Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106-4965
Address all correspondence and requests for reprints to: Dr. John Nilson, Department of Pharmacology, Case Western Reserve University School of Medicine, W319, 2109 Adelbert Road, Cleveland, Ohio 44106-4965.
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ABSTRACT |
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INTRODUCTION |
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LH is a heterodimeric glycoprotein hormone consisting of an -subunit
(
GSU) common to all glycoprotein hormones, and a unique
ß-subunit (3). The genes that encode these subunits
reside on separate chromosomes and are controlled by entirely different
5'-flanking regions (4). Although common hormones regulate
GSU and LHß subunit gene expression, we postulate that different
mechanisms are used. In this paper we will focus on the
mechanisms underlying androgen-dependent suppression of
GSU promoter
activity whereas the companion paper (2) will focus on
LHß.
We have previously shown, using transient transfection assays in
T31 cells, that androgens can directly suppress
GSU gene
activity while estrogens have no effect (5). Studies with
various AR mutants identified the DNA-binding domain (DBD) and
adjoining hinge region as the minimal domains necessary and sufficient
for suppression of the
GSU promoter (6). However, in
the context of the entire receptor, both the DBD and ligand-binding
domain (LBD) are required for AR-mediated suppression of the
GSU
gene, highlighting the necessity of its ligand (6). While
a high-affinity binding element for AR is present in the proximal 111
bp of the
GSU promoter, mutation of this element had no effect on
AR-mediated suppression (6). Instead, block mutation
studies indicated that two regulatory elements, the tandem cAMP
regulatory elements (CREs) and the
basal element (
BE), which are
critical for targeting expression of the
GSU gene to gonadotropes,
are each also essential for androgen-mediated suppression
(6). Together, these data suggest that AR may exert its
effect by binding directly to the proteins that occupy one or both of
these sites.
Because the identity of proteins that bind BE are unknown
(7), we have concentrated on those that can bind the
tandem CREs. Several members of the bZip family of transcription
factors bind the tandem CREs in the
GSU promoter, including CRE
binding protein (CREB), CRE modulator (CREM), activating
transcription factors 1 and 2 (ATF1, ATF2), and cJun (8).
Here we investigate whether specific members of the bZip protein family
can be implicated in AR suppression of
GSU promoter activity. In
addition, a number of direct and indirect mechanisms for
transcriptional repression by nuclear receptors (NR) have been
described (9, 10). Therefore, we also determined whether
additional indirect mechanisms were contributing to androgen-dependent
suppression of
GSU promoter activity. Assessment included changes in
critical protein expression, phosphorylation status of the AR-DBD, and
alterations in histone acetyltransferase (HAT) activity.
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RESULTS AND DISCUSSION |
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While cJun and ATF2 had little effect on basal promoter activity when
transfected in the absence of AR, both bZip proteins rescued the
reporter construct from AR-mediated suppression even when cotransfected
at a 1:1 ratio (Fig. 1). Addition of both
cJun and ATF2 together resulted in the same
GSU promoter activity as
each alone, indicating that the endogenous proteins are not limiting
(data not shown). In contrast, overexpression of CREB alone markedly
attenuated activity of the
GSU promoter. This negative effect of
CREB appears specific for the
GSU promoter since its overexpression
did not compromise activity of rous sarcoma virus (RSV), SV40, or LHß
promoters (data not shown). More importantly, overexpression of CREB
failed to rescue the
GSU promoter from the suppressive effect of AR,
suggesting that not all bZip family members are functionally
equivalent. In this regard, overexpression of constitutively active
SF-1 (SF-1
LBD) (11), SF-1, Egr-1, or Pitx1 also failed
to rescue the
GSU promoter from androgen-negative regulation (data
not shown) providing further support for specific roles of cJun and
ATF2. Importantly, no significant difference in promoter activity was
found when cDNA from individual expression vectors was increased from
60 to 600 ng/well (Fig. 1
). This suggests that rescue activity is
caused by specific interactions with AR, and is not due to an increase
in the amount of transfected cDNA representing the transcription
factors.
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We established the specificity of interactions between AR and cJun/ATF2
through the use of a mutant AR harboring an alanine substitution for
cysteine in the first zinc finger region (C576A) of the DBD. This
mutant AR was shown previously to be unable to suppress activity of the
GSU promoter, suggesting that the cysteine substitution alters a
critical protein-protein interaction (6). This notion is
supported by other studies showing that mutations in the GR-DBD (C500Y,
L501P) abrogated octamer factor binding and their recruitment to DNA
(16). Another level of functional specificity is achieved
by the binding of cJun/ATF2, since cJun alone bound both the wild-type
and mutant AR-DBD while ATF2 bound only to the wild-type construct.
