating a
significant role for this isozyme in the cytotoxicity of these drugs,
whereas the degree of resistance to etoposide or doxorubicin in
topoisomerase IIβ null cells was smaller. Similarly, we found the
for mitoxantrone in growth inhibition assays is five times
higher in topoisomerase IIβ null Nalm-6 cells than in their wild
type counterparts while the ratio is only 2.8 for etoposide (data
not shown). Thus, these data support previous work
ing a functional interaction between topoisomerase IIβ and
HDAC inhibitors such as VPA in the synergistic action of histone
Figure 4. Quantication of topoisomerase IIβ, HP1β and DNA content in C127I cells exposed for 5 days
to 50 nM TSA. (A–C) Quantitative immunouorescence images were collected at 10x. Horizontal bars =
median values. (D) Western blot analysis of topoisomerase IIβ and HP1β. 5, 2.5 or 1.25 μg of protein from
whole cells extracts were loaded as shown. Con, no treatment; TSA, 50 nM TSA 5 days.
www.landesbioscience.com Nucleus 67
leading to increased DNA damage. For example, SAHA
and VPA cause chromatin decondensation in MCF7 cells
as viewed by EM and DNase sensitivity.
This deconden-
sation may allow greater access or accumulation of topoi-
somerase poisons in chromatin. Similarly, prolonged VPA
treatment (48 hr) has been shown to result in reduction in
HP1, DNMT1, SMC1 and SMC3 protein levels in MCF7
cells, though this was not observed in mouse cells for any
of the HP1 isotypes.
Our findings add to this chromatin-
mediated mechanism by demonstrating a redistribution of
topoisomerase IIβ within the nucleus following HDAC
inhibition analogous to that previously observed for HP1β.
Specifically we find that in mouse C127I cells, which pos-
sess easily identifiable heterochromatic foci, topoisomerase
IIβ is concentrated in heterochromatin and redistributes to
a speckled pan-nuclear pattern upon TSA-treatment (Figs.
1–3 and S1). Previous photo-bleaching studies have shown
that topoisomerase IIβ is relatively mobile in the nucleus,
thus association with heterochromatic or other structures
is likely to be transient with exchange between compart-
ments the equilibrium of which is altered by hyperacety-
lation of heterochromatin during HDACI treatment.
Human cells do not contain the prominent chromo-
centres that are present in mouse cells, but topoisomerase
IIβ was concentrated in the perinucleolar region and in
foci co-localizing with HP1α in A549 human lung car-
cinoma cells. Notably though, topoisomerase IIβ was not
found in the barr-body in female human cells (E. Heard,
personal communication), suggesting that its observed
accumulation is limited to constitutive heterochromatin.
As was observed in mouse cells, topoi-
somerase IIβ was redistributed follow-
ing TSA or VPA treatment (Fig. 8). TSA
also altered the distribution of etoposide-
induced topoisomerase IIβ-DNA com-
plexes. In non-TSA treated C127I cells,
adducts were concentrated in heterochro-
matic regions, reflecting the distribution
of the enzyme in these cells, while after
TSA treatment, adducts were distributed
more evenly throughout the nucleus.
We have not addressed the HDAC
specificity of the observed topoisomerase
II redistribution, but we note that HDAC2
was found to be the predominant target in
MCF7 cells for HDACIs with regards to
chromatin decondensation, downregula-
tion of HP1 and other chromatin proteins
and potentiation of epirubicin cytotoxic-
We have employed TSA and VPA
in the work reported here. Both inhibit
HDAC2, although VPA but not TSA
selectively induces proteasomal degrada-
tion of HDAC2.
