From the Department of Pharmacology and Cancer
Center, Case Western Reserve University School of Medicine, Cleveland,
Ohio 44106 and the § Department of Biochemistry and Cancer
Center, Case Western Reserve University, School of Medicine
Cleveland, Ohio 44106
Received for publication, October 22, 2002, and in revised form, January 13, 2003
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ABSTRACT |
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Tip60 (Tat-interactive
protein, 60 kDa), a cellular protein with
intrinsic histone acetyltransferase activity, is involved in DNA
damage repair and apoptosis. Recent studies have suggested that Tip60
acts either as a co-activator or a co-repressor to modulate
transcription. In this study, we demonstrate that Tip60 represses
reporter gene expression when it is fused to the Gal4 DNA binding
domain. We also show that Tip60 associates with histone deacetylase 7 (HDAC7) through its N-terminal zinc finger-containing region and that
HDAC7 activity is required for the repressive effect of Tip60. Because
endogenous Tip60 interacts with STAT3, we hypothesized that Tip60 might
complex with STAT3 and HDAC7 and modulate STAT3-mediated
trans-activation. Consistent with this hypothesis, the overexpression
of Tip60 represses STAT3-driven reporter gene expression, which can be
further potentiated by the co-transfection of HDAC7. Furthermore,
interleukin-9-induced c-myc expression, which depends on
STAT3 activity, is abrogated by exogenous expression of Tip60. This is
the first demonstration of which Tip60 represses STAT3 activity in part
through the recruitment of HDAC7.
Tip601
(Tat-interactive protein,
60 kDa) is a member of the MYST family of proteins,
which are highly conserved from yeast to human and play diverse
physiological functions (1). Several members among this family, such as
SAS3 (Something About Silencing), Esa1 (Essential sas2-related
acetyltransferase), and Tip60, possess intrinsic histone
acetyltransferase (HAT) activity (2-4), suggesting their potential
roles in chromatin remodeling and gene regulation. Tip60 is expressed
in a variety of tissues and cell lines, and its homologues have been
identified in chicken, mouse, and human (5-7). Tip60 is mainly
localized in the nucleus; however, cytoplasmic and perinuclear
localization has been reported previously (4, 8-11). Tip60 forms
stable nuclear complexes, which possess ATPase and DNA helicase
activities, that promote histone acetylation in nucleosomes (12). It
associates with transcriptional activators, such as HIV-1 Tat, type I
nuclear hormone receptors, and APP ( Eucaryotic genomic DNA is packaged with histones into nucleosomes,
which are the primary structural units of chromatin. The packaging of
DNA into chromatin inhibits transcription in part by hindering the
binding of transcription factors and basal transcriptional machinery.
Many transcriptional co-activators possess intrinsic HAT activity that
provides a link between histone acetylation and transcriptional
regulation (17). The identification and characterization of histone
deacetylases provide support for the view that reversible acetylation
of histones plays an important role in gene regulation (18). Mammalian
class I HDACs composed of HDAC1-3, HDAC8, and HDAC11 are highly
homologous to the yeast gene, Rpd3 (19, 20). This class of
deacetylases is present in multisubunit complexes such as Sin3/HDAC
(21) and NuRD/Mi2/NRD (22) as well as SMRT- and nuclear
receptor co-repressor-containing complexes (23, 24). Class II HDACs,
including HDAC4-7 and HDAC9/10, are homologous to the yeast gene,
Hda1 (20). Class II HDACs have been implicated in gene
regulation through association with co-repressors such as SMRT
(silencing mediator for retinoic acid and thyroid hormone receptor),
nuclear receptor co-repressor, BCoR, and transcription factors of the
MEF2 and POK family members (25-29).
Many cytokines, hormones, and growth factors utilize STAT signaling
pathways to induce diverse biological responses including development,
cell proliferation, differentiation, and survival (30). STAT3 is a STAT
family member involved in normal cellular responses and oncogenesis
(31, 32). Through the induction of genes involved in cell cycle
progression, apoptosis, and cell motility, STAT3 plays a crucial
role in mediating the physiological effects of many cytokines. STAT3
null mice die early in embryogenesis prior to gastrulation, possibly
because of deficient leukemia inhibitory factor signaling (33).
Tissue-specific STAT3 knock-out studies suggest a role for STAT3 in
IL-10-mediated anti-inflammatory responses, growth factor-mediated
migration of epidermal cells, and IL-6-mediated effects on T cells
(31). We and others (34, 35) demonstrated that STAT3 acts in synergy
with insulin receptor substrate-1/2 signaling to induce the
proliferative and anti-apoptotic effects of IL-9. In addition, STAT3
has been implicated in tumor initiation and progression. Constitutive
activation of STAT3 is observed in a wide variety of human cancers, and
blockade of constitutive STAT3 signaling results in growth inhibition
and apoptosis of tumor cells (36). Furthermore, a constitutively active
mutant form of STAT3 induces oncogenic transformation of NIH 3T3 cells (37).
