[Epigenetics 2:2, 92-103; April/May/June 2007]; ©2007 Landes Bioscience
Research Paper
Ubiquitin-Dependent Distribution of the Transcriptional Coactivator
p300 in Cytoplasmic Inclusion Bodies
Jihong Chen1
Sabina Halappanavar1
John P.H. Th’ ng2
Qiao Li1,*
1Department of Pathology and Laboratory Medicine; Faculty of Medicine;
University of Ottawa; Ottawa, Ontario Canada
2Northwestern Regional Cancer Thunder Bay; Ontario, Canada
*Correspondence to: Qiao Li; Department of Pathology and Laboratory Medicine;
University of Ottawa; 451 Smyth Road, Room 4155; Ottawa, Ontario, Canada,
K1H 8M5; Tel.: 613.562.5422; Fax: 613.562.5442; Email: qiaoli@uottawa.ca
Original manuscript submitted: 12/13/06
Manuscript accepted: 04/23/07
Previously published online as an Epigenetics E-publication:
http://www.landesbioscience.com/journals/epigenetics/abstract.php?id=4326
Key words
transcriptional coactivator, the 26S prote-
asome, ubiquitin, aggresome, histone
deacetylase inhibitor
Acknowledgements
We thank Dr. M-A Ikeda for the p300WT and
p300ΔM plasmids. This work is supported by
operating grant from Canadian Institutes of
Health Research (CIHR). During the course
of this study, Q.L. holds an investigator award
from CIHR.
92 Epigenetics 2007; Vol. 2 Issue 2
Abstract
The protein level of transcriptional coactivator p300, an essential nuclear protein,
is critical to a broad array of cellular activities including embryonic development, cell
differentiation and proliferation. We have previously established that histone deacetylase
inhibitor such as valproic acid induces p300 degradation through the 26S proteasome
pathway. Here, we report the roles of cellular trafficking and spatial redistribution in
valproic acid‑induced p300 turnover. Our study demonstrates that p300 is redistributed
to the cytoplasm prior to valproic acid‑induced turnover. Inhibition of proteasome‑de‑
pendent protein degradation, does not prevent nucleo‑cytoplasmic shuttling of p300,
rather sequesters the cytoplasmic p300 to a distinct perinuclear region. In addition, the
formation of p300 aggregates in the perinuclear region depends on functional micro‑
tubule networks and correlates with p300 ubiquitination. Our work establishes, for the
first time, that p300 is also a substrate of the cytoplasmic ubiquitin‑proteasome system
and provides insight on how cellular trafficking and spatial redistribution regulate the
availability and activity of transcriptional coactivator p300.
Introduction
Transcriptional coactivator p300 plays an essential role in integrating multiple
signal‑transduction events through the regulation of gene expression. The function of
p300 is often mediated by bridging the sequence‑specific DNA binding transcription
factors with the basal transcription machinery and providing a scaffold for nucleation
of multi‑components of transcriptional regulatory complex.1 The impact of p300 on
transcriptional regulation can also be achieved through histone acetylation and chromatin
remodeling as p300 contains an intrinsic histone acetyltransferase activity.2,3 Additionally,
p300 also regulates the functions of many transcription regulators such as p53, NF‑Y,
GATA‑1, TR‑RXR, E2F and Rb through its acetyltransferase activity.4‑9
Somatic mutations of p300 gene has been found in a variety of malignancies such
as colorectal, gastric and breast cancers, indicating a tumor suppressor‑like activity for
p300.10,11 In addition, cells devoid of p300 exhibit defects in proliferation and specific
transcription, underscoring how critical the gene dosage of p300 is for normal embryonic
development.12 Many lines of evidence have demonstrated that the levels and activities of
p300 are essential to a wide range of cellular processes including embryonic development,
cell differentiation and proliferation. However, the molecular mechanisms that regulate
p300 activity per se are poorly understood.
Protein degradation through ubiquitin‑proteasome pathway is a specifically regulated
event which involves covalent ubiquitination of lysine residues of the target protein
and degradation of the ubiquitin‑tagged substrate through the 26S proteasome, a large
multi‑protein complex found in all eukaryotic cells.13‑15 Growing evidence suggests that
the ubiquitin‑proteasome pathway is not just for eliminating misfolded or damaged
proteins, but is actively involved in the regulation of fundamental cellular processes such
as cell cycle progression, differentiation and apoptosis.16‑20 The 26S proteasome mediated
p300 degradation has been documented in F9 embryonic carcinoma cells during retinoic
acid‑induced differentiation and in cardiocytes exposed to doxorubicin.21,22 We previously
reported that PI3K inhibitors enhance p300 degradation through the 26S proteasome,
which seems to be mediated by the nuclear proteasome system.23 We have also established
that histone deacetylase (HDAC) inhibitors are able to induce p300 degradation through
the 26S proteasome pathway as well.24,25 However, whether the selective p300 turnover is
mediated exclusively by the nuclear proteasome system is still unclear.
 Spatial Regulation of Transcriptional Coactivator p300
Figure 1. Effects of valproic acid, butyrate and MG132 on cellular localiza‑
tion of transcriptional coactivator p300. Cells were treated with valproic
acid (VPA, 2 mM) or sodium butyrate (5 mM) in the absence or presence of
MG132 (MG, 5 mM) for 8 hours. The cells were then immunostained with
a p300 antibody and a fluorescence labeled secondary antibody. Hoechst
(25 ng/ml) was also used to visualize the nuclei. Images were captured
by optical sectioning. Shown are representatives of single 0.25 mm optical
sections.