Interestingly, when ATF2 and cJun were added together, cJun no longer
bound the mutant AR (Fig. 2
). Together, these data suggest that the
cJun/ATF2 heterodimers expose a specific protein interface necessary
for mediating the suppressive effects of AR. Alternatively, the loss of
binding between cJun and the AR mutant (C576A) could be due to ATF2
physically pulling cJun from its interaction with C576A because of the
high affinity between the heterodimer pair. Importantly, these data
identify a new binding partner for AR, namely ATF2.
We found it noteworthy that overexpression of CREB alone repressed
activity of the GSU promoter in a gonadotrope-derived cell line,
while overexpression of cJun or ATF2 alone had little effect on
transcription (Fig. 1
). These findings contrast with earlier work
indicating that overexpression of cJun in a cell line derived from
trophoblasts repressed activity of the
GSU promoter through its
tandem CREs (17). Together these observations suggest that
the activity of individual CRE-binding proteins may vary between cell
types. For example, in an earlier study, we replaced the tandem CREs
with AP-1 elements that bind only cJun-containing heterodimers. The
activity of this mutant
GSU promoter was severely abrogated when
examined in trophoblasts, but not in gonadotropes (18).
This differential activity of the
GSU promoter is further supported
by the fact that most other nonprimates harbor a single variant CRE
that binds only cJun/ATF2 (8, 19). Interestingly, these
nonprimates express their
GSU gene in gonadotropes, but fail to
express it in trophoblasts (19, 20, 21). For these reasons, we
focused on cJun, ATF2, and CREB rather than CREM or ATF1. Thus, there
appears to be a strong correlation between the binding of heterodimers
that contain cJun and gonadotrope-specific expression of the
GSU
gene. Since androgens negatively regulate expression of
GSU in all
mammals, our data suggest that cJun is a critical target of AR and that
its heterodimer partner, ATF2, adds yet another level of
selectivity.
The suppression of GSU transcription by CREB suggests that it
competes with endogenous cJun/ATF2 for CRE binding and inhibits gene
activity. Interestingly, the addition of both CREB and AR
completely abrogated
GSU promoter activity (Fig. 1
). AR has no
effect on CREB expression as tested by immunoblot assay in androgen-
treated
T31 cells (data not shown). Perhaps the loss in
GSU
promoter activity occurs as a result of CREB and AR competing for
additional adaptor proteins such as CREB-binding protein (CBP), or
because CREB inhibits cJun and ATF2 from binding the CREs along with AR
binding the remaining heterodimers, or a combination of these events.
The addition of CBP in transient transfection assays had no effect on
AR suppression of
GSU promoter activity (data not shown). However,
cotransfection with both CREB and CBP may be what is required to
relieve the
GSU promoter from the suppressive effects of AR.
AR-Mediated Suppression of GSU Promoter Activity Does Not Depend
On Altered Protein Concentrations
While assessing AR protein-protein interactions, we evaluated
whether additional indirect mechanisms were contributing to the
suppression of the GSU promoter. For example, complete negative
regulation by androgens could require a concerted set of
transcriptional and posttranscriptional mechanisms including altered
expression of critical factors. Whole-cell lysates, from
T31 cells
transiently transfected with AR, were prepared at specific time points
after treatment. For AR measurements, lysates were prepared after
1 h, based on reports of ligand-induced degradation of ER
(22), and after 24 h, since all transfection assays
were harvested at this time point. Expression of AR appeared to be
stabilized in the presence of ligand (Fig. 3A
). Western blot analyses of cJun, ATF2,
and CREB, however, did not indicate any change in protein expression
after overexpression of AR (data not shown).
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AR-Mediated Suppression of the GSU Promoter Does Not Require
Histone Deacetylase (HDAC) Activity
Histone deacetylation has been associated with repressor activity
of nuclear receptors as chromatin returns to a highly packaged,
transcriptionally inert state (29, 30, 31). A number of the
transcriptional proteins that regulate activity of the GSU promoter
are also known to affect the acetylation status of histones. For
example, ATF2 has intrinsic HAT activity that specifically acetylates
histones H2B and H4 in vitro (32). The middle
portion of ATF2 (residues 112350) has significant similarity to the
HAT domain of p300/CBP-associated factor (PCAF), particularly
motif A, which binds acetyl coenzyme A (32).