Considering the importance of STAT3 in transmitting the biological
effects of extracellular stimuli and the potential relevance of STAT3
to oncogenesis, it is not surprising that many interacting partners may
regulate STAT3 activity. Co-activators CBP/p300 and NCoA/SRC1a,
which possess intrinsic HAT activity, interact with STAT3 and enhance
its transcriptional activity (38, 39). Transcriptional activators such
as c-Jun, Sp1, and GR associate with and act in synergy with
STAT3 to activate gene expression (40). Tyrosine phosphatases and
suppressor of cytokine signaling #3 negatively regulate STAT3
activation (41). PIAS3 (protein inhibitor of activated STAT3), a
protein that specifically binds to activated STAT3, inhibits
STAT3 transcriptional activity by reducing DNA binding (42). Therefore,
studies of STAT3 regulatory mechanisms will not only further our
understanding of how STAT3 exerts its physiological functions but may
provide treatments for cancers in which STAT3 activity is dysregulated.
In this study, we demonstrate that Tip60 associates with STAT3. Tip60
acts as a co-repressor for STAT3 to regulate gene expression in
cytokine signaling and exerts a repressive effect on gene expression in
part through the recruitment of histone deacetylase 7. Thus, this study
provides a novel mechanism involved in the regulation of STAT3 activity.
Reagents and Antibodies--
Anti-Myc (9E10) and
anti-phospho-tyrosine (PY99) were from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA). Anti-FLAG (M2) and anti-phopho-STAT3 (Y704) were
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Anti-STAT3 antibody was from R&D Systems, Inc. (Minneapolis, MN).
Anti-HA antibody was from BAbCo (Richmond, CA). Tricostatin A (TSA) was
from Sigma. Murine IL-9 and IL-6 were from R&D Systems Inc.
Plasmid Construction--
Tip60 N- and C-terminal deletion
mutants were generated by PCR and confirmed by sequencing. The 5'
primer used for C-terminal deletion mutants is
5'-ATGGACATCAGTGGCCGG-3'. For N-terminal deletion mutant N1, the 5'
primer is 5'-TCCCCGTACCCACAG-3'. The 3' primers used for Tip60 deletion
mutants are 5'-CACGGCTTGAGGCG-3' (for Tip60C3);
5'-ATTGTAGTCTTCCGTTG-3' (for Tip60C2); 5'-TTGATGCTGGTGAT-3' (for Tip60C1); and 5'-AGTGTCTGGTCACC-3' (for
Tip60N1). Tip60 deletion mutants were cloned into
pFLAG-CMV2 vector at EcoRI and BamHI sites.
Tip60HAT Cell Culture and Transfection--
Human embryonic kidney (HEK)
293 cells and hepatoma HepG2 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum (FBS).
Transient transfection was carried out by calcium phosphate
co-precipitation. T lymphoma TS1 cells were maintained in Click's
medium (Irvine Scientific, Santa Ana, CA) supplemented with 10% FBS
and murine IL-9 (0.1 ng/ml). Electroporation was performed to establish
stable cell lines expressing wild-type or mutant forms of Tip60 as
described previously (43).
Cytokine Stimulation, Immunoprecipitation, and
Immunoblotting--
TS1 cells were serum-starved in Dulbecco's
modified Eagle's medium in the absence of IL-9 for 2-6 h. Cells
(2×107/ml) were then stimulated with IL-9 (50 ng/ml) at
37 °C and lysed in 1 ml of TNE lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5% glycerol,
5 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 2 mM
sodium vanadate) containing 1% Nonidet P-40 for immunoprecipitation or
0.5% Nonidet P-40 for co-immunoprecipitation. Solubilized proteins
were collected for immunoprecipitation and immunoblotting according to
procedures described previously (44). All of the experiments were
repeated at least twice with similar results.
Chloramphenicol Acetyltransferase (CAT) Assay--
HepG2 cells
were transfected with 0.5 µg of pCMV-HA-STAT3, 2 µg of p4×SIE CAT
(45) (or pCAT as a negative control), 0.2 µg of
pCMV- Tip60 Possesses Transcriptional Repression Activity--
Tip60
possesses intrinsic HAT activity and enhances the activity of
transcriptional activators, supporting its role as a co-activator (7,
13, 14). However, some studies argue that Tip60 may act as a
co-repressor for certain transcriptional regulators (15, 16, 46). In
addition, yeast homologues of Tip60, SAS2, and SAS3 are involved in
gene silencing (47, 48). Here, transient transfection assays were
performed to test whether Tip60 can repress basal transcription in HEK
293 cells. In these experiments, we used pMH100-TK-LUC, which contains
a luciferase gene driven by a thymidine kinase promoter with four
copies of Gal4 binding site as a reporter plasmid (49). When pM-Tip60,
which expresses Tip60 in-frame with the Gal4 DNA-binding domain (DBD),
was co-transfected into HEK 293 cells with pMH100-TK-LUC, Tip60
repressed reporter gene expression in a dose-dependent
manner (Fig. 1A).
