From the Graduate Institute of Life Sciences,
National Defense Medical Center, the § Division of Molecular
and Genomic Medicine, National Health Research Institutes, Taipei 11529 Taiwan, Republic of China, the ¶ Institute of Molecular Biology,
Academia Sinica, Taipei 11529, Taiwan, Republic of China, and the
Department of Molecular Pharmacology and Toxicology, University
of Southern California, Los Angeles, California 90033
Received for publication, January 14, 2003
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ABSTRACT |
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Daxx has been reported to function as a
transcriptional modulator in the nucleus. In the present study, we have
explored the role of Daxx in regulating the transcriptional activity of
the glucocorticoid receptor (GR). Overexpression of Daxx suppressed GR-mediated activation of the mouse mammary tumor virus promoter in
COS-1, HeLa, and 293T cells. In vitro and in
vivo studies revealed that Daxx could directly bind to GR. The
mapping analysis further demonstrated that the C-terminal region of
Daxx-(501-740) mediates the interaction and transcriptional repression
of GR. The repressive effect of Daxx and Daxx-(501-740) on GR could be
alleviated by co-expression of promyelocytic leukemia protein (PML).
Furthermore, immunofluorescence analysis showed that overexpression of
wild-type PML results in the translocation of Daxx and Daxx-(501-740)
to the PML oncogenic domains (PODs). By contrast, a PML
sumoylation-defective mutant failed to recruit Daxx to PODs and to
reverse the Daxx repression effect on GR. Accordingly,
As2O3 treatment rendered the sequestration of
endogenous Daxx to the PODs, leading to an enhancement of GR
transactivation in COS-1 cells. Taken together, these findings suggest
that recruitment of Daxx into the subnuclear POD structures sequesters
it from the GR/co-activators complex, thereby alleviating its
repressive effects. Our present studies provide the important link
between Daxx/PML interaction and GR transcriptional activation.
The glucocorticoid receptor
(GR),1 a member of the
ligand-dependent nuclear receptor superfamily, plays
important roles in governing development, growth, apoptosis, and
anti-inflammatory processes (1-5). Like other superfamily members, GR
contains the N-terminal transactivation domain, central DNA
binding domain, and the C-terminal ligand binding domain (6). Prior to
exposure to its respective ligand, the GR and other receptors in this
family form complexes with heat shock proteins (HSPs), restricting the unliganded-receptor in its inactive state. Upon ligand binding, the
receptor is dissociated from HSPs and translocates to the nucleus where
the liganded receptor binds to the glucocorticoid-responsive element
and recruits co-factors to regulate transcription (7). A diverse group
of proteins have emerged as potential cofactors for nuclear receptors
through direct protein-protein interactions. For instances, several
co-activators have been identified that bind to GR and modulate GR
transcriptional activity, including CBP/p300, p160 family co-activators
(SRC-1, GRIP1, and p/CIP), and chromatin remodeling complexes (BRG-1
(SWI/SNF) complex and P/CAF (ADA/SAGA) complex) (8, 9). These
co-activators appear to be assembled in a multiprotein complex,
facilitating the access of nuclear receptors and the RNA polymerase II
core machinery to their target DNA by a chromatin remodeling process
(10).
In addition to regulating GR transcriptional activity via direct
protein-protein interactions, some co-factors appear to modulate GR
activity through indirect fashion. PML, a protein located at the
promyelocytic oncogenic domains (PODs), has previously been reported to
stimulate GR and RXR Daxx, a PODs-associated protein, was identified as an interacting
protein of PML (19-21). Recent studies implied that Daxx might
function as a transcriptional coregulator. Daxx has been shown to
exhibit transcriptional repression activity by inhibiting the
transcription factor Pax3 and ETS1 through direct protein-protein interactions (22, 23). Most recently, Emelyanov et al.
reported that Daxx acts as a transcriptional co-activator or
co-repressor of Pax5 in different cellular contexts (24). Although the
exact mechanism accounting for these observations is still unclear, the
recruitment of nuclear factors possessing either histone
acetyltransferase or histone deacetylase activity by Daxx to modulate
Pax5 transcriptional activity was proposed (24). In addition, the
transcriptional repression effect of Daxx could be modulated by
subnuclear compartmentalization through protein-protein interactions.