Cytoplasmic inclusion bodies are found in many neurodegen-
erative diseases including Huntington’s disease, spinocerebellar
ataxia type 3 disease, amyotrophic lateral sclerosis and alzheimer’s
disease.26‑29 Inclusion bodies are intracellular foci containing various
protein aggregates. In many cases, the formation of cytoplasmic
inclusion bodies at the perinuclear region requires retrograde although it is able to enhance p300 degradation through the 26S
transport of the aggregated protein on microtubules. Hence, aggresome
is used to define these microtubule‑dependent cytoplasmic inclusion
bodies which are often formed as a result of expression of mutant
proteins or overwhelmed proteasome system.30,31 The components
of aggresome include ubiquitin, subunits of the 20S proteasome and
intermediate filament vimentin, suggesting that aggresome could
serve as an alternative proteolysis center of the cellular system.31,32
Recent studies have demonstrated that aggresome is indeed not just a
sequester area for misfolded protein aggregates, rather an integral part
of normal cellular regulatory process to control the levels of targeted
proteins in response to specific stimuli.33,34
In this report, we describe the molecular mechanisms that regulate
the activity of transcriptional coactivator p300. We show that intra-
cellular redistribution of p300 is important for valproic acid‑induced
p300 degradation through the 26S proteasome pathway. In addition,
the distribution of p300 in cytoplasmic inclusion bodies is microtu-
bule‑dependent and correlates with p300 ubiquitination. Our study
suggests that intracellular redistribution and cytoplasmic degradation
of p300 are integral regulatory mechanisms employed by cellular
systems to control the availability and the activity of transcriptional
coactivator p300 in the nucleus for chromatin acetylation and was detected in 89.7% ± 6.7% of the cells upon double treatments
transcriptional control.
Results
Valproic acid and butyrate induce cytoplasmic distribution of
p300. Intracellular trafficking has been implicated in the regula-
tion of the activity and turnover of several nuclear proteins.35 We
previously reported that histone deacetylase inhibitors such as
valproic acid and butyrate induce the degradation of transcriptional
coactivator p300 through the 26S proteasome pathway.24,25 To
further delineate the mechanisms by which p300 is signaled for the this, treatment of the cells with proteasome inhibitor MG132 did not
selective turnover, we examined the effect of valproic acid and butyrate
on cellular distribution of p300 by using immunofluorescence acid or butyrate, rather sequestered the cytoplasmic p300 at the
www.landesbioscience.com Epigenetics 93
microscopy coupled with ApoTome optical sectioning. The intra-
cellular localization of endogenous p300 protein in HeLa cells was
examined following a time course of treatments with valproic acid
or butyrate, since the reduction of the steady‑state level of p300
protein in the HeLa cells can only be observed after 16 hours of
treatments.25
In untreated cells, the endogenous p300 resided predominantly
in the nucleus with 13.6% ± 10.8% of cells displaying very faint
cytoplasmic staining (Fig. 1), which is in accordance with other reports
that p300 is mainly a nuclear protein.23,36 However, upon 8 hours
of valproic acid treatment, the p300 staining became fairly diffused
throughout the nucleoplasm and 80.3% ± 6.5% of cells showed
certain degree of cytoplasmic p300 staining (Fig. 1). Interestingly, the
cytoplasmic p300 staining was mainly observed around the nuclear
envelope (Fig. 1). The altered spatial distribution of p300 protein was
detected as early as 4 hours of treatment, prior to the reduction of
p300 protein and apoptotic cell death induced by valproic acid,25,37
and the intensity of cytoplasmic p300 staining was correlated with
the duration of valproic acid treatments (data not shown). Upon 16
hours of valproic acid treatment, the level of nuclear p300 staining
was significantly reduced, since about 70% of cellular p300 has been
degraded.25 In addition, butyrate, another HDAC inhibitor, was
also able to induce the redistribution of p300 to the cytoplasm in
83.0% ± 3.5% of cells (Fig. 1). In contrast, LY294002, a PI3K
inhibitor,38 does not induce cytoplasmic distribution of p300,
proteasome.23 Taken together, these data indicate that intracellular
trafficking of p300 may be involved in the control of p300 activity
by HDAC inhibitors such as valproic acid and butyrate.
Inhibition of proteasome activity sequesters p300 to a distinct
perinuclear region. Nucleo‑cytoplasmic shuttling and cytoplasmic
degradation could well serve as a regulatory mechanism to control
p300 activity in response to different intracellular signals. To deter-
mine whether redistribution of p300 to the cytoplasm is an integral
part of the selective p300 degradation‑mediated by the 26S protea-
some, we studied the effect of MG132, a peptide aldehydes reversibly
inhibiting the 26S proteasome activity,39 on the cytoplasmic redis-
tribution of p300 induced by valproic acid and butyrate. The HeLa
cells were treated with MG132 alone or in combination with valproic
acid or butyrate, and then subjected to ApoTome optical sectioning
analysis.
As shown in (Fig. 1), some punctuated p300 staining pattern was
captured at a distinct perinuclear region following 8 hours of MG132
treatment in 86.3% ± 6.7% of the HeLa cells, and the distinct
perinuclear p300 staining was intensified by cotreatment with
valproic acid. In addition, the distinct perinuclear p300 staining
with valproic acid and MG132. Similarly, butyrate treatment also
enhanced the distinct perinuclear p300 staining in 89.9% ± 4.4%
cells in the presence of MG132 (Fig. 1). These data indicate that
valproic acid and butyrate induce the cytoplasmic redistribution
of p300, which may lead to the degradation of p300 through the
cytoplasmic proteasome system.
The distinct p300 staining pattern at the perinuclear region
induced by valproic acid or butyrate in the presence of MG132 highly
resembles the structure of aggresome. Formation of aggresome can be
accelerated by inhibition of the 26S proteasome activity. In line with
prevent the cytoplasmic redistribution of p300 induced by valproic
 Spatial Regulation of Transcriptional Coactivator p300
Figure 2. Colocalization of p300 with g‑tubulin at perinuclear region. Cells
were treated with MG132 (MG, 5 mM) in the absence or presence of
valproic acid (VPA, 2 mM) for 8 hours. The cells were then immunostained
with antibodies specific to p300 (green) and g‑tubulin (red). Images were
acquired by optical sectioning. Shown are representatives of single 0.25
mm optical sections.