Overexpression of Gal4DBD-Tip60HAT
Tip60 acts as a co-activator for Tat in HeLa cells but as a
co-repressor for Tat in Jurkat cells (16). To examine the repressive effect of Tip60 in other cell lines, we co-transfected pM, pMTip60, or
pMTip60HAT Histone Deacetylase 7 Potentiates the Repressive Effect of
Tip60--
One of the mechanisms by which repressors or co-repressors
regulate gene expression is through the recruitment of histone deacetylases (18). To test whether Tip60 can recruit other repressive molecules such as HDACs to regulate gene expression, we included the
class I histone deacetylase member, HDAC1, and class II histone deacetylase member, HDAC7, in transient transfection assays.
Co-transfection of pCMX-HA-HDAC7 together with pMTip60 greatly reduced
reporter gene expression (Fig.
2A, columns 9-12)
compared with co-transfection of pCMX-HA-HDAC7 with pM (Fig.
2A, column 8). Thus, HDAC7 potentiates the
repressive effect of Tip60 in a dose-dependent manner.
Unlike HDAC7, HDAC1 did not increase the repressive effect of Tip60
(Fig. 2A, columns 3-7), indicating that HDAC1
and HDAC7 have distinct roles in the regulation of Tip60-mediated
transcriptional repression.
To test whether histone deacetylase activity is required for HDAC7 to
potentiate the repressive effect of Tip60, we treated transfected cells
with TSA, an inhibitor of class I and II histone deacetylases, for
12 h before harvesting cells for luciferase assays. As shown in
Fig. 2B, TSA abrogated the ability of HDAC7 to enhance
Tip60-mediated repression (Fig. 2B, columns
7-10). As in HEK 293 cells, overexpression of HDAC7 enhanced the
repressive activity of Tip60 in DF1 cells (Fig. 2C).
Tip60 Interacts with HDAC7--
To test whether Tip60 and HDAC7
complex in cells, pCMX-HA-HDAC7 and pCMV2-FLAG-Tip60 were
co-transfected or transfected together with an empty vector into HEK
293 cells. The lysates of transfected cells were immunoprecipitated
with anti-HA or anti-FLAG antibodies. Immunoblots probed with anti-HA
indicated that immunoprecipitation with anti-FLAG pulled down HA-tagged
HDAC7 only when pCMV2-FLAG-Tip60 but not empty vector was present (Fig.
3A). By probing blots of anti-HA immunoprecipitates with anti-FLAG, it was also demonstrated that Tip60 and HDAC7 formed a complex (Fig. 3A).
To define the regions in Tip60 essential for association with HDAC7,
Tip60 deletion mutants were generated and cloned into the pCMV2-FLAG
vector (Fig. 3B). Plasmids encoding wild-type or mutant
Tip60 were co-transfected into HEK 293 cells together with pCMX-HA-HDAC7. Anti-HA immunoprecipitates and whole cell lysates were
resolved by SDS-PAGE, and immunoblots were probed first with anti-FLAG
and then with anti-HA. Although the C-terminal deletion mutants C1
(amino acids 1-451) and C2 (amino acids 1-366) and the N-terminal
deletion mutant N1 (amino acids 261-513) co-immunoprecipitated with
HDAC7, the C-terminal deletion mutant C3 (amino acids 1-255) did not
associate with HDAC7 (Fig. 3C). Because Tip60C3
is well expressed in whole cell lysates, we conclude that the region
between amino acids 261 and 366 that contains a zinc finger motif is
essential for Tip60 association with HDAC7. To map the interaction
region for Tip60 in HDAC7, we performed mammalian two-hybrid assays. They showed that amino acids 241-533 in HDAC7 are essential for Tip60
association (Fig. 3D).