Interestingly, motif A of the HAT domain was also found to be
responsible for stimulation of CRE-dependent transcription
(32). In addition, p300/CBP has been shown to either
stimulate or repress promoter activity by acetylating non-histone
proteins, including transcription factors (33, 34, 35, 36, 37).
Finally, AR is also a target for acetylation, with lysine residues
within the carboxy terminus affected by either trichostatin A (TSA) or
the HAT-containing transcriptional coactivators p300 and PCAF. This
acetylation may be functionally significant, depending on the cell
type, given the recent demonstration that this posttranscriptional
modification was important for hormone-dependent transactivation in
prostate cell lines (38).
Nuclear receptors such as retinoic acid and thyroid hormone receptors
function as potent transcriptional repressors by interacting with
corepressors that recruit HDAC complexes to the promoter
(39). To address whether histone deacetylation was
associated with AR-mediated suppression of the GSU promoter,
transient transfection assays were performed in the presence of
increasing amounts of TSA, a specific inhibitor of class I and II HDAC
enzymes. Specific members of class I and II HDAC enzymes are known to
interact with the corepressor SMRT (signal mediator and regulator of
transcription) (40). A reporter vector containing the
minimal thymidine kinase (TK) promoter was used as a positive control;
its activity was stimulated by increasing concentrations of TSA
treatment (inset, Fig. 3B
) (41, 42). In
contrast, TSA had no impact on the activity of the
GSU promoter,
either in the presence or absence of AR (Fig. 3B
). While we acknowledge
that we have not tested for class III HDAC enzyme activity, our data
suggest that class I and II HDAC enzymes have no ability to alter the
transcriptional properties of the
GSU promoter in
T31 cells.
This makes it less likely that AR interacts with a nuclear corepressor
such as SMRT to suppress gonadotropin gene expression in these
cells.
Phosphorylation Status of the AR-DBD Does Not Determine Its Ability
to Suppress the GSU Promoter
Phosphorylation of AR has been suggested to be important for two
steps of receptor activation: acquisition of the ability to bind ligand
and enhancement of DNA binding and subsequent transactivation
(43). Indeed, mutation of serine to alanine in the hinge
region (S650A) resulted in decreased phosphorylation and
transactivation (44).1 To
determine whether this also holds for transrepression, we examined
activity of the GSU promoter in transient transfection assays using
two AR mutants. One carries a double mutation in the amino-terminal
domain, AR S81, 94A, whereas the other harbors the mutation in the
hinge region described above (AR S650A). While specific serine residues
(S650) in the AR-DBD and hinge region have been shown to be required
for AR activation (43, 44), we did not find this residue
or others in the amino terminus (81, 94) to be important
for AR-mediated suppression of
GSU (Fig. 3C
). As no other
phosphorylation sites have been detected in the AR-DBD-hinge region
(554660) (43, 44), these data suggest that the
phosphorylation status of AR is not important for its repressive effect
on the
GSU promoter.
In summary, we propose a model whereby AR suppresses GSU promoter
activity through direct protein-protein interactions with a specific
subset of CRE-binding transcription proteins, including cJun and ATF2
(Fig. 4
). Because AR suppression requires
functional CRE sequences (6), we conclude that the
protein-protein interactions occur within the promoter sequences. While
cJun and ATF2 play critical roles in mediating the suppressive effects
of androgens, we have also shown in previous studies (6)
that essential contributions also come from an adjacent regulatory
element,
BE. These combined data suggest that shared adaptor
proteins may be involved in transducing the signal from the proteins
that bind
BE and the CREs. We are currently identifying proteins
that bind
BE and predict that they will probably form a higher order
complex with cJun and ATF2. While AR may potentially interact with
proteins that bind
BE or those involved in bridging
BE and the
CREs, our data suggest that AR-mediated suppression of
GSU promoter
activity requires at least the selective participation of cJun and
ATF2. We suggest that AR interacts with cJun and ATF2 via its DBD
leaving other regions such as the LBD free to interact with other
adaptor proteins. These interactions most likely disrupt the synergy
that is required between proteins that bind
BE and cJun/ATF2 on the
CREs and consequently attenuate activity of the
GSU promoter.