In addition, topoisom-
erase IIα and IIβ physically interact with
HDAC1 and HDAC2 and topoisomerase
Figure 5. Eect of TSA pre-treatment on the quantity of etoposide induced
topoisomerase IIβ-DNA complexes. Cells grown on glass coverslips were either
cultured in control medium or in medium containing 50 nM TSA for 5 days
after which etoposide (50 μM) was added to the medium. Two hours later cells
were extracted in DRT buer (see Materials and Methods). Quantitative immu-
nouorescence was carried out using the anti-topoisomerase IIβ antibodies
Table 1. Potentiation data obtained in A549 lung cancer cells for mitoxantrone, mAMSA,
etoposide, doxorubicin and epirubicin, and the effect of the HDAC inhibitor VPA
TopoII poison
Topoisomerase II
VPA pre-treatment
(1.6 mM)
α and β
1.6, 8
α and β
α > β
α > β
+ NO
- NO
Epirubicin N/D
+ NO
- NO
Cells were either plated directly or pre-incubated with 1.6 mM VPA before plating into 96 well
plates in media containing a range of topoisomerase poisons and either 0, 1.6 or 8 mM VPA.
Growth inhibition assays were performed and IC
values were determined for each topoisom-
erase poison/VPA combination. Experiments were carried out at least in triplicate and potentia-
tion values (PF
) were calculated (see Table S1). Where potentiation is scored as “YES”, IC
obtained with VPA were significantly lower than that obtained with topoisomerase poison alone
(p < 0.05 one tailed paired t-test). Topoisomerase II preference pertains to evidence for the rela-
tive involvement of topoisomerase IIα or –β in mediating the cytotoxic effects in cell line systems
(Errington, et al. 1999; Toyoda, et al. 2008).
68 Nucleus Volume 2 Issue 1
release of topoisomerase IIβ from heterochromatic sites and a
shift in the distribution of resulting topoisomerase IIβ adducts
towards the euchromatic portion of the nucleus that is induced
by HDACIs.
Materials and Methods
Cell culture and reagents. Mouse C127I cells and A549 human
lung carcinoma cells were acquired from European Collection of
Cell Cultures (ECACC). Both cell lines were cultured in DMEM
containing 10% FCS and antibiotics. Trichostatin A (Sigma) was
dissolved in water and was added to cell cultures at between 2 nM
and 80 nM. VPA (Sigma) was added to cultures at the concentra-
tions indicated in the text. Rabbit polyclonal antibodies 18511,
3535 and 30400 were generated in house and their speci-
ficity was confirmed by western blotting with whole cell extracts
and purified recombinant topoisomerase IIα and β. Anti topoi-
somerase IIβ mab H-8 was from Santa Cruz, mouse MAB anti-
fibrillarin 18380 and anti-HP1β rat MAB MAC353 were from
Abcam, mouse anti-γH2AX MAB was from Millipore.
Growth inhibition assays. Control (untreated) cells or cells
incubated in medium containing VPA at 1.6 or 8 mM (10%
and 50% of IC
respectively) for 72 hours were trypsinized and
IIβ is associated with HDAC1 in the NuRD complex.
Previous descriptions of the intranuclear distribution of topoi-
somerase IIβ have varied in the literature.
This may reflect
the dynamic nature of topoisomerase IIβ. Topoisomerase IIβ
is recruited to the promoters of certain genes during transcrip-
tional activation
and it is possible that the HDACI-induced
redistribution of topoisomerase IIβ reported here reflects the
normal dynamic behaviour of the protein during the execution
of transcriptional programs, though this possibility remains to
be explored. We have consistently observed topoisomerase IIβ to
be concentrated in chromocentres in untreated fixed mouse cells
(Figs. 1–3 and S1). Notably, this distribution is mirrored in the
pattern of topoisomerase IIβ-DNA adducts formed in living cells
by etoposide (Fig. 6), arguing against the observed pattern being
an artifact of fixation.
The cytotoxic potential of topoisomerase poisons such as
etoposide is mediated predominantly through topoisomerase
HDACIs sensitize cells to topoisomerase poisons
including etoposide, but the sensitization is reported to be depen-
dent upon topoisomerase IIβ.