We have recently demonstrated that overexpression of a nucleolar
protein MSP58 alleviates the transcriptional repression elicited by
Daxx, correlating well with sequestration of Daxx to the nucleolus via
Daxx/MSP58 protein-protein interactions (25). Furthermore, Lehembre
et al. has shown that SUMO-1-modified PML could relieve the
transrepression effect of Daxx on Pax3 transcriptional activity through
sequestering Daxx into the PODs (26). Taken together, PML may exert its
regulatory effect on transcription factor(s) through altering the
compartmentalization of Daxx or other co-regulator(s). This notion led
us to raise a question as to whether GR is also regulated by PML, at
least in part, through Daxx sequestration to the PODs.
In the present study, we have characterized both biochemical and
functional interactions between Daxx and GR. Furthermore, the
Daxx-mediated GR repression could be alleviated by the co-expression of
wild-type PML but not by a sumoylation-defective PML mutant, which also
fails to recruit Daxx to the PODs. Our findings thus provide the first
evidence that Daxx could modulate the transcriptional activity of
nuclear hormone receptor through protein-protein interactions and sheds
light on the mechanism underlying the PML role in modulating nuclear
hormone receptor function.
Plasmids Construction--
LexA-Daxx, its deletion
mutants LexA-MST3, LexA-lamin, HA-Daxx, and its derived mutant
constructs have been described before (25). A mammalian GST-Daxx
expression vector was generated by insertion of Daxx into pDEST 26 (Invitrogen). MMTV-Luc and pCMX-PML were kind gifts from Drs.
Chawnshang Chang and Ronald Evan, respectively. pGal-AD-GR was
engineered by inserting GR coding region into the BamHI and
XhoI sites of yeast vector pACT2, which expresses the Gal4 activation domain (Clontech). pCMX-PML- Two-hybrid Assay and Cell Culture and Transient Transfection--
All mammalian cell
lines were obtained from the American Type Culture Collection (ATCC).
COS-1, HeLa, and 293T cells were maintained in Dulbecco's minimal
essential medium supplemented with 10% fetal bovine serum (FBS) and
antibiotics. Cells were seeded into 10-cm plates the day before
transfection. Transient transfections were carried out using a
LipofectAMINE transfection kit (Invitrogen). Thirty-six hours after the
transfection, cells were harvested for co-immunoprecipitation assays
and Western blot analyses. For the reporter assays, 2-3 × 105 cells were seeded on 6-well plates for transfection.
Four hours prior to transfection, cells received fresh medium with 10%
dextran-coated charcoal-stripped FBS. A total of 2 µg of DNA were
transfected, including GST Pull-down Assay--
For purification of GST and GST-Daxx
fusion proteins, 293T cells were transfected with pDEST26 (Invitrogen)
or pDEST26-Daxx. Cells were lysed 48 h later in buffer EM2
containing 50 nM HEPES (pH 7.6), 50 mM NaCl,
1% Nonidet P-40, 5 mM EDTA, 10% glycerol, and
4-(2-aminoethyl)-benzenesulfonyl fluoride (Sigma) and centrifuged at
17,000 × g for 10 min at 4 °C. Supernatants were
incubated with glutathione-agarose beads for 1 h at 4 °C. The
bound proteins were then analyzed by SDS-PAGE followed by Western
blotting using antibody against GST protein. 35S-labeled GR
protein was made with the TNT reticulocyte lysate system (Promega).
35S-labeled proteins were incubated with GST or
GST-Daxx-agarose beads, which contained equal amounts of protein in 0.2 ml of binding buffer (10 mM HEPES, pH 7.5, 50 mM NaCl, 0.1% Nonidet P-40, 0.5 mM
dithiothreitol, and 0.5 mM EDTA) for 2 h, washed four
times, and analyzed by SDS-PAGE and autoradiography.
Immunoprecipitation and Western Blotting Assays--
To test the
interaction in mammalian cells, wild-type GR along with HA-tagged Daxx
expression construct was transfected into COS-1 cells by the
lipofection method. Thirty-six hours after cotransfection, cells were
solubilized in 1 ml of modified RIPA buffer (50 mM
Tris-HCl, pH 7.8, 150 mM NaCl, 5 mM EDTA, 0.5%
Triton X-100, 0.5% Nonidet-P40, 0.1% sodium deoxycholate, and
protease inhibitor mixture (Complete, Roche Molecular Biochemicals).