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MATERIALS AND METHODS |
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DNA
All plasmid DNAs were prepared from overnight bacterial
cultures using QIAGEN DNA plasmid columns according to
manufacturers protocol (QIAGEN, Chatsworth, CA). The
human GSU promoter construct has been described previously (7, 8, 45). The wild-type human AR (hAR) expression vector consists
of the full-length AR cDNA fused to the cytomegalovirus (CMV) promoter
(46). CMVGH (47) was described previously and
has been used as a control expression vector that encodes a protein
unrelated to gonadotrope activity. AR mutants S81, 94A, and S650A were
generously provided by Elizabeth Wilson (University of North Carolina,
Chapel Hill, NC) (44). CMVCREB was constructed by
inserting the EcoRI/XhoI fragment of CREB
into
pcDNA3 (Invitrogen, San Diego, CA) (48).
pCMV2-cJun was kindly supplied by Paul Dobner (University of
Massachusetts Medical Center, Worcester, MA) (49).
pCMV2-cJun was digested with EcoRI, treated with calf
alkaline phosphatase, and ligated into pGEM-4Z (Promega Corp., Madison, WI) for TnT reactions. ATF2 (50)
was constructed by inserting the full-length 1,900-bp BamHI
fragment from RSV-ATF2, kindly donated by Michael Green (University of
Massachusetts Medical Center, Worcester, MA), into CMV5 and pcDNA3
expression vectors. GST-hAR-DBD encoding amino acids 554644 of hAR
inserted into pGEX-5X-1 (Amersham Pharmacia Biotech,
Uppsala, Sweden) was generously provided by Drs. Olli Janne and Jorma
Palvimo (University of Helsinki, Helsinki, Finland) (15).
GST-hAR-DBD-C576A was made by inserting the PCR fragment containing
residues 554644 of full-length mutant AR-C576A (46) (PCR
primers 5' with BamHI linker
5'-GCGCGGATCCTTTCCACCCCAGAAGACCTGC-3', 3' with EcoRI linker
5'-GCGCGAATTCCTCTCCTTCCTCCTGTAGTTTCAG-3') into pGEX-2T
(Amersham Pharmacia Biotech).
Cell Culture and Transient Transfections
T31 cells were maintained in high-glucose DMEM supplemented
with 5% FBS, 5% horse serum, penicillin, and streptomycin (Life Technologies, Inc.). Twenty-four hours before transfection,
180,000 cells were plated per 35-mm well in six-well plates. Cells were
transfected with the indicated DNAs using LipofectAMINE (Life Technologies, Inc.) according to the manufacturers guidelines.
Reporter constructs (luciferase, 1.25 µg/well) were cotransfected
with expression vectors as indicated. The amount of transfected cDNAs
was kept constant in the dose-response transfections by adding empty
CMV expression vector (CMV5). The lipofectamine/DNA solution was
replaced with complete medium containing charcoal-stripped serum along
with various treatments after 1216 h. Treatments included 100
nM DHT, 100% EtOH vehicle, MG132 (as indicated), or TSA
(as indicated). Cells were harvested 24 h later using 150 µl of
reporter lysis buffer (Promega Corp., Madison, WI).
Luciferase activity was quantified by luminescence using 15 µl lysate
and 100 µl luciferase assay reagent (Promega Corp.).
ß-Galactosidase activity was quantified also by luminescence using
the Galacto-light assay system (Tropix, Bedford, MA). The values were
averaged over a minimum of three independent experiments.
Western Blot Analysis
Cells were rinsed twice with ice-cold PBS solution and harvested
from the plates, and then resuspended in cell lysis buffer (1% Triton
X-100, 0.1% SDS, 150 mM NaCl, 50 mM Tris-Cl,
pH 8, with protease inhibitors PMSF, aprotinin, and leupeptin) and
incubated on ice with frequent vortexing for 30 min. The cell debris
was pelleted and the supernatant was analyzed for protein content by
Bradford analysis (Bio-Rad Protein Assay, Bio-Rad Laboratories, Inc. Hercules, CA). Lysate (3040 µg) was resolved on an
SDS-PAGE gel using a 4% stacking gel and a 10% separating gel as
described elsewhere (7). Proteins were then transferred to
a nitrocellulose membrane (Protran, Schleicher & Schuell, Inc., Keene, NH) and rinsed in a solution of PBS-Tween (0.1%
Tween-20 in PBS). The membrane was blocked in PBS-Tween with 2.5% dry
milk at 4 C overnight, and then incubated with primary antibodies, as
indicated, in PBS-Tween-2.5% dry milk for 23 h at room temperature.