This apparent contradiction can
be resolved if HDACIs somehow recruit topoisomerase IIβ as
a target for topoisomerase poisons. A possible mechanism for
this recruitment is provided by the chromatin decondensation,
Figure 6. TSA alters the distribution of etoposide-induced topoisomerase IIβ-DNA complexes in the nucleus. (A) Cells were cultured either in control
medium or in medium containing 50 nM TSA for 5 days, after which etoposide (50 μM) was added to the medium. Cells were extracted in situ 2 hours
later in DRT buer before xing (shown as “E”) or were immediately xed with paraformaldehyde and permeabilized (shown as “F”, see Materials and
Methods). Immunouorescence was carried out using the anti-topoisomerase IIβ antibody 18513. Image stacks were collected. Deconvolved equato-
rial sections are shown. Circled nuclei are enlarged and shown below. Bar = 10 μm. (B) Enlarged nucleus indicated by the circled nucleus in the rst
column of A, 18513-stained. Bar = 5 μm. (C and D) Enlarged image of nucleus shown in the third column of A, stained with DAPI and 18513 (TopoIIβ)
respectively. (E and F) Enlarged image of nucleus shown in the fth column of A, stained with DAPI and 18513 (TopoIIβ) respectively.
www.landesbioscience.com Nucleus 69
plated into 96 well plates containing combinations of VPA (0,
1.6 or 8 mM) and either vehicle or a dose range of topoisomer-
ase II poison. Plates were incubated for 72 hours under normal
conditions, after which relative cell growth was determined by
XTT assay (Roche) according to the manufacturer’s instructions.
Optimum cell plating density was determined from growth
curves. Data was normalized to the growth obtained with VPA
alone. Assays were performed at least in triplicate and IC
values were calculated. Potentiation factors (PF
) are the
ratio of the concentration of topoisomerase poison required to
achieve a 50% growth inhibition (IC
) in the absence of VPA to
that in the presence of VPA.
Immunofluorescence analysis. Cells were grown on glass
cover slips. For standard paraformaldehyde fixation, coverslips
Figure 7. TSA alters the background pattern of histone H2AX phosphorylation. (A) TSA-treated or control cells were plated onto coverslips and sub-
sequently exposed to medium containing 0 or 1 μM etoposide. Cells were xed and processed for immunouorescence using anti-γH2AX and anti
topoisomerase IIβ (18513). Images were collected with a 40x objective, maximum intensity projections derived from deconvolved images are shown.
In the rst two columns, greyscale image levels were adjusted such that background (signal present in the absence of a DSB-inducing agent) γH2AX
was clearly visible. In the third column levels were adjusted as for the counting of induced γH2AX foci, such that etoposide-induced γH2AX foci are
clearly visible but with minimal contribution from the background. (B) Quantitative immunouorescent analysis of background γH2AX intensity in
control cells and cells treated with 50 nM TSA for 5 days.
were briefly washed in PBS, immersed in 4% paraformaldehyde
in PBS for 10 minutes at room temperature and then permeabi-
lized by incubation in KCM+T [120 mM KCl, 20 mM NaCl,
10 mM TRIS-HCl, pH 7.5, 0.5 mM EDTA, 0.1% (v/v) Triton
X-100] for 5 minutes. Coverslips were then blocked for 1 hour
at room temperature with 10% dried milk powder, 5% BSA in
KCM+T. First and second antibody incubations were in blocking
buffer for 1 hour each at room temperature. Washes were carried
out using KCM+T. Second antibodies were Alexa Fluor-488 or
Alexa Fluor-594 conjugated (Invitrogen). After second antibody
incubation and washing, coverslips were mounted on slides with
Vectashield containing DAPI and were sealed with nail varnish.