Cell lysates were mixed with antiserum against HA (Babco, Richmond, CA), Daxx (Santa Cruz Biotechnology, Santa Cruz, CA, catalogue number
sc-7152), or GR (Santa Cruz Biotechnology, catalogue number sc-1004),
and the immunocomplexes were collected on protein A-Sepharose beads
(Amersham Biosciences). Immunoblot analyses of precipitated proteins
were performed as described previously (25). The anti-PML antibody was
purchased from Santa Cruz Biotechnology (catalogue number sc-966).
Immunofluorescence--
COS-1 cells were plated onto
collagen-coated (10 µM) coverslips the day before
transfection. The expression vectors of HA-tagged Daxx or its deletion
mutants or GR along with PML or PML sumoylation-defective mutant were
transiently transfected into COS-1 cells. Twenty-four hours after
transfection, the cells received fresh 0.05% stripped FBS and were
cultured for an additional 12 h in the presence or absence of 100 nM Dex or 1 µM As2O3
as indicated. Cells were fixed in 4% paraformaldehyde in
phosphate-buffered saline and permeabilized with 0.4% Triton X-100.
The permeabilized cells were then incubated with an anti-HA monoclonal
antibody and an anti-GR polyclonal antibody (Santa Cruz Biotechnology,
catalog number sc-1004) for 1 h at room temperature. Following
this incubation, cells were washed three times for 10 min with
phosphate-buffered saline at room temperature and then incubated at 8 µg/ml with fluorescein isothiocyanate and Texas Red-conjugated
anti-rabbit IgG (DAKO) for 1 h at 20 °C. Nuclei were revealed
by DAPI staining (10 µg/ml). The coverslips were inverted, mounted on
slides, and sealed with nail polish. Pictures were taken using a
fluorescent microscopy.
Overexpression of Daxx Suppresses GR-mediated Transactivation of
the MMTV Promoter--
To test whether Daxx is involved in regulating
GR transcriptional activation, the Daxx expression construct was
cotransfected into COS-1 cells along with the GR expression construct
and the MMTV-Luc reporter. As shown in Fig.
1A, Daxx suppressed
GR-mediated transactivation in a dose-dependent fashion. By
contrast, a transcriptional co-activator, GRIP1, potentiated the
transactivation ability of GR. Furthermore, the repressive effect of
Daxx on GR was also observed in other cell lines, such as HeLa and 293T
cells (Fig. 1B), indicating that the
Daxx-dependent GR repression is not a cell type-specific
event.
Daxx Binds to GR Both in Vitro and in Vivo--
To explore whether
the transcriptional repression of GR by Daxx is through the direct
protein-protein interaction between Daxx and GR, we first performed the
co-immunoprecipitation experiments. COS-1 cells were cotransfected with
expression constructs encoding GR and HA-Daxx and followed by treatment
of dexamethasone. Forty-eight hours after transfection, cell lysates
were subjected to immunoprecipitation assays with an anti-GR antibody
followed by Western blot analyses with an anti-HA antibody. As shown in
Fig. 2A, Daxx was detected in
the immunoprecipitated complexes of GR (top panel,
lane 4). This interaction was also validated in a reciprocal
co-immunoprecipitation assay (data not shown). These results suggest
that Daxx and GR can associate to form complexes in mammalian
cells.
To further test whether this association is through direct
protein-protein interaction, GST pull-down assays were performed using
GST-Daxx fusion protein and in vitro translated
[35S]methionine-labeled GR. As shown in Fig.
2B, GR was specifically pulled down by GST-Daxx but not by
GST protein, indicating that GR binds to Daxx directly. It should be
noted that the interaction between GR and Daxx was not affected by the
presence or absence of dexamethasone. To further substantiate the
observed direct protein-protein interaction between Daxx and GR, we
also carried out the yeast two-hybrid assays. The GR cDNA clone was
subcloned in frame into pACT2 vector (Gal AD-GR) and subsequently
analyzed with different bait proteins (Daxx, MST3, and lamin) for the
ability to activate His3 and LacZ reporter genes.