Tip60 Interacts with STAT3--
We previously showed that Tip60
binds to the IL-9R
To further characterize the Tip60-STAT3 interaction following cytokine
stimulation, we generated an IL-9-dependent TS1 cell line
that constitutively expresses Myc-tagged Tip60. Tip60-STAT3 complexes
were in anti-Myc or anti-STAT3 immunoprecipitates (Fig. 4E,
lane 3 or 4), further demonstrating that Tip60
complexes with STAT3 in cells. To test whether the Tip60-STAT3
interaction in TS1 cells is induced by IL-9, we examined their
co-immunoprecipitation in IL-9-stimulated or unstimulated cells
following serum starvation. The Tip60-STAT3 interaction is constitutive
and unaffected by IL-9 induction (Fig. 4E, lanes
1 and 2). Because serum starvation promotes degradation
of Tip60 (data not shown), a much lower level of Tip60 was recovered
from serum-starved cells (Fig. 4E, lanes 1,
2, 5, and 6) than unstarved cells
(Fig. 4E, lanes 3 and 7). Endogenous
STAT3·Tip60 complexes were also detected in anti-Tip60 immunoprecipitates from IL-9-stimulated or unstimulated TS1 cells, further supporting the finding that their association is not
phosphorylation-dependent (Fig. 4F).
Tip60 Represses the Transcriptional Activity of STAT3--
To test
whether Tip60 regulates STAT3 activity, a CAT reporter plasmid
containing four tandem repeats of the STAT3 binding site (SIE) was
transfected into HepG2 cells together with plasmids expressing
Tip60 Represses STAT3-mediated Primary Response Gene Expression
following IL-9 Stimulation--
Because Tip60 regulates STAT3-driven
reporter expression (Fig. 5), we tested whether Tip60 also regulates
STAT3-mediated expression of primary response genes induced by IL-9. We
established TS1 stable cell lines, which express Myc-tagged wild- type
Tip60, Tip60HAT In this study, we provide strong evidence that Tip60 acts as a
co-repressor for basal and transcriptional activator-mediated gene
expression, in part through the recruitment of HDAC7. The finding
supports the notion that Tip60 and other MYST members may repress gene
expression and provides a possible mechanism for the repressive effect
of Tip60 on STAT3-mediated activation following cytokine stimulation.
Recent studies have shown that Tip60 and HBO1, another MYST family
member, regulate gene expression by acting as co-repressors (15, 16,
46). Because Tip60 possesses intrinsic HAT activity, it has been
difficult to explain how Tip60 exerts its repressive function. Several
studies have made it possible to begin to understand the co-repressor
role of Tip60. First, although recombinant Tip60 acetylates histone
subunits H2A, H3, and H4 in vitro using purified histones as
substrates, the HAT activity of Tip60 is much weaker than that of p300
in the same assay (4). Second, a purified Tip60-containing nuclear
multisubunit complex but not purified Tip60 alone promotes histone
acetylation in nucleosomes (12), suggesting that Tip60 acetylates
histones in chromatin when complexed with other accessory proteins such as TAP54 Our study shows that Tip60 interacts with HADC7 in cells, and
co-transfection of HDAC7 potentiates the repressive effect of Tip60 in
Gal4 DBD or STAT3 reporter assays. This raises the possibility that
Tip60 may repress gene expression via complexes with HDAC7. Through
association with transcriptional factors involved in diverse physiological functions in vivo, Tip60 may direct HDAC7 to
modulate a subset of genes regulated by Tip60-associated transcription factors. Unlike class I HDACs, HDAC7 expression is tissue-specific (25,
54) and its localization is cell type-specific and signal-regulated (28, 54). These properties of HDAC7 may explain the cell type-specific effect of Tip60 on gene expression. Our study reveals that Tip60 possesses modest repressive activity in HEK 293 and HepG2 cells but
potently represses transcription in DF1 cells. This suggests that the
expression level and/or the localization of HDAC7 may be different in
these cells and may explain the different effects of Tip60 on
transcription in different cells (16, 46). In addition, the expression
level and localization of Tip60 can be regulated by extracellular
signals (9, 11, 15). In cell lines that poorly express HDAC7 or in
which HDAC7 is cytoplasmic, Tip60 would tend to form complexes with
other partners, which may result in positive regulation of gene
expression. However, if the Tip60·HDAC7 complexes predominate over
others, Tip60 may repress gene expression. Interestingly, the zinc
finger region responsible for association with HDAC7 is highly
conserved within MYST family members, indicating that similar
mechanisms may mediate the repressive effect of other MYST family members.
In this study, we identified Tip60 as a STAT3-interacting protein. We
show that Tip60 interacts with STAT3 in a phosphorylation-independent manner. Non-phosphorylated STAT3 is predominantly located in the cytoplasm. After phosphorylation by activated Jaks and other kinases, STAT3 forms dimers and translocates into nucleus (55). Tip60 is in the
cytoplasm and nucleus in TS1 cells, and IL-9 stimulation induces the
nuclear translocation of
Tip60.2 This suggests
that most Tip60·STAT3 complexes are in the cytoplasm prior to
cytokine induction but are nuclear following stimulation. Tip60 exerts
its repressive effect in a STAT3 reporter assay (Fig. 5A)
and STAT3-mediated gene expression following IL-9 stimulation (Fig.
