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INTRODUCTION |
The AR1 is a member of
the nuclear hormone receptor superfamily of
ligand-dependent transcription factors. In the nucleus, the
ligand-activated receptors bind to their cognate response elements in
or near promoter regions of target genes to positively or negatively
regulate gene expression (reviewed in Ref. 1). The mechanism by which
specific modulation of target gene expression is achieved by nuclear
receptors (NRs) is not clearly defined. Early research indicated that
there might be factors common to different receptors required for
efficient transcriptional activation. This was demonstrated by the
observation that different ligand-bound steroid receptors affected or
inhibited transactivation by other NRs. This suggested the
sequestration of mutually required cofactors (2, 3).
Subsequent work has led to the identification of several coactivators
of NR-mediated transactivation. These coactivators enhance levels of NR
transactivation severalfold (reviewed in Refs. 4 and 5) and display a
broad specificity across the superfamily, with the exception of ARA70,
which appears to be specific for the AR (6). The NR-bound coactivator
proteins are believed to enhance the stability of the pre-initiation
complex, resulting in increased rates of transcription initiation.
Direct contacts between pre-initiation complex components and
coactivators have been demonstrated, supporting this hypothesis; for
example, SRC-1 has been shown to interact with TATA box-binding protein
and TFIIB (7). In addition, members of the NR superfamily have also
been shown to interact with components of the pre-initiation complex (8-10).
The discovery that the coactivators SRC-1, p300, and CBP (CREB-binding
protein) possess intrinsic histone acetyltransferase (HAT) activity
provided further insight into the regulation of NR transactivation (11,
12). Histone acetylation has long been associated with transcriptional
activity (13), and the subsequently altered nucleosomal structure is
believed to facilitate access for additional factors necessary for
activation (14). Histone deacetylases can reverse the effects on
nucleosomal disruption of HAT proteins, leading to repression of gene
expression (15-17). The subsequent demonstration that a histone
deacetylase, HDAC1, interacted with the NR corepressors SMRT (18) and
NCoR (19) provoked the