As summarized in Fig. 3B,
yeast co-transformed with GalAD-GR and LexA-Daxx was able to form
colonies in the medium plate lacking histidine, indicating a positive
interaction between GR and Daxx. Again, this interaction is specific
since no interaction between GR and lamin or MST3 was detected. The
interaction was further verified by liquid The Functional Interaction between the C-terminal Domain of Daxx
and GR--
To delineate the region(s) of Daxx that is involved in
Daxx/GR interaction, various deletion constructs of Daxx were
engineered (Fig. 3A) and subjected to analyses in yeast
two-hybrid assays. The strength of interaction was scored by the colony
growth in histidine-auxotroph-selective medium and quantified by liquid
To establish the correlation between interaction and GR transcriptional
repression by Daxx, these Daxx mutants were analyzed for their
respective abilities to repress GR transactivation. As illustrated in
Fig. 3D, overexpression of Daxx-(501-740), like full-length
Daxx, was capable of suppressing GR-mediated transcriptional activation, whereas both Daxx-(1-501) and Daxx-(1-625) failed to do
so, indicating that the C-terminal region of Daxx is sufficient for
Daxx-mediated GR transrepression. Taken together, these findings provide direct evidence that the interaction between Daxx and GR
correlates well with Daxx function as a GR transrepressor.
PML Reverses Daxx Transrepression of GR by Recruiting Daxx to the
PODs--
Recent studies indicated that PML overexpression reverses
the Daxx-dependent transcriptional repression and such a
de-repression correlates well with the ability of sumoylated PML to
sequester Daxx to the PODs (21). In this scenario, we anticipated that PML should be able to relieve the Daxx transrepression on GR activity. To test this possibility, COS-1 cells were cotransfected with PML
expression construct along with the expression constructs harboring
either full-length Daxx or Daxx-(501-740) and assayed for GR
transactivation potential. As shown in Fig.
4A, overexpression of PML
resulted in a reduction of transrepression elicited by both Daxx and
Daxx-(501-740) on GR in a dose-dependent manner. To
further unveil whether this de-repression by PML correlates with the
recruitment of Daxx to the PODs, immunofluorescent analyses were
performed. COS-1 cells were transiently transfected with a combination
of GR, PML, HA-Daxx, HA-Daxx-(1-625), and HA-Daxx-(501-740) as
indicated and then subsequently stained with the appropriate antibodies, followed by immunofluorescence analysis. When HA-Daxx or
HA-Daxx-(501-740) was overexpressed alone, a fairly diffuse and evenly
distributed staining pattern was observed through the nucleus (Fig.
4B, a and i). By contrast,
HA-Daxx-(1-625) gave an exclusively cytoplasmic staining pattern
(e), suggesting that the C-terminal region of
Daxx-(626-740) is required for the nuclear localization. Notably,
co-expression of PML resulted in a dramatic change of HA-Daxx and
HA-Daxx-(501-740) distribution patterns (b and
j), as evidenced by the Daxx-PML colocalized to the PODs (d and l). Conversely, the cytoplasmic
distribution of HA-Daxx-(1-625) was not altered by PML, supporting the
previous findings that PML fails to interact with Daxx-(1-625)
(h). Taken together, these findings evidently suggest that
the de-repression by PML is through its ability to sequester Daxx to
PODs. It is very worthy of knowing that the distribution of GR was not
altered upon PML overexpression (Fig. 4B, m),
consistent with the notion that PML did not directly bind to GR
(11).2
To further substantiate that the recruitment of Daxx by PML to PODs
plays an important role in regulating GR transcriptional activity, a
PML sumoylation-defective mutant was subjected to the same analyses for
its ability to recruit Daxx to the PODs and to reverse the
transrepression elicited by Daxx on GR. Notably, co-expression of
sumoylation-defective PML mutant failed to recruit Daxx from
nucleoplasm to PODs (Fig. 5A).
As expected, sumoylation-defective PML mutant, unlike the wild-type
PML, was unable to relieve the Daxx transrepression effect on GR (Fig.
5B), indicating that the Daxx-mediated GR transrepression
and the recruitment of Daxx to PODs by PML are an interdependent event.
To further demonstrate the effect exerted by PML on the
Daxx-dependent GR transrepression and the Daxx nuclear
compartmentalization, we treated COS-1 cells with
As2O3. As2O3 has been
shown to promote the SUMO-1 modification of PML, which in turn induces
the recruitment of PML and Daxx to the PODs (19, 21, 26). As shown in
Fig. 6A,
As2O3 treatment caused a marked increase of the
number of POD formation, as evidenced by immunofluorescent analysis of
endogenous PML (a and d). This treatment also
rendered the redistribution of endogenous Daxx from nucleoplasm to the
PODs, leading to the co-localization of Daxx with PML (b and
e). Coordinately, As2O3 treatment
resulted in an increment of the GR-mediated transcriptional activity
(Fig. 6B). Taken together, the results of transient
cotransfection assays and immunofluorescence studies unequivocally
indicate that PML potentiates GR transcriptional activation by
compartmentalizing Daxx from nucleoplasm to the PODs.