6B). Although overexpression of Tip60 only produces a 2-fold repression of the transcriptional activity of STAT3 in HepG2 cells, co-transfection of HDAC7 greatly enhances repression mediated by Tip60
(Fig. 5B). STAT3 and HDAC7 associate with the C terminus (Fig. 4A) and the central region (Fig. 3C) of
Tip60, respectively, raising the possibility that Tip60 may recruit
HDAC7 to STAT3 proteins. Therefore, Tip60 inhibits STAT3 activity
through a mechanism distinct from that used by PIAS3, which inhibits
STAT3 activity by reducing its DNA binding (42).
Consistent with the essential role of co-activators with HAT activity
in transcription regulation, CBP/p300 and NCoA/SRC1a also play
essential roles in STAT3-mediated gene expression (38, 39).
Furthermore, the HAT activity of CBP/p300 is required for STAT3
activation (56). Interestingly, a STAT-interacting protein, Nmi,
enhances STAT activity by increasing the association of p300 with STATs
(57), supporting the essential role of p300 in regulating STAT
activity. TSA treatment dramatically increases STAT3 activity induced
by IL-6, indicating that HDACs are involved in regulation of STAT3
activity (56). Thus, HDAC7 recruited via Tip60 provides an efficient
way to directly antagonize STAT3 activity conferred by the HAT activity
of p300 or NCoA, probably through deacetylation of histones. However,
it is also possible that Tip60-HDAC7 regulates the reversible
acetylation of proteins other than histones. Tip60 and HDAC7 interact
with endothelin-A in response to endothelin-1 induction (11),
and it will be interesting to test whether endothelin-A is a substrate
for these two proteins.
Although our study suggests that Tip60 represses gene expression mainly
through the recruitment of HDAC7, we cannot exclude the possibility
that other mechanisms are involved. Because the HAT activity of Tip60
is partially required for Tip60 to mediate basal and STAT3-mediated
gene expression (Figs. 1C and 6C), Tip60 may
acetylate histones and/or other cellular proteins to negatively regulate gene expression. SAS2, a yeast homologue of Tip60, acetylates H4-Lys-16 in gene silencing (58, 59). Chameau, a new Drosophila member
of the MYST family, is required for the maintenance of Hox
gene silencing and can partially substitute the SAS2 HAT in yeast (60).
It is probable that the HAT of Tip60 plays a role in repressing gene
expression through acetylation of histones. Because Tip60 promotes the
acetylation of androgen receptor to regulate its transcriptional
activity (53), it is also possible that Tip60 may acetylate
transcription factors such as STAT3 to negatively regulate their
transcriptional activity.
Our data suggest that Tip60 negatively regulates STAT3 activity,
possibly in concert with PIAS3 and SOCS3. Dysregulation of STAT3
activity is involved in tumor development, and many studies are aimed
at effective therapeutics to attenuate the signaling pathways involved.
Whether dysregulated expression of Tip60 or HDAC7 is involved in
abnormal activation of STAT3 in cancer has not yet been investigated.
Our finding that a Tip60 C-terminal mutant loses the ability to repress
STAT3 activity suggests that loss-of-function mutants of Tip60 may
contribute to the development of disease states. Further studies will
help in the development of Tip60 as a target for the treatment of
various diseases including cancer.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Amyloid Precursor Protein), to activate gene expression
(7, 13, 14). Tip60 has also been implicated in the negative regulation
of gene expression through binding to CREB or the transcriptional
repressor ZEB (Zinc Finger E
Box-binding Protein) (15, 16). Interestingly, Tip60
interacts with membrane receptors for IL-9 and endothelin (10, 11),
suggesting its involvement in signal transduction in response to
extracellular stimuli. Cytosolic phospholipase A2-interacting
protein, a differentially spliced form of Tip60, interacts with
cytosolic phospholipase A2 to enhance cytosolic phospholipase
A2-mediated cell death and prostaglandin E2 production (9).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was obtained by PCR using pcDNA3.1 plasmid
containing Tip60HAT
(15) as the template and cloned into
pMT2 vector with a Myc tag at the EcoRI site.