Figure 3. Localization of p300 and a subunits of the 20S proteasome. Cells
were treated with MG132 (MG, 5 mM) in the absence or presence of val‑
proic acid (VPA, 2 mM) for 8 hours. The cells were then immunostained with
antibodies specific to p300 (green) and a subunits of the 20S proteasome
(red). Images were acquired by optical sectioning. Shown are representa‑
tives of single 0.25 mm optical sections.
distinct perinuclear region as punctuated staining (Fig. 1). Therefore,
we wished to determine whether the perinuclear accumulation of
p300 is related to aggresome structure.
The perinuclear p300 colocalizes with g‑tubulin and with
subunits of the 20S proteasome. The cytoplasmic inclusion bodies of
mammalian cells, termed as aggresome, are microtubule‑dependent
and have a distinct perinuclear localization.30 To determine whether
aggresome is in fact the structural basis for the perinuclear accumu-
lation of p300 induced by MG132, we used immunofluorescence
optical sectioning to characterize the spatial distribution of p300 in
relation to two well‑characterized aggresome markers, g‑tubulin and
a subunits of the 20S proteasome.31
As shown in (Fig. 2), in untreated cells, g‑tubulin was distrib-
uted around the nuclear envelope as reported by others30 and it did
not colocalize with p300. However, following 8 hours of valproic
94 Epigenetics 2007; Vol. 2 Issue 2
Figure 4. Colocolization of p300 with ubiquitin. Cells were treated with
MG132 (MG, 5 mM) and valproic acid (VPA, 2 mM) in the absence (top
panels) or presence (bottom panels) of nocodazole (10 mM) for 8 hours.
Following the treatments, the cells were fixed and stained with antibodies
specific for p300 (green) and ubiquitin (red). Images were captured by opti‑
cal sectioning. Each image represents optical section of 0.25 mm depth.
acid treatment, colocalization of p300 with g‑tubulin was observed
around the nuclear envelope as yellow speckles on the overlay image
(Fig. 2). Upon double treatments with valproic acid and MG132,
the colocalization p300 with g‑tubulin was more evident and became
localized to a distinct perinuclear region (Fig. 2). In addition, the
distinct perinuclear p300 staining induced by valproic acid in the
presence of MG132 localized to the same region as a subunits of
the 20S proteasome (Fig. 3). Taken together, these data suggest that
the distinct perinuclear p300 is likely sequestered into the aggresome
following inhibition of the 26S proteasome activity.
The perinuclear p300 colocalizes with ubiquitin. Aggresome,
in addition to containing g‑tubulin and subunits of the 20S protea-
some, is also rich in ubiquitin since the inclusion body often serves
as a assembly for components of the 26S proteasome system.31,40
To further assess the relationship of the distinct perinuclear p300
with the aggresome structure, we studied colocalization of p300 with
ubiquitin in response to treatment with valproic acid in the absence
or presence of MG132 by using ApoTome optical sectioning. As
shown in (Fig. 4), inhibition of the 26S proteasome activity with
MG132 sequestered ubiquitin into aggresome structure as previ-
ously reported.41 Importantly, p300 colocalized with ubiquitin at
the distinct perinuclear region following double treatments with
MG132 and valproic acid, while the ubiquitin staining is more
punctuated in the aggresome like structure (Fig. 4). This suggests that
the perinuclear p300 may be modified by ubiquitin and targeted for
degradation through the cytoplasmic proteasome system.
Experimentally, the formation of aggresome can be induced
by inhibition of the 26S proteasome activity but be blocked by
depolymerization of microtubules with nocodazole.31 To determine
whether the colocalization of p300 with ubiquitin at the distinct
perinuclear region is microtubule‑dependent, we treated the cells
with nocodazole in parallel experiments. As shown in (Fig. 4),
nocodazole treatment alone did not affect the cellular distribution
 Spatial Regulation of Transcriptional Coactivator p300

Figure 5. Effects of leptomycin‑B on valproic acid‑induced p300 degrada‑

tion. (A) Cells were treated with valproic acid (VPA, 2 mM) alone or in
combination with leptomycin‑B (LMB, 10 nM) for 16 hours. Cell lysates were
prepared and subjected to Western blot analysis with antibodies specifically
against p300 and SRC. (B) Quantitative analysis of the Western blot results
is expressed as fold variations compared to the untreated control. SRC bands
were used as internal controls. Error bars represent the standard deviations
of three independent experiments. (C) Following 8 hours of various treat‑
ments, cells were fixed and stained with a p300 antibody for immunofluo‑
rescence microscopy. The cells were also stained with hoechst (25 ng/ml) to
visualize the nuclei.

of p300 comparing to untreated control cells. However, in cells

treated with MG132 in combination with valproic acid, the addi-
tion of nocodazole prevented the distribution of p300 to the distinct
perinuclear region and led to p300 scattered around the cytoplasm
while still localizing with ubiquitin (Fig. 4). Together, these data
indicate that microtubule‑dependent retrograde transport mecha-
nism is involved in the formation of p300 aggresome during valproic
acid‑induced p300 turnover.
Leptomycin B inhibits valproic acid‑induced p300 degradation.
Intracellular trafficking are effective means to control the level and
activity of nuclear proteins. Many transcription regulators shuttle
between nucleus and cytoplasm by energy‑dependent transport
across the nuclear membrane. For example, nuclear export and
cytoplasmic degradation of p53 is fundamental for inactivating its
transcriptional activity during normal cell growth.35,42 Exportin1
or CRM1 (chromosomal region maintenance‑1) is one of the best
characterized export receptor engaged in transporting many cellular
proteins from the nucleus to cytoplasm.43 To discern whether
intracellular trafficking is involved in valproic acid‑induced p300
degradation, we employed leptomycin B, a nuclear export inhibitor
which binds directly to CRM1 and blocks the CRM1‑mediated
nuclear protein export.43
The levels of endogenous p300 protein were assessed by using
quantitative Western analysis following treatment of HeLa cells with
valproic acid in the absence or presence of leptomycin B. As shown in
(Figs. 5A and B), valproic acid treatment reduced the levels of endog-
enous p300 by about 70% as compared to the untreated control cells
in agreement with early report,25 while leptomycin B treatment alone
Figure 6. Domain specific ubiquitination of p300. Cells were transfected with
expression plasmids for various M2‑tagged p300 truncations spanning the
full length of p300 together with expression plasmid for HA‑tagged ubiqui‑
tin. Following treatments with MG132 (MG, 5 mM), the M2 tagged p300
were immunoprecipitated by using a M2‑conjugated agarose and subjected
to Western blot analysis with a M2 antibody. The blot was then stripped
and reprobed with a HA antibody to detect the p300‑ubiquitin conjugates.