In the present studies, we have identified Daxx as a
GR-interacting protein and demonstrated that Daxx regulates the GR
transcriptional activation on MMTV promoter. We provided biochemical
evidence that Daxx associates with GR in yeast, in vitro,
and in mammalian cells. The interaction between GR and Daxx is mediated
through the C-terminal region of Daxx. Furthermore, co-expression of
PML relieves the transcriptional repression by Daxx, correlating well with the recruitment of Daxx from the nucleoplasm to the PODs. Our
findings not only provide the first direct link between Daxx expression
and GR activity but also shed some light on the molecular mechanism
underlying the role of PML in regulating GR transcriptional potential.
Daxx was initially identified as the adaptor molecule between Fas
receptor and Jun N-terminal kinase in Fas-mediated apoptosis (27).
However, accumulated evidence suggested that Daxx might function as a
transcriptional repressor through its interaction with a growing number
of transcription factors. Daxx has been shown to repress the basal
activity by a heterologous TK promoter through Gal4DBD-Daxx fusion (19,
21, 25). Daxx was also reported to inhibit the activity of the
transcriptional activators Pax3 and ETS1 (22, 23). While Daxx
overexpression inhibited the endogenous Pax5 transcriptional activity
in the human HS-Sultan cell line and the murine plasmacytoma J558L cell
line, Daxx potentiated Pax5 transcriptional activity in M12.4.1 and A20
murine B cells (24). In the present study, we further demonstrated that
Daxx could suppress the GR-mediated transcriptional activation of MMTV reporter. The transrepressional effect of Daxx on GR activity has also
been demonstrated in several cell lines, indicating that this
repressive effect is likely not a cell type-specific event. Furthermore, we also provided biochemical and cell biological evidence
that the C-terminal region of Daxx is necessary and sufficient to
interact with GR and repress GR transcriptional activity. Intriguingly, this C-terminal region of Daxx has also been reported to interact with
other transcription factors, such as Pax3, Pax5, and ETS1 (19, 23, 24)
as well as a wide variety of molecules, including Fas receptor, PML,
centromeric protein CENP-C, DNA methyltransferase I, HSP27,
Glut4, Ubc9, and SUMO-1 (21, 27-32). Conceivably, the C-terminal
region of Daxx acts as a major docking domain for protein-protein interactions. To our knowledge, it is not yet demonstrated how this
domain provides a binding surface for these proteins with such
distinctive functions. However, we postulate that Daxx is likely
involved in the signaling pathway cross-talks through competitive binding by distinct factors to the specific motif(s) located within the
C-terminal domain of Daxx.
Although Daxx functions as a transcriptional co-repressor, the
molecular mechanism as to how it suppresses the transcriptional activation remains largely unknown. Previous report showed that the
treatment with a deacetylase inhibitor, trichostatin A, efficiently reversed the repressive effect of Daxx (21), suggesting that histone
deacetylation may be involved in Daxx-mediated transcriptional repression. Consistently, interaction between the Daxx and HDAC1 has
been demonstrated by in vitro pull-down assay and in
vivo overexpression experiments (21). Furthermore, Hollenbach
et al. has recently shown that endogenous hDaxx eluted from
size exclusion chromatography column has an apparent molecular
mass of 360 kDa and associates with multiple proteins that are
critical for transcriptional repression, such as histone deacetylase
II, components of chromatin such as core histone H2A, H2B, H3, and H4,
and a chromatin-associated protein Dek (33). The findings of the hDaxx
association with histone deacetylase are consistent with our notion
that HDACs play an essential role in Daxx-mediated transcriptional
modulation via chromatin remodeling processes. Moreover, that Daxx was
reported to be associated with condensed chromatin in the cells lacking
PML (20) also supports the role of Daxx in the establishment and/or
maintenance of a transcriptionally silenced chromatin structure.
Whether overexpressed Daxx results in silencing the chromatin structure
of GR-regulated genes remains to be explored.