The membrane was rinsed three times in PBS-Tween, and then incubated
with PBS-Tween plus 2.5% dry milk containing the secondary antibody
for 12 h at room temperature. After three 15-min rinses in PBS-Tween,
the antibody-labeled proteins were visualized by chemiluminescence
using the Renaissance Western Blot Chemiluminescence Reagent Plus
(NEN Life Science Products, Boston, MA).
Antibodies
The following antibodies were used. Primary: p53, AR
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA);
secondary: antirabbit horseradish peroxidase (Santa Cruz Biotechnology, Inc., or Amersham Pharmacia Biotech).
Purification of GST Fusion Constructs and GST-Pull-Down
Assays
Empty GST construct (pGEX-5X-1), GST-hAR-DBD, or
GST-hAR-DBD-C576A was transformed into the DH5 strain of
Escherichia coli. A single colony was inoculated into 2 ml
LB + ampicillin (100 µg/ml) and incubated in a 37 C shaker for
5 h. This inoculation was then diluted 1:15 in fresh LB-amp broth
and incubated at 37 C overnight. Sixteen hours later, a further
dilution (1:100) was incubated for 3 h at 37 C. Isopropyl
ß-D-thiogalactopyranoside was added to a final
concentration of 1 mM, and culture was grown for
another 4 h at 37 C. Bacteria were pelleted at 7500 x
g for 10 min at 4 C and then frozen overnight at -80 C. The
pellet was resuspended in 3 ml NET (150 mM NaCl,
50 mM Tris-Cl, pH 7.4, 5 mM
EDTA, PMSF, pepstatin A, leupeptin, and aprotinin protease inhibitor
solution) with 1 mg/ml lysozyme added. The pellet was vortexed
frequently during the 15- to 30-min incubation. The cells were
disrupted by one freeze-thaw cycle, a 30 sec sonication, and two
additional freeze-thaw cycles. The suspension was centrifuged at 35,000
rpm for 1 h at 4 C, and the supernatant was aliquoted and stored
at -80 C.
GST-protein extracts (75 µl) were incubated with 25 µl Glutathione Sepharose Beads (Amersham Pharmacia Biotech) for at least 2.5 h at 4 C and then washed twice with 1 ml NENT buffer (20 mM Tris-Cl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% IGEPAL, 0.5% dry milk, and the protease inhibitor solution) and twice with 1 ml binding buffer (20 mM HEPES, pH 7.9, 10% glycerol, 60 mM NaCl, 1 mM dithiothreitol, 6 mM MgCl2, 1 mM EDTA, and the protease inhibitor solution). The beads were resuspended in 200 µl binding buffer and incubated with 46 µl in vitro translated products at 4 C overnight. Indicated expression vectors were used to make [35S] methionine-labeled in vitro translated products with TnT Coupled Reticulocyte Lysate reaction system according to manufacturers instructions (Promega Corp.). The matrix was washed three times with 1 ml NENT buffer, and then twice with binding buffer and resuspended in 20 µl elution buffer (3 mg/ml glutathione in 50 mM Tris-Cl, pH 7.5). After a 10-min incubation at room temperature, the suspension was centrifuged, and 18 µl of the eluant were loaded onto a 10% SDS-PAGE gel for analysis. GST-hAR-DBD, GST-hAR-DBD-C576A, or GST-bound radiolabeled protein products were visualized after exposure to film for 5 d (Biomax MR, Eastman Kodak Co., Rochester, NY).
Statistical Analysis
Differences in luciferase activity compared with control were
analyzed by a two-tailed Students t test (Fig. 3B).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ATF, Activation transcription factor; AR,
androgen receptor; ARE, androgen response element; BE,
basal
element; CRE, cAMP response element; CBP, CREB binding protein; CMV,
cytomegalovirus; CREB, CRE binding protein; CREM, CRE modulator; DBD,
DNA-binding protein; DHT, dihydrotestosterone; ER, estrogen receptor;
hAR, human AR; HAT, histone acetyltransferase; HDAC, histone
deacetylase; LBD, ligand-binding domain; PMSF, phenylmethylsulfonyl
fluoride; RSV, rous sarcoma virus; TK, thymidine kinase; TSA,
trichostatin A.
1 Full-length AR numbering is based on 919
residues.
Received for publication March 13, 2001. Accepted for publication May 14, 2001.
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REFERENCES |
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