Shown are the representatives of three independent experiments. Solid
arrow indicates the position of M2‑tagged full‑length p300 (A‑C). Schematic
presentation of various p300 expression plasmids used in the experiments
are also shown (D).
did not have much impact on the steady‑state level of p300 protein.
Most intriguingly, the reduction of p300 induced by valproic acid
was efficiently prevented by cotreatment of the cells with leptomycin
B (Figs. 5A and B). These data suggest that CRM1 pathway may be
important for intracellular redistribution and cytoplasmic degrada-
tion of p300 and the shuttling of p300 from the nucleus to the
cytoplasmic region may be involved in valproic acid‑induced p300
degradation.

www.landesbioscience.com Epigenetics 95
 Spatial Regulation of Transcriptional Coactivator p300
Figure 7. Localization of p300‑M to the vimentin structure is microtubule‑de‑
pendent. Cells were transfected with expression plasmids for p300‑M or
p300‑N, and treated with MG132 (MG, 5 mM) and valproic acid (VPA, 2
mM) in the absence or presence of nocodazole (Noco, 10 mM). The cells
were then stained with a M2 antibody for detecting the truncated forms of
p300 (green) and an antibody for vimentin (red). Images were captured by
optical sectioning. Each image represents optical section of 0.25 mm depth.
Next, we examined the effect of leptomycin‑B on valproic
acid‑induced cellular redistribution of p300 by using immuno-
fluorescence microscopy. As shown in (Fig. 5C), treatment with
leptomycin‑B alone did not change the nuclear staining pattern p300
in comparison with untreated control cells. However, cotreatment of
the cells with leptomycin‑B impaired the cytoplasmic distribution
of p300 induced by valproic acid and restored the intensity of p300
nuclear staining to a similar degree of the untreated cells (Fig. 5C).
Collectively, these data suggest that nuclear export may be involved
in the cytoplasmic redistribution of p300, and CRM1 pathway is
important for nucleo‑cytoplasmic shuttling and cytoplasmic degra-
dation of p300 induced by valproic acid.
Domain specific ubiquitination of p300. Next, we wished to
further delineate whether valproic acid‑induced intracellular redis-
tribution of p300 is linked to ubiquitin‑dependent pathway. To
identify the p300 domain harbouring ubiquitinated lysine residues,
we first performed in vivo ubiquitination assay by using M2‑tagged
truncated forms of p300 spanning the full length of p300 (Fig. 6).
The M2‑tagged p300 truncations and HA‑tagged ubiquitin were
coexpressed in HeLa cells which were then treated for 16 hours with
MG132 prior to immunoprecipitation of the truncated forms of
p300 with a M2 antibody. Inhibition of the proteasome‑dependent
protein degradation with MG132 is essential in this assay since
accumulation of ubiquitinated p300 is required for detection of the
ubiquitin conjugates by Western blotting. The blot was first probed
with the M2 antibody to examine the expression of truncated forms
of p300, then stripped and reprobed with antibody specific for the
HA‑tag to detect p300‑ubiquitin conjugates. As shown in (Fig. 6A),
the p300‑M671‑1194 truncation which contains 19 lysine residues is
highly ubiquitinated, but not the p300‑N1‑670 which contains 27
Lysine residues, suggesting that ubiquitination of p300 is regulated
by a domain specific mechanism. In addition, the ubiquitination
96 Epigenetics 2007; Vol. 2 Issue 2
of a M2‑tagged full‑length p300 was also detected in the assay
(Fig. 6C). However, the full‑length p300 protein was barely visible
even after high amount of expression plasmid transfection and
extended Western film exposure in comparison to the truncated
forms of p300, indicating that p300 activity is tightly regulated in
cellular system (Fig. 6C).
The ubiquitination of p300 is important for the cytoplasmic
distribution of p300. In aggresome containing cells, vimentin, a
type III intermediate filaments, is wrapped around the inclusion
body, which is believed to contribute to stability of aggresome and
is a well established marker for the inclusion body.30,34 Next, we
examined the intracellular localization of vimentin and the truncated
forms of p300‑N1‑670 and p300‑M671‑1194 in response to MG132
treatment in the absence or presence of valproic acid. ApoTome
optical sectioning microscopy revealed that the p300‑N1‑670 and
p300‑M671‑1194 stained similar and are both nuclear in untreated
cells (Fig. 7). Interestingly, the p300‑N1‑670 staining pattern did
not change and remained nuclear despite of various treatments
(Fig. 7). However, the p300‑M671‑1194 which is highly ubiquitinated
was stained visibly at the distinct perinuclear region upon the
treatments with MG132 and the perinuclear accumulation of
p300‑M671‑1194 was enhanced by cotreatment with valproic acid
(Fig. 7). In addition, the perinuclear p300‑M671‑1194 localized to
the vimentin region (Fig. 7). Taken together, these data suggest that
p300‑M671‑1194 harbors the specific signals required for aggresome
distribution of p300.
Importantly, nocodazole, a microtubule toxin, was able to disrupt
the localization of p300‑M671‑1194 and vimentin to the distinct
perinuclear region. As shown in (Fig. 7), treatment with nocodazole
alone did not change the staining pattern of p300‑M671‑1194 in
untreated control cells. However, the cytoplasmic p300‑M671‑1194
stained as speckles scattered around the nuclear envelope and
vimentin distributed around perinuclear region as rope‑like filaments
in response to treatment with MG132 and valproic acid in the pres-
ence of nocodazole, (Fig. 7). The effect of nocodazole on the cellular
distribution of p300‑M671‑1194 inclusion was similar to that of
endogenous wild type p300, suggesting a possible link between p300
ubiquitination and formation of cytoplasmic inclusion bodies.