Another main feature of the present study is to explore the mechanism
as to how PML regulates GR transcriptional activity. PML has been
proposed to function as a transcription cofactor through recruiting
co-activators (13, 34). For example, PML has been shown to potentiate
the transactivation activity of AP-1 and progesterone receptor (35,
36). Further studies revealed that PML does not interact directly with
either Jun/Fos or progesterone receptor but instead targets
transcriptional co-activators, such as CBP and TIF1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
transcriptional activity through its
interaction with CBP (11). These results demonstrated that a
portion of CBP is recruited to the PODs through its association with
PML, suggesting that PML and/or POD-associated proteins may function as
an important cofactor in governing nuclear hormone receptor
transcriptional activity and function. Besides PML and CBP, several
cellular proteins have been reported to be associated with PODs,
including Sp100, Daxx, p53, Rb, small ubiquitin-like modifier (SUMO-1),
and BLM (12, 13). Results from many elegant studies have revealed that
overexpression of p53 suppressed GR-mediated reporter gene activation
(14, 15) and Rb potentiated GR transcriptional activity through its
interaction with hBRM (16), respectively. Recently, SUMO-1 modification
of GR has been shown to regulate its transcriptional activity (17, 18).
These findings suggest that POD-associated proteins play important
roles in modulating GR transcriptional activity. Whether other
POD-associated proteins are also involved in regulating GR
transcriptional activity remains to be explored.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SUMO
containing a lysine-to-arginine mutation at amino acid positions
65, 160, and 490 was created in three rounds consecutively of
site-directed mutagenesis on the same template pCMX-PML. Site-directed
mutagenesis was conducted by using QuikChange site-directed
mutagenesis kit according to the manufacturer's instruction (Stratagene).
-Galactosidase Assay--
Yeast
two-hybrid assays were performed as described (25). Briefly, L40 yeast
strain was first transformed with individual bait and followed by the
prey construct transformation. Yeast transformants were selected on
medium lacking histidine, leucine, and tryptophan for four days.
His+ colonies were further tested for
-galactosidase
activity. Quantitative X-gal assays were performed with yeasts
containing pairs of bait and prey plasmids as indicated. The X-gal
activities were determined from three separate liquid yeast cultures
according to the instructions of the Galacto-light Plus kit (Tropix
Inc., Bedford, MA).
-galactosidase expression construct as an
indicator for normalization of transfection efficiency. Following
transfection, cells were cultured in the medium containing
charcoal-stripped 0.05%(V/V) FBS with or without ~10-100
nM dexamethasone and/or further treatment of 1 µM As2O3. Cell lysates were
harvested 16 h later and assayed for relative activity (firefly
luciferase for the reporter and
-galactosidase activity for the
indicator) as the manufacture instructed (PerkinElmer Life Sciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Repression of glucocorticoid-stimulated
MMTV-LTR-driven luciferase activity by Daxx. A, COS-1
cells were transiently transfected in 6-well plates with 500 ng of
pMMTV-Luc, 50 ng of pSV40- -galactosidase, 500 ng of pKSR-GR, and
increasing amounts of HA-Daxx plasmids as indicated or 500 ng of
pCMV-GRIP1. The total amount of plasmid transfected per well were kept
constant by addition of empty pcDNA3 vector as needed. Transfected
cells were incubated in complete medium for 16-18 h, then washed, and
incubated in Dulbecco's modified Eagle's medium with 0.05%
charcoal-stripped fetal calf serum with or without 100 nM
dexamethasone (Dex) for another 24 h. Relative
luciferase activity is represented as mean ± S.D. (luciferase
light units/
-galactosidase). B, the effect of Daxx on
GR-mediated transcription of pMMTV-Luc reporter was assayed in COS-1,
HeLa, and 293T cells. Five hundred nanograms of luciferase reporters,
50 ng of pSV40-
-galactosidase, 500 ng of pKSR-GR, and 500 ng of
HA-Daxx plasmids per well were used in these experiments.
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Fig. 2.
Interaction between GR and Daxx in
vitro and in vivo. A, GR
interacts with Daxx in transfected COS-1 cells. COS-1 cells were
transiently transfected with the indicated combinations of expression
constructs encoding GR and HA-tagged Daxx, respectively. Following
treatment with 100 nM Dex for 30 h, total cell lysates
were analyzed by Western blot (WB) to check expression of GR
and Daxx proteins (bottom panel). Equal amounts of cell
lysates were immunoprecipitated (IP) with an anti-GR
antibody, followed by blotting with an anti-HA antibody (upper
panel), whereas reciprocal co-immunoprecipitation experiments were
performed using an anti-HA antibody, followed by blotting with an
anti-GR antibody (data not shown). B, interaction of
GST-Daxx with in vitro translated GR. The GST or GST-Daxx
fusion proteins were immobilized on the glutathione-agarose beads and
then incubated with in vitro translated
[35S]methionine-labeled GR protein. After wash, the bound
components were resolved on a 10% SDS-polyacrylamide gel. The 20%
amount of input GR was shown as a positive control for GR.