-galactosidase in combination with different amounts of
plasmids encoding wild-type or mutant Tip60, or pCMX-HA-HDAC7. Following 24 h of transfection, medium containing DNA-calcium phosphate precipitates was replaced by fresh medium and cells continued
to grow for 24 h. Cells were washed twice with phosphate-buffered saline and then incubated in FBS-deprived medium with or without IL-6
(10 ng/ml). After 16 h, cells were harvested and the CAT assay was
performed at 37 °C for 1 h in a 125-µl reaction containing 5 µl of n-butyryl-CoA (5 mg/ml) and 3 µl of
D-threo-[dichloroacetyl-1-14C]chloramphenicol
(CFA754, 54 mCi/mmol, 25 µCi/ml, Amersham Biosciences). Reactions
were stopped by the addition of 300 µl of TMPD-xylene (2:1)
and vortexed vigorously for 30 s. After centrifugation for 5 min,
10 µl of upper phase solution from each sample was taken for
scintillation counting. The
-galactosidase assay was performed to
normalize the CAT activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, a Tip60 HAT-deficient
mutant with substitutions of two amino acids in the acetyl-CoA motif
(15), also modestly repressed transcriptional activity (Fig.
1A).
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Fig. 1.
Tip60 represses basal transcription.
Repressive effect of wild-type or mutant Tip60 in HEK 293 (A), HepG2 (B), or DF1 (C) cells.
Various combinations of plasmids were co-transfected into HEK 293, HepG2, or DF1 cells in 6-well plates by calcium-phosphate method.
Transfection mixture was replaced with fresh medium after transfection
for 12 h. Following transfection for 36-48 h, cell lysates were
prepared and the luciferase assay was performed with dual-luciferase
assay kit (Promega) according to the manufacturer's manual.
RLU, relative luciferase units.
together with pMH100-TK-LUC into HepG2 or DF1
cells. As shown in Fig. 1B, wild-type and HAT-Tip60
repressed transcription in HepG2 cells. Tip60 repressed reporter gene
expression at a greater extent (up to 8-fold) in DF1 cells (Fig.
1C, columns 2-5), suggesting that the repressive
effect of Tip60 is cell type-specific. In DF1 cells, the repressive
effect of HAT-Tip60 was less than that of wild-type Tip60 (Fig.
1C, columns 6-9).
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Fig. 2.
HDAC7 potentiates the repressive effect of
Tip60. A, co-transfection of HDAC7 but not HDAC1
enhances the repressive effect of Tip60 in HEK 293 cells. pCMX-HA-HDAC7
or pCMX-HA-HDAC1 (0.1-1.2 µg) was co-transfected with
pM-Tip60 (0.4 µg) or empty vector into HEK 293 cells in
6-well plates by calcium-phosphate method. Total DNA amount for each
transfection was kept the same by adjusting the amount of empty vector.
B, TSA abrogates HDAC7-mediated repressive effect on Tip60.
Following transfection for 24 h, TSA was added to transfected
cells and incubated overnight before harvest for luciferase assays.
C, HDAC7 modestly potentiates the repressive effect of Tip60
in DF1 cells. RLU, relative luciferase units.
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Fig. 3.
Tip60 interacts with HDAC7.
A, co-immunoprecipitation of Tip60 and HDAC7 in HEK-293
cells. 5 µg of plasmids pFLAG-CMV2-Tip60 and pCMV-HA-HDAC7 was
co-transfected or transfected alone into HEK 293 cells, and cell
lysates were immunoprecipitated with anti-FLAG (2 µl) or anti-HA (5 µl). Immunoblotting was performed with anti-FLAG or anti-HA.
B, schematic presentation of FLAG-tagged Tip60 deletion
mutants. C, pCMV-HA-HDAC7 (5 µg) was co-transfected with
FLAG-tagged wild-type or various deletion mutants of Tip60 in
pFLAG-CMV2 (5 µg) or empty vector into HEK 293 cells by
calcium-phosphate method. Transfected cells were incubated for 36-48 h
before harvest, and cell lysates were subjected to immunoprecipitation
with 5 µl of anti-HA. After extensive wash, immunoprecipitated
proteins and whole cell lysates were separated by 10% SDS-PAGE. After
proteins were transferred onto a PVDF membrane and immunoblotted with
anti-FLAG, the membrane was stripped and re-blotted with anti-HA.
D, interaction of Tip60 with HDAC7 mutants in mammalian
two-hybrid assay. pM-Tip60 (0.1 µg) was co-transfected
with various HDAC7 deletion mutants in pVP16 (0.1 µg) or pVP16 into
HEK 293 cells in 24-well plates. A firefly luciferase gene driven by
four tandem Gal4 binding sites and a TATA motif was used as a reporter.
Following transfection for 36-48 h, cell lysates were prepared and the
luciferase assay was conducted by dual luciferase assay kit. Firefly
luciferase activity was normalized with Renilla luciferase activity.
RLU, relative luciferase units.