Furthermore, p300DM, a mutant p300 with the potential ubiqui-
tination sites deleted in the context of full length p300, stained as a
nuclear protein regardless of valproic acid and MG132 treatments
and did not localize to the vimentin region (Fig. 8). On the other
hand, the full‑length p300 was detected in the vimentin region upon
inhibition of proteasome‑dependent protein degradation (Fig. 8).
These data again support a link between p300 ubiquitination and
cytoplasmic inclusion body formation. Our study, for the first time,
establishes that p300 is a substrate of the cytoplasmic proteasome
and demonstrates the role of spatial redistribution in the regulation
of p300 function.
Discussion
The main conclusion from this work is that nucleo‑cytoplasmic
shuttling is involved in the control of the availability and activity of
transcriptional coactivator p300 in the nucleus and the cytoplasmic
degradation of p300 is mediated through ubiquitin‑proteasome
pathway. In addition, the cytoplasmic distribution of p300 is
independent of the 26S proteasome activity.
The nuclear level of transcriptional coactivator p300 is tightly
regulated to maintain normal cell proliferation and development.12,21
Effective elimination of p300 activity following histone acetylation
 Spatial Regulation of Transcriptional Coactivator p300
Figure 8. Localization of p300 to the vimentin structure is domain‑dependent.
Cells were transfected with expression plasmids for the full‑length p300
(p300WT) or for a mutant p300 with the ubiquitination domain deleted
(p300DM), and treated with MG132 (MG, 5 mM) and valproic acid (VPA, 2
mM). The cells were then stained with a M2 antibody for detecting the tran‑
siently expressed p300 (green) and an antibody for vimentin (red). Images
were captured by optical sectioning. Shown are representatives of single
0.25 mm optical sections.
and gene activation is essential for cells to ensure precisely controlled
transcription. Generally speaking, treatment of cells with HDAC
inhibitors, such as valproic acid and butyrate, renders histones in
hyperacetylated status, making the cells more transcription active, or
lesser of p300‑dependent.44,45 In response, p300 is shuttled from the
nucleus to the cytoplasm upon the treatments (Fig. 1). The physical
removal of p300 from the nucleus, its site of action, may serve as an
integral mechanism to control the function and availability of the
coactivator in response to cellular challenge, limiting its opportunity
to interact with sequence‑specific transcription factors, to acetylate
histones or transcriptional regulators and to coordinate gene activa-
tion. Interestingly, it has been shown recently that p300 and CBP are
distributed in the cytoplasm of oocytes within primordial follicles and
enter the nucleus during different stages of oocyte growth.46
We also provide evidence that CRM1 pathway is involved
in valproic acid‑induced p300 turnover (Fig. 5). Previously, we
reported that valproic acid induces p300 degradation by augmenting
gene expression of the B56g3 regulatory subunit of PP2A.25 The
PP2A‑mediated p300 turnover may depend on the nuclear proteasome
system since the B56g3 regulatory subunit is a nuclear protein.47,48 In
addition, PI3K inhibitor‑enhanced p300 degradation does not induce
the cytoplasmic distribution of p300.23 Here, we show that cytoplasm
appears to be also a site of p300 degradation, or the fate of cytoplasmic
p300 is indeed for a selective turnover. First, cytoplasmic localization
of p300 is observed prior to the reduction of the coactivator induced
by valproic acid (Figs. 1–4). Second, inhibition of CRM1 activity
prevents effectively the selective p300 turnover (Fig. 5). Collectively,
these results suggest that nucleo‑cytoplasmic shuttling may be required
for the selective p300 turnover through the cytoplasmic proteasome.
However, the cytoplasmic distribution of p300 does not require the
26S proteasome activity since proteasome inhibitor is able to block
valproic acid‑induced p300 degradation,25 but not the cytoplasmic
distribution of the coactivator (Figs. 1–4). The mechanism of this
spatial control of p300 degradation is seemingly different from that of
p27kip1 turnover. The presence of proteasome inhibitor can block both
nuclear export and degradation of p27kip1, an indication that the 26S
proteasome is involved in the regulation of p27kip1 export.49
www.landesbioscience.com Epigenetics 97
Proteins shuttle in and out of nucleus through the nuclear core
complex, which is a highly regulated event to rapidly change the
cellular localization of key signaling components during signal
transduction. Many studies have established transcriptional coacti-
vator p300 as a nuclear protein fitting to its function.23,36 Multiple
nuclear localization signal (NLS) reside in p300 to safeguard its
nuclear localization. Our study suggests that nuclear export may
also be involved in the control of cellular localization and nuclear
activity of p300, which may be mediated by the CRM1 pathway.
The cytoplasmic redistribute of p300 correlates with ubiquitina-
tion and with the p300 domain containing multiple lysine residues
(Figs. 6–8). Modification of these lysine residues by acetylation may
evoke potential nuclear export signals or mechanisms of coupling
p300 to the CRM1 receptor. It is known that treatment of cells with
HDAC inhibitors increase p300 acetylation.50 Therefore, it is most
likely that HDAC inhibitor such as valproic acid may also induce
selective p300 turnover through nucleo‑cytoplasmic shuttling of
p300 or mechanism of p300 acetylation.
Ubiquitination has versatile roles in regulation of cellular activity
as it can activate protein kinases, mediate protein‑protein interaction,
signal nuclear export and mark protein for endocytosis. Nonetheless,
targeting protein for proteasome degradation remains the most
known function for ubiquitination. The 26S proteasome is localized
to both cytoplasmic and nuclear compartments of the cell and is
responsible for the clearance of ubiquitin‑tagged proteins in general.