Immunoblotting analysis of GST and GST-Daxx protein using an antibody
against GST were aligned to show protein levels (bottom
panel).
-galactosidase assay
(Fig. 3C). Taken together, our results clearly demonstrated that GR interacts with Daxx through direct protein-protein
interactions.
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Fig. 3.
The COOH domain of Daxx mediates the
interaction and transcriptional repression of GR. A,
schematic presentation of different Daxx deletion mutants tested in
yeast two-hybrid and mammalian transfection assays. The LexA constructs
of Daxx(FL), Daxx-(1-625), Daxx-(1-501), and Daxx-(501-740) used in
yeast two-hybrid assays represent the fusion proteins between LexA and
various Daxx deletion mutants. All Daxx deletion mutants were also
fused to a HA tag for the purpose of detection in the mammalian cells.
Proteins reported to interact with different regions of Daxx are
indicated. B, domain mapping studies of Daxx associated with
GR. The wild-type and the deletion mutants of Daxx as indicated (amino
acid residue in parentheses) were fused to the LexA DNA
binding domain. The LexA-lamin and LexA-MST3 serve as negative
controls. GR fused to Gal4 activation domain functions as prey for
interaction. Yeast transformants were streaked on histidine-auxotroph
selection plates and scored after a 4-day incubation at 30 °C.
Growth is indicated as " " to "++++" based on the size of the
colonies. C, liquid X-gal assay for Daxx-GR interactions.
The relative strength of protein interactions was determined by
measuring
-galactosidase activities using the Galacto-light Plus kit
(Tropix Inc.) and normalized by cell density
(A600). The bar graph shows the average activity
observed from two independent experiments performed in triplicate.
D, the effect of Daxx mutants on glucocorticoid-stimulated
MMTV-LTR-driven luciferase activity in COS-1 cells. Transient
transfection was conducted as described in Fig. 1A. COS-1
cells were cotransfected with MMTV-Luc reporter, the expression vector
for GR, together with expression plasmids for either of the different
Daxx mutants. Data presented here are the means of at least three
independent experiments. E, Western blot showing the
expression of different Daxx mutant whole cell extracts were analyzed
on a 10% SDS-polyacrylamide gel transferred to Immobilon-P membrane
and probed by the anti-HA antibody. Numbers indicate size
markers.
-galactosidase assay. As shown in Fig. 3B, the C
terminus-deleted Daxx (amino acids 1-501 and 1-625) failed to
interact with GR. In contrast, the mutant with a deletion of amino acid
residues 1-501 of Daxx was still capable of interacting with GR,
albeit the strength of interaction with GR was less than that from the full-length Daxx. Additionally, consistent results were also observed with the liquid
-galactosidase assay. Altogether, these results implicated that the C-terminal region of Daxx is necessary and sufficient for its interaction with GR.
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Fig. 4.
PML activates GR transactivation
through sequestration of Daxx. A, PML relieves the Daxx
transrepression effect on GR transcriptional activity. COS-1 cells were
transfected in six-well plates with MMTV-Luc, -galactosidase, GR,
Daxx(FL), or Daxx-(501-740), and indicated amounts of PML expression
plasmids. Transfected cells were incubated in complete medium for
16-18 h and then grown in the absence or presence of 10 nM
Dex for another 24 h. Luciferase activity was reported as relative
light units and represented as the mean ± S.D. B, PML
overexpression induces the recruitment of Daxx but not that of GR to
the PODs. COS-1 cells were transiently transfected with plasmid
constructs as indicated. The cells transfected with GR and PML were
treated with 100 nM Dex for 3 h before fixation. The
primary antibodies used in immunostaining analyses were the PG-M3
monoclonal antibody against PML, the rabbit polyclonal anti-GR antibody
(M-20) against GR, and a rabbit polyclonal anti-HA antibody (Y-11)
against the HA-tagged Daxx and its derivative proteins. The secondary
antibodies for these studies were fluorescein isothiocyanate-conjugated
anti-mouse immunoglobulin G and Texas Red-conjugated anti-rabbit
immunoglobulin G. After immunostaining and washing procedures, the
samples were analyzed by immunofluorescence microscopy.
4',6'-Diamidino-2-phenylindole (DAPI) staining revealed the
positions of the nucleus. Immunofluorescence micrographs of
immunolabeled proteins are presented with the respective colors in the
lower corners of the image. Superimposing the two colors
(Merged) results in a yellow signal.