-chain, suggesting that Tip60 may be involved in
IL-9 signaling. STAT3 plays an essential role in IL-9-induced
expression of primary response genes such as c-Myc and
Cited2 (43, 50). To explore the possibility that Tip60 may
regulate STAT3 activity, we examined the association between proteins
by immunoprecipitation. pCMV2-FLAG-Tip60 was co-transfected into HEK
293 cells with an empty vector or a plasmid encoding Myc-tagged STAT3.
Immunoprecipitation with anti-Myc followed by immunoblotting with
anti-FLAG showed that Tip60 associates with STAT3 (Fig.
4A). Although
Tip60N1-(261-513) interacts with STAT3,
Tip60C1-(1-451) does not (Fig. 4A). This indicates that the C-terminal region of Tip60 (amino acids 452-513) is
necessary for STAT3 association. To map the Tip60-interacting domain in
STAT3, we co-transfected plasmids expressing FLAG-Tip60 and Myc-tagged
wild-type or mutant STAT3 (Fig. 4B) into HEK 293 cells. Fig.
4, C and D, shows that the central region of
STAT3, which contains the DNA binding domain, is necessary and
sufficient for Tip60 association.
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Fig. 4.
Tip60 interacts with STAT3.
A, Tip60 and STAT3 co-immunoprecipitate in HEK 293 cells.
pcDNA-Myc-STAT3 (5 µg) was co-transfected with 5 µg of
pFLAG-CMV2-Tip60, pFLAG-CMV2-Tip60C1,
pFLAG-CMV2-Tip60N1, or empty vector into HEK 293 cells.
Cell lysates were immunoprecipitated with 20 µl of anti-Myc and
immunoblotted with anti-FLAG or anti-Myc. B, schematic
illustration of Myc-tagged STAT3 mutants. C and
D, interaction of Tip60 with STAT3 mutants in HEK 293 cells.
pCMV-FLAG-Tip60 (5 µg) was co-transfected with various STAT3 mutants
in pcDNA-Myc (5 µg) into HEK 293 cells. Cell lysates were
immunoprecipitated with anti-Myc (20 µl) and immunoblotted with
anti-FLAG or anti-Myc. E, 2 × 107 log-phase
TS1/Myc-Tip60 cells were harvested and lysed before or after starvation
for 3 h followed by IL-9 (50 ng/ml) stimulation for 30 min. Cell
lysates were immunoprecipitated with anti-Myc (20 µl) or anti-STAT3
(5 µl). Immunoblots were probed with anti-STAT3 or anti-Myc.
NT, normal treatment. F, TS1 cells (1 × 108) were deprived of serum and IL-9 for 3 h prior to
stimulation by IL-9 (50 ng/ml) for 15 min. Cells were harvested, and
cell lysates were immunoprecipitated with 5 µl of anti-Tip60.
Immunoblots were probed with anti-phosphotyrosine, anti-STAT3, and
anti-Tip60, sequentially.
-galactosidase, STAT3, and wild-type or mutant Tip60. Cell lysates
from IL-6-stimulated or unstimulated cells were used for the CAT assay,
which was normalized for
-galactosidase activity. STAT3-mediated
reporter gene expression was induced ~10-fold by IL-6. Overexpression
of wild-type Tip60 attenuated the transcriptional activity of STAT3 in
a dose-dependent manner, whereas the overexpression of
Tip60C1, which does not interact with STAT3 (Fig.
4A), enhanced STAT3 activity (Fig.
5A). To further characterize
the role of HDAC7 in Tip60-mediated repression of STAT3 transcriptional
activity, we included pCMX-HDAC7 along with pCMV2-FLAG-Tip60 in these
experiments and found that HDAC7 potentiates the repressive effect of
Tip60 on STAT3-responsive reporter activity (Fig. 5B). In
contrast, the overexpression of Tip60 could not repress the
transcriptional activity of p53 (Fig. 5C) or Smad3 (data not
shown), suggesting that Tip60 functions as a specific co-repressor for
STAT3 through the recruitment of HDAC7.
View larger version (31K):
[in a new window]
Fig. 5.
Tip60 acts as a co-repressor of STAT3 through
the recruitment of HDAC7. A, Tip60 represses
STAT3-driven reporter expression. B, co-transfection of
HDAC7 potentiates the repressive effect of Tip60 on STAT3 activity.
Various combinations of plasmids were co-transfected into HepG2 cells
in 6-well plates, and the CAT assay was performed as described under
"Materials and Methods." C, overexpression of Tip60 does
not affect the transcriptional activity of p53. pG13-Luc (2 µg), a
p53 specific reporter plasmid, was co-transfected into HepG2 cells with
various combinations of pCMV2-p53, pCMV2-Tip60, and pCMX-HDAC7 as
indicated in Fig. 5C by calcium-phosphate method. Following
24 h of transfection, medium containing DNA-calcium phosphate
precipitates was replaced by fresh medium and cells continued to grow
for 24 h. Cell lysates were prepared, and luciferase assay was
performed with dual-luciferase kit. These experiments were repeated at
least twice with similar results.