Our previous study shows that nuclear proteasome system may
participate in the regulation of p300 turnover.23 In this study, we
demonstrate that the cytoplasmic proteasome system is also impor-
tant for valproic acid‑induced p300 turnover.
The formation of p300 aggresome depends on functional micro-
tubule networks and the perinuclear p300 aggregates colocalize
with g‑tubulin, ubiquitin and a subunits of the 20S proteasome
(Figs. 2–4). Aggresome can be formed as the consequence of over-
whelmed proteasome system or of cellular response to mutant or
misfolded proteins.30,32 Apparently, the formation of aggresome is a
cellular protective mechanism to sequester potentially toxic protein
aggregates that are scattered around the intracellular milieu or to
deliver them for disposal through an alternative pathway such as
autophagosome. Many nuclear proteins undergo cytoplasmic degra-
dation as an integral part of normal cellular regulatory process. One
such example is the inducible NO synthase (iNOS), a host defense
protein. It has been reported that cells regulate the levels of iNOS
through aggresome formation.33 We, for the first time, provide
the evidence that p300, a nuclear protein, is also a substrate of the
cytoplasmic ubiquitin‑proteasome and of aggresome.
HDAC6 is a microtubule‑associated deacetylase and plays an
important role in aggresome formation.51,52 Inhibition of HDAC6
activity by HDAC inhibitors such as TSA and SAHA impairs tubulin
acetylation and aggresome formation.51,53 However, valproic acid
does not inhibit the activity of HDAC6 and HDAC10, although it
is able to impede the activity of several other members of class I and
class II HDACs.54 In agreement with these observations, Treatment
of the cells with valproic acid does not affect cellular distribution of
HDAC6 (data not shown). Thus, valproic acid does not impair the
formation of aggresome, rather enhances the deposition of p300 in
the aggresome when the proteasome system is challenged.
In conclusion, our study defines a novel mechanism that
regulates the availability and activity of p300 in the nucleus
and provides evidence for the roles of cellular trafficking and
spatial distribution in the regulation of gene expression through
transcriptional coactivator p300.
 Spatial Regulation of Transcriptional Coactivator p300
Materials and Methods
Plasmids and reagents. Plasmids for HA‑tagged ubiquitin, M2
tagged wild type or mutant forms of p300 have been described
previously.3,5,55,56 Valproic acid, sodium butyrate and MG132
were purchased from sigma. Leptomycin‑B was purchased from
LC laboratories. M2‑conjugated agarose and antibodies against
M2‑tag were from Sigma. Antibodies against p300, SRC, DM, Eckner R. Gene dosage‑dependent embryonic development and proliferation defects
g‑tubulin, vimentin, ubiquitin, a subunits of the 20S proteasome
and HA‑tag were purchased from Santa Cruz. Hoechst and secondary J 1998; 17:7151‑60.
antibodies conjugated with fluorescent dyes were supplied by
Molecular Probes.
Cell culture and western analysis. Cells were maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal
bovine serum (Invitrogen). Cell lysates were prepared first by
suspending cell pellets in whole cell extraction buffer containing 50
mM Tris‑HCl (pH 7.6), 400 mM NaCl, 10% glycerol, 1 mM dithio-
threitol, 1 mM phenylmethylsulfonyl fluoride, and 1% nonidet P‑40
(NP‑40). The suspension was then incubated at 4˚C for 30 minutes
and centrifuged at 14,000 g for ten minutes at 4˚C. Protein concen-
tration was determined by Bradford assay (Bio‑Rad) using bovine
serum albumin as the standard.
Transfection and immunoprecipitation. Transfection was
performed by using ExGen 500 as described previously.23 For immu-
noprecipitation, the NaCl concentration of the whole‑cell extracts
was adjusted to 150 mM and the NP‑40 concentration was adjusted
to 0.1%.25 The extracts were incubated with M2 conjugated agarose
at 4˚C for 2 hours, washed, and subjected to Western blot analysis.
Immunofluorescence optical sectioning microscopy. Cells were
grown on coverslips for minimum of 16 hours before various treat-
ments. At the end of the treatment, the cells were washed with
phosphate buffered saline (PBS), fixed in 4% paraformaldehyde,
permeabilized in 0.2% NP‑40, and blocked in PBS containing
0.2% tween 20 and 10% nonfat milk powder.23 The cells were then
incubated with various primary antibodies at 4˚C for overnight
and with corresponding fluorescence labeled secondary antibodies
for 30 minutes at room temperature. For DNA staining, the cells
were incubated with hoechst (25 ng/ml) for 5 minutes in darkness
before mounted in 90% glycerol. The cells were scanned with a Zeiss
ApoTome system attached to an Axiovert microscope using a 63×
Plan Apochromat oil objective (Carl Zeiss Inc). The Z sections were
collected at an optical depth of 0.25 mm. Images were optimized
by an ApoTome software (Applied Precision). All experiments were
performed at least three times.
References
1. Chan HM, La Thangue NB. p300/CBP proteins: HATs for transcriptional bridges and
scaffolds. J Cell Sci 2001; 114:2363‑73.
2. Brownell JE, Allis CD. Special HATs for special occasions: Linking histone acetylation to
chromatin assembly and gene activation. Curr Opin Genet Dev 1996; 6:176‑84.
3. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y. The transcriptional coacti-
vators p300 and CBP are histone acetyltransferases. Cell 1996; 87:953‑9.
4. Gu W, Roeder RG. Activation of p53 sequence‑specific DNA binding by acetylation of the
p53 C‑terminal domain. Cell 1997; 90:595‑606.
5. Li Q, Herrler M, Landsberger N, Kaludov N, Ogryzko VV, Nakatani Y, Wolffe AP. Xenopus
NF‑Y presets chromatin to potentiate p300 and acetylation‑ responsive transcription from
the Xenopus hsp70 promoter in vivo. EMBO J 1998; 17:6300‑15.
6. Boyes J, Byfield P, Nakatani Y, Ogryzko V. Regulation of activity of the transcription factor
GATA‑1 by acetylation. Nature 1998; 396:594‑8.