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Fig. 5.
Correlation between Daxx
compartmentalization and GR transrepression. A,
sumoylation-defective PML did not recruit the Daxx into the PODs.
Double immunofluorescence staining of Daxx and PML or
sumoylation-defective PML was performed on the transfected COS-1 cells.
The staining pattern was determined as in Fig. 4A.
B, SUMO-1 modification of PML is essential for
suppressing Daxx-mediated repression of GR transcriptional
activity. PML or sumoylation-defective PML, GR, and Daxx(FL) expression
constructs were cotransfected in COS-1 cells as indicated with the
MMTV-Luc reporter construct. Expression levels of PML and
sumoylation-defective PML were immunoblotted with an anti-PML antibody
from the whole cell lysates.
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Fig. 6.
As2O3
stimulates the translocation of Daxx to PODs and potentiates the
GR-mediated transactivation. A, COS-1 cells were
treated with 1 µM As2O3 for
12 h, and then double immunofluorescence staining of endogenous
Daxx and PML was performed. The staining pattern was determined as in
Fig. 4A. B, COS-1 cells were transfected with the
MMTV-Luc reporter and GR expression constructs. Following the
transfection, cells were pretreated with 100 nM Dex for
12 h and then incubated in medium with or without 1 µM As2O3 for another 12 h.
Luciferase activity was then determined as described above.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(11, 36, 37).
PML binds to CBP in vitro, promotes CBP localization to
PODs, and enhances transcriptional activity of nuclear receptors such
as GR and retinoid-X receptor in transient transfection assays (11).
Similarly, a co-activator complex, composed of PML, TIF1
, and CBP,
is recruited to retinoic acid response element, suggesting that
PML is a co-activator for retinoic acid receptors (37). In this study,
we propose an alternative regulatory route for PML in modulating GR
transactivation function. The recruitment of Daxx to PODs by PML may
represent another mechanism whereby the activity of the GR is
activated. Our results clearly demonstrated that wild-type but not
sumoylation-defective PML is critical for attenuating transrepression
effect of Daxx on GR by translocating Daxx to the PODs. Likewise, that
As2O3-treatment enhanced GR transcriptional
activation utilizes a similar mechanism by modulating Daxx
localization. These findings are consistent with the established notion
from recent studies that SUMO-1 modification of PML is required for
both the sequestration of Daxx to the PODs and efficient inhibition of
Daxx-mediated repression. One may hypothesize that retention of Daxx
into PODs through PML overexpression would prevent the access of Daxx
to GR, subsequently impeding its repressive action. Likewise, Lehembre
et al. have recently reported that SUMO-1-modified PML could
derepress Pax3 transcriptional activity through sequestration of the
Daxx repressor into the PODs (26). Taken together, these findings
suggest that PML may modulate the activities of a variety of
transcription factors by altering the compartmentalization of its
respective co-repressor(s) to the PODs. Comparable to this scenario, we
have recently identified that nucleolar protein MSP58 can enhance the
transactivation potential by sequestering Daxx to the nucleolus (25).
In light of these observations, PODs and/or nucleolus may participate
to the transcriptional activation of specific target genes by tethering
co-repressors, such as Daxx, from the diffuse nuclear fraction where
transcription takes place.
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FOOTNOTES |
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* This work was supported by intramural funds of National Health Research Institutes (to H.-M. S.) and in part by National Institute of Health Research Grants R01 DE10742 and DE14183 (to D. K. A.).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.
** To whom correspondence should be addressed: Div. of Molecular and Genomic Medicine, National Health Research Institutes, 128 Sec2, Yen-Chiu-Yuan Rd., Taipei 11529, Taiwan. Tel.: 886-2-2652-4122; Fax: 886-2-27890484; E-mail: shihh@nhri.org.tw.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M300387200
2 D.-Y. Lin and H.-M. Shih, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
GR, glucocorticoid
receptor;
MSP58, 58-kDa microspherule protein;
Dex, dexamethasone;
PML, promyelocytic leukemia protein;
PODs, PML oncogenic domains;
SUMO, small ubiquitin-like modifier;
CBP, CREB binding protein;
FBS, fetal
bovine serum;
GST, glutathione S-transferase;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
HA, hemagglutinin;
DAPI, 4',6-diamidino-2-phenylindole;
MMTV, murine
mammary tumor virus.
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