, or Tip60C1 at comparable
levels (Fig. 6A). Following
serum starvation, total RNA were prepared from unstimulated or
IL-9-stimulated cells. The samples were analyzed for the expression of
c-myc, a target gene for STAT3 in cells transformed by Src
or induced by IL-9 or platelet-derived growth factor (43, 51). As shown
in Fig. 6B, c-myc was induced by IL-9 in parental
TS1 cells, and ectopic expression of Tip60 or Tip60HAT
significantly attenuated c-myc induction following IL-9
treatment for 30 min. The difference of c-myc induction in
Tip60 and Tip60HAT
-transfected cells became significant
90 min following cytokine stimulation (Fig. 6C). Ectopic
expression of Tip60C1 slightly increased c-myc
expression in 30 and 90 min, which is consistent with the ability of
Tip60C1 to up-regulate the STAT3-mediated reporter gene
expression following IL-6 stimulation (Fig. 5A). These data
suggest that Tip60C1 may function as a dominant-negative mutant for Tip60 to regulate STAT3 activity. Similar results were also
obtained for Cited2 expression (data not shown), indicating that Tip60 down-regulates STAT3-induced primary response gene expression in IL-9-stimulated cells.
View larger version (31K):
[in a new window]
Fig. 6.
Tip60 represses STAT3-mediated gene
expression in IL-9-stimulated cells. A, schematic
presentation and detection of Myc-tagged wild-type and mutant Tip60
expressed in TS1 cells. 2 × 107 log-phase TS1 cell
lines stably transfected with wild-type or mutant Tip60 were harvested.
Cell lysates were immunoprecipitated with anti-Myc, and immunoblot was
probed with anti-Myc. B, overexpression of wild-type Tip60
represses IL-9-induced expression of c-myc. Cells were
starved for 5 h prior to stimulation by IL-9 (50 ng/ml) for 30 or
90 min. Total RNA samples were isolated by TriZOL reagent from
Invitrogen following manufacturer's instructions. 10 µg of total RNA
of each sample was used for Northern blotting. The same nylon membrane
was probed with c-myc and 36B4 sequentially.
C, quantitation of c-myc expression.
36B4, a ribosome RNA-encoding housekeeping gene, was used as
an internal control to normalize the expression of c-myc in
TS1 cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
, which possesses ATPase activity (12) and DNA helicase. However, TAP54
/
and DNA helicase are not detectable in
Tip60-containing complexes recruited to the KAI1
promoter via NF
B p50 (52), suggesting that Tip60 exerts its
functions through interaction with different protein partners. Third,
Tip60 may promote acetylation of cellular proteins other than histones
to regulate gene expression. For example, Tip60 enhances the activity
of wild-type but not an acetylation-deficient mutant of androgen
receptor, demonstrating that Tip60 acts as a co-activator by promoting
the acetylation of androgen receptor but not histones (53).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Daniel Sliva for pMT2-Myc-Tip60;
Dr. James Kamine for providing Tip60HAT mutant construct;
Dr. Heinz Baumann for pCAT and pSIECAT plasmids; Dr. Ronald M. Evans for pCMX-HA-HDAC7, pCMX-HA-HDAC1, and pMH100; and Dr. David
Donner for reading the paper.
![]() |
FOOTNOTES |
---|
* This study was supported by National Institutes of Health Grants DK50570, CA78433, and HL48819 (to Y.-C. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of the James T. Pardee-Carl A. Gerstacker Assistant Professor of Cancer Research Faculty Chair in Cancer Research at Case Western Reserve University Cancer Center.
To whom correspondence should be addressed: Dept. of
Pharmacology, School of Medicine, Case Western Reserve University, 2109 Adelbert Rd., W353, Cleveland, OH 44106-4965. Tel.: 216-368-6931; Fax:
216-368-3395; E-mail: yxy36@po.cwru.edu.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210816200
2 H. Xiao, J. Chung, H.-Y. Kao, and Y.-C. Yang, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Tip60, Tat-interactive protein, 60 kDa; Esa, Essential sas2-related acetyltransferase; HAT, histone acetyltransferase; HIV, human immunodeficiency virus; CREB, cAMP-response element-binding protein; IL, interleukin; HDAC, histone deacetylase; SAS, Something About Silencing; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; STAT, signal transducers and activators of transcription; CBP, CREB-binding protein; HA, hemagglutinin; TSA, tricostatin A; HEK, human embryonic kidney; FBS, fetal bovine serum; SIE, STAT3 binding site; CAT, chloramphenicol acetyltransferase.
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