For CSK extraction, coverslips were incubated on ice for 5 min-
utes in CSK buffer [10 mM Pipes (pH 7.0), 100 mM NaCl, 300
70 Nucleus Volume 2 Issue 1
detected using the primary antibodies indicated and Alexa Fluor
594-conjugated second antibodies (Invitrogen). Microscopy was
performed using an Olympus IX81 motorized microscope with
a 10x objective (UPlanFLN, NA 0.3) and employing a 120 W
X-Cite illumination system. Greyscale images were collected
with a Hamamatsu Orca-AG camera with 2 x 2 binning. Images
were background and shade corrected as described previously in
reference 41 and 42. Fluorescence intensity (integrated fluores-
cence) per nucleus was determined for DAPI and Alexa Fluor 488
channels using Volocity Quantitation software (Improvision).
Data was plotted using Graphpad Prism.
This work was supported by a grant (05-179) from the Association
for International Cancer Research.
Supplemental materials can be found at:
mM sucrose, 3 mM MgCl
, 1 mM EGTA 0.5% Triton-X100]
containing protease inhibitors, before paraformaldehyde fixa-
tion and immunostaining. For DRT extraction, coverslips were
extracted once on ice in [30 mM HEPES, 65 mM Pipes, 10 mM
EGTA, 2 mM MgCl
, pH 6.9 with 350 mM NaCl, 0.5% Triton
X-100] containing protease inhibitors for two minutes with occa-
sional gentle agitation. Cells were then fixed for 10 minutes in
4% paraformaldehyde in PBS before immunostaining as above.
Images were obtained using an Olympus IX81 motorized micro-
scope fitted with a Hamamatsu Orca-AG monochrome camera.
Z-stacks were collected using a 40x objective (PlanS Apo, NA
0.95) at 0.2 μm intervals or using a 60x objective and OptiGrid
confocal (Qioptiq) at 0.3 μm intervals. Iterative deconvolution
was performed using Volocity software (Improvision).
Western blotting. Cells were grown in 9 cm tissue culture
flasks. Whole cell extracts were prepared, separated by SDS
PAGE and blotted onto nitrocellulose membranes as described
previously in reference 40.
Quantitative immunofluorescence. Cells were grown on glass
coverslips and immunostained as above. Topoisomerase II was
Figure 8. Topoisomerase IIβ distribution and HDACI-induced redistribution in A549 lung cancer cells. (A) A549 cells were xed with paraformalde-
hyde, permeabilized and immunostained with rabbit anti-topoisomerase IIβ (green) and anti-brillarin MAB (red). In the lower row, cells were exposed
to 2 mM VPA for 24 hours prior to xation. (B) Control cells or cells treated with 2 mM VPA or 50 nM TSA for 48 hours were permeabilized prior to xa-
tion as described in Materials and Methods (CSK extraction). Antibodies are as in (A). (C) Control, TSA treated or VPA treated cells were CSK extracted
as in (B) and then xed and immunostained with anti-rabbit topoisomerase II (18513β) and mouse anti HP1α. In all cases equatorial confocal planes are
shown. In the right-hand column of part (C) x-z and y-z confocal sections are also displayed for the topoisomerase II β/HP1α merged images.
Scale bar = 10 μm.
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    • "Notably, overexpression of histone acetylase hMOF, known to target H4K16, can mimic HDACi treatment, while overexpression of SIRT1 deacetylase counteracted the effect, pointing to hMOF and SIRT1 activities as critical parameters in HDACi-mediated sensitization to etoposide. Moreover, TSA treatment displaces TopoII from its binding to heterochromatin (Cowell et al., 2011). Although the underlying molecular mechanism is till to be defined, the displacement can convert TopoII to an effective relevant target for topoisomerase poisons. "
    [Show abstract] [Hide abstract] ABSTRACT: Etoposide derives from podophyllotoxin, a toxin found in the American Mayapple. It was first synthesized in 1966 and approved for cancer therapy in 1983 by the U.S. Food and Drug Administration (Hande, 1998). Starting from 1980s several studies demonstrated that etoposide targets DNA topoisomerase II activities thus leading to the production of DNA breaks and eliciting a response that affects several aspects of cell metabolisms. In this review we will focus on molecular mechanisms that account for the biological effect of etoposide.