7. Li Q, Imhof A, Collingwood TN, Urnov FD, Wolffe AP. p300 stimulates transcription
instigated by ligand‑bound thyroid hormone receptor at a step subsequent to chromatin
disruption. EMBO J 1999; 18:5634‑52.
8. Martinez‑Balbas MA, Bauer UM, Nielsen SJ, Brehm A, Kouzarides T. Regulation of E2F1
activity by acetylation. EMBO J 2000; 19:662‑71.
98 Epigenetics 2007; Vol. 2 Issue 2
9. Chan HM, Krstic‑Demonacos M, Smith L, Demonacos C, La Thangue NB. Acetylation
control of the retinoblastoma tumour‑suppressor protein. Nat Cell Biol 2001; 3:667‑74.
10. Muraoka M, Konishi M, Kikuchi‑Yanoshita R, Tanaka K, Shitara N, Chong JM, Iwama
T, Miyaki M. p300 gene alterations in colorectal and gastric carcinomas. Oncogene 1996;
12:1565‑9.
11. Gayther SA, Batley SJ, Linger L, Bannister A, Thorpe K, Chin SF, Daigo Y, Russell P,
Wilson A, Sowter HM, Delhanty JD, Ponder BA, Kouzarides T, Caldas C. Mutations
truncating the EP300 acetylase in human cancers. Nat Genet 2000; 24:300‑3.
12. Yao TP, Oh SP, Fuchs M, Zhou ND, Ch’ng LE, Newsome D, Bronson RT, Li E, Livingston
in mice lacking the transcriptional integrator p300. Cell 1998; 93:361‑72.
13. Ciechanover A. The ubiquitin‑proteasome pathway: On protein death and cell life. EMBO
14. Lam YA, Lawson TG, Velayutham M, Zweier JL, Pickart CM. A proteasomal ATPase sub-
unit recognizes the polyubiquitin degradation signal. Nature 2002; 416:763‑7.
15. Kleijnen MF, Shih AH, Zhou P, Kumar S, Soccio RE, Kedersha NL, Gill G, Howley PM.
The hPLIC proteins may provide a link between the ubiquitination machinery and the
proteasome. Mol Cell 2000; 6:409‑19.
16. Sherman MY, Goldberg AL. Cellular defenses against unfolded proteins: A cell biologist
thinks about neurodegenerative diseases. Neuron 2001; 29:15‑32.
17. Iwai K, Yamanaka K, Kamura T, Minato N, Conaway RC, Conaway JW, Klausner RD,
Pause A. Identification of the von Hippel‑lindau tumor‑suppressor protein as part of an
active E3 ubiquitin ligase complex. Proc Natl Acad Sci USA 1999; 96:12436‑41.
18. Lam YA, Pickart CM, Alban A, Landon M, Jamieson C, Ramage R, Mayer RJ, Layfield R.
Inhibition of the ubiquitin‑proteasome system in Alzheimer’s disease. Proc Natl Acad Sci
USA 2000; 97:9902‑6.
19. Petersen BO, Wagener C, Marinoni F, Kramer ER, Melixetian M, Denchi EL, Gieffers C,
Matteucci C, Peters JM, Helin K. Cell cycle‑ and cell growth‑regulated proteolysis of mam-
malian CDC6 is dependent on APC‑CDH1. Genes Dev 2000; 14:2330‑43.
20. Kamura T, Sato S, Iwai K, Czyzyk‑Krzeska M, Conaway RC, Conaway JW. Activation of
HIF1alpha ubiquitination by a reconstituted von Hippel‑ Lindau (VHL) tumor suppressor
complex. Proc Natl Acad Sci USA 2000; 97:10430‑5.
21. Brouillard F, Cremisi CE. Concomitant increase of histone acetyltransferase activity and
degradation of p300 during retinoic acid‑induced differentiation of F9 cells. J Biol Chem
2003; 278:39509‑16.
22. Poizat C, Sartorelli V, Chung G, Kloner RA, Kedes L. Proteasome‑mediated degradation of
the coactivator p300 impairs cardiac transcription. Mol Cell Biol 2000; 20:8643‑54.
23. Chen J, Halappanavar SS, St‑Germain JR, Tsang BK, Li Q. Role of Akt/protein kinase B in
the activity of transcriptional coactivator p300. Cell Mol Life Sci 2004; 61:1675‑83.
24. Li Q, Su A, Chen J, Lefebvre YA, Hache RJ. Attenuation of glucocorticoid signaling
through targeted degradation of p300 via the 26S proteasome pathway. Mol Endocrinol
2002; 16:2819‑27.
25. Chen J, St‑Germain JR, Li Q. B56 regulatory subunit of protein phosphatase 2A mediates
valproic acid‑induced p300 degradation. Mol Cell Biol 2005; 25:525‑32.
26. Cooper JK, Schilling G, Peters MF, Herring WJ, Sharp AH, Kaminsky Z, Masone J, Khan
FA, Delanoy M, Borchelt DR, Dawson VL, Dawson TM, Ross CA. Truncated N‑terminal
fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic
aggregates in cell culture. Hum Mol Genet 1998; 7:783‑90.
27. Ross CA, Pickart CM. The ubiquitin‑proteasome pathway in Parkinson’s disease and other
neurodegenerative diseases. Trends Cell Biol 2004; 14:703‑11.
28. Burnett BG, Pittman RN. The polyglutamine neurodegenerative protein ataxin 3 regulates
aggresome formation. Proc Natl Acad Sci USA 2005; 102:4330‑5.
29. Taylor JP, Hardy J, Fischbeck KH. Toxic proteins in neurodegenerative disease. Science
2002; 296:1991‑5.
30. Johnston JA, Ward CL, Kopito RR. Aggresomes: A cellular response to misfolded proteins.
J Cell Biol 1998; 143:1883‑98.
31. Kopito RR. Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 2000;
10:524‑30.
32. Taylor JP, Tanaka F, Robitschek J, Sandoval CM, Taye A, Markovic‑Plese S, Fischbeck KH.
Aggresomes protect cells by enhancing the degradation of toxic polyglutamine‑containing
protein. Hum Mol Genet 2003; 12:749‑57.
33. Kolodziejska KE, Burns AR, Moore RH, Stenoien DL, Eissa NT. Regulation of inducible
nitric oxide synthase by aggresome formation. Proc Natl Acad Sci USA 2005; 102:4854‑9.
34. Garcia‑Mata R, Gao YS, Sztul E. Hassles with taking out the garbage: Aggravating
aggresomes. Traffic 2002; 3:388‑96.
35. O’Keefe K, Li H, Zhang Y. Nucleocytoplasmic shuttling of p53 is essential for
MDM2‑mediated cytoplasmic degradation but not ubiquitination. Mol Cell Biol 2003;
23:6396‑405.
36. Yaciuk P, Moran E. Analysis with specific polyclonal antiserum indicates that the E1A‑ asso-
ciated 300‑kDa product is a stable nuclear phosphoprotein that undergoes cell cycle
phase‑specific modification. Mol Cell Biol 1991; 11:5389‑97.
37. Chen J, Ghazawi FM, Bakkar W, Li Q. Valproic acid and butyrate induce apoptosis in
human cancer cells through inhibition of gene expression of Akt/protein kinase B. Mol
Cancer 2006; 5:71.
38. Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol
3‑kinase, 2‑(4‑ morpholinyl)‑8‑phenyl‑4H‑1‑benzopyran‑4‑one (LY294002). J Biol Chem
1994; 269:5241‑8.
 Spatial Regulation of Transcriptional Coactivator p300

39. Lee DH, Goldberg AL. Proteasome inhibitors: Valuable new tools for cell biologists. Trends
Cell Biol 1998; 8:397‑403.
40. Wigley WC, Fabunmi RP, Lee MG, Marino CR, Muallem S, DeMartino GN, Thomas
PJ. Dynamic association of proteasomal machinery with the centrosome. J Cell Biol 1999;
145:481‑90.
41. Wojcik C, Yano M, DeMartino GN. RNA interference of valosin‑containing protein (VCP/
p97) reveals multiple cellular roles linked to ubiquitin/proteasome‑dependent proteolysis. J
Cell Sci 2004; 117:281‑92.
42. Stommel JM, Marchenko ND, Jimenez GS, Moll UM, Hope TJ, Wahl GM. A leucine‑rich
nuclear export signal in the p53 tetramerization domain: Regulation of subcellular localiza-
tion and p53 activity by NES masking. EMBO J 1999; 18:1660‑72.
43. Kudo N, Matsumori N, Taoka H, Fujiwara D, Schreiner EP, Wolff B, Yoshida M,
Horinouchi S. Leptomycin B inactivates CRM1/exportin 1 by covalent modification
at a cysteine residue in the central conserved region. Proc Natl Acad Sci USA 1999;
96:9112‑7.
44. Wolffe AP, Collingwood TN, Li Q, Yee J, Urnov F, Shi YB. Thyroid hormone receptor,
v‑ErbA, and chromatin. Vitam Horm 2000; 58:449‑92.
45. Li Q, Sachs L, Shi YB, Wolffe AP. Modification of chromatin structure by the thyroid
hormone receptor. Trends Endocrinol Metab 1999; 10:157‑64.
46. Kwok RP, Liu XT, Smith GD. Distribution of coactivators CBP and p300 during mouse
oocyte and embryo development. Mol Reprod Dev 2006; 73:885‑94.
47. McCright B, Rivers AM, Audlin S, Virshup DM. The B56 family of protein phosphatase
2A (PP2A) regulatory subunits encodes differentiation‑induced phosphoproteins that target
PP2A to both nucleus and cytoplasm. J Biol Chem 1996; 271:22081‑9.
48. Tehrani MA, Mumby MC, Kamibayashi C. Identification of a novel protein phosphatase
2A regulatory subunit highly expressed in muscle. J Biol Chem 1996; 271:5164‑70.
49. Tomoda K, Kubota Y, Kato J. Degradation of the cyclin‑dependent‑kinase inhibitor
p27Kip1 is instigated by Jab1. Nature 1999; 398:160‑5.
50. Thompson PR, Wang D, Wang L, Fulco M, Pediconi N, Zhang D, An W, Ge Q, Roeder
RG, Wong J, Levrero M, Sartorelli V, Cotter RJ, Cole PA. Regulation of the p300 HAT
domain via a novel activation loop. Nat Struct Mol Biol 2004; 11:308‑15.
51. Kawaguchi Y, Kovacs JJ, McLaurin A, Vance JM, Ito A, Yao TP. The deacetylase HDAC6
regulates aggresome formation and cell viability in response to misfolded protein stress. Cell
2003; 115:727‑38.
52. Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, Yoshida M, Wang XF, Yao
TP. HDAC6 is a microtubule‑associated deacetylase. Nature 2002; 417:455‑8.
53. Koeller KM, Haggarty SJ, Perkins BD, Leykin I, Wong JC, Kao MC, Schreiber SL.
Chemical genetic modifier screens: Small molecule trichostatin suppressors as probes of
intracellular histone and tubulin acetylation. Chem Biol 2003; 10:397‑10.
54. Gurvich N, Tsygankova OM, Meinkoth JL, Klein PS. Histone deacetylase is a target of
valproic acid‑mediated cellular differentiation. Cancer Res 2004; 64:1079‑86.
55. Suganuma T, Kawabata M, Ohshima T, Ikeda MA. ����������������������������
Growth suppression of human
carcinoma cells by reintroduction of the p300 coactivator. Proc Natl Acad Sci USA 2002;
99:13073‑8.
56. Treier M, Staszewski LM, Bohmann D. Ubiquitin‑dependent c‑Jun degradation in vivo is
mediated by the delta domain. Cell 1994; 78:787‑98.

www.landesbioscience.com Epigenetics 99