    Full-text · Article · Nov 2015
    • "Human TOP2B protein directly interacts with a number of proteins including CD3ε UBC9, TOPBP1, p53, pRB, SNF2H, HDAC1 and HDAC2 (Cowell et al., 2000; Johnson et al., 2001; LeRoy et al., 2000; Mao et al., 2000; Nakano et al., 1996; Tsai et al., 2000; Xiao and Goodrich, 2005; Yamane et al., 1997; Yuwen et al., 1997) several of which are involved in transcriptional regulation. Inhibition of HDACs by TSA redistributes TOP2B from heterochromatin to euchromatin in mouse epithelial cells (Cowell et al., 2011). TOP2B is also found associated with several complexes involved in the regulation of transcriptional initiation. "
    [Show abstract] [Hide abstract] ABSTRACT: We report the whole genome ChIP seq for human TOP2B from MCF7 cells. Using three different peak calling methods, regions of binding were identified in the presence or absence of the nuclear hormone estradiol, as TOP2B has been reported to play a role in ligand-induced transcription. TOP2B peaks were found across the whole genome, 50% of the peaks fell either within a gene or within 5 kb of a transcription start site. TOP2B peaks coincident with gene promoters were less frequently associated with epigenetic features marking active promoters in estradiol treated than in untreated cells. Significantly enriched transcription factor motifs within the DNA sequences underlying the peaks were identified. These included SP1, KLF4, TFAP2A, MYF, REST, CTCF, ESR1 and ESR2. Gene ontology analysis of genes associated with TOP2B peaks found neuronal development terms including axonogenesis and axon guidance were significantly enriched. In the absence of functional TOP2B there are errors in axon guidance in the zebrafish eye. Specific heparin sulphate structures are involved in retinal axon targeting. The glycosaminoglycan biosynthesis-heparin sulphate/heparin pathway is significantly enriched in the TOP2B gene ontology analysis, suggesting changes in this pathway in the absence of TOP2B may cause the axon guidance faults.
    Full-text · Article · Oct 2015
    • "Topo II has been long involved in high-order organization of chromatin (9), centromere configuration (82) and genome compaction in sperm cells (83–85). In mammalian cells, topo IIβ is implicated in heterochromatin transitions that depend on histone deacetylase (81). Therefore, yeast topo II and mammalian topo IIβ may use similar mechanisms to repress transcription by inducing or stabilizing condensed chromatin states. "
    [Show abstract] [Hide abstract] ABSTRACT: Eukaryotic topoisomerase II (topo II) is the essential decatenase of newly replicated chromosomes and the main relaxase of nucleosomal DNA. Apart from these general tasks, topo II participates in more specialized functions. In mammals, topo IIα interacts with specific RNA polymerases and chromatin-remodeling complexes, whereas topo IIβ regulates developmental genes in conjunction with chromatin remodeling and heterochromatin transitions. Here we show that in budding yeast, topo II regulates the expression of specific gene subsets. To uncover this, we carried out a genomic transcription run-on shortly after the thermal inactivation of topo II. We identified a modest number of genes not involved in the general stress response but strictly dependent on topo II. These genes present distinctive functional and structural traits in comparison with the genome average. Yeast topo II is a positive regulator of genes with well-defined promoter architecture that associates to chromatin remodeling complexes; it is a negative regulator of genes extremely hypo-acetylated with complex promoters and undefined nucleosome positioning, many of which are involved in polyamine transport. These findings indicate that yeast topo II operates on singular chromatin architectures to activate or repress DNA transcription and that this activity produces functional responses to ensure chromatin stability.
    Full-text · Article · Aug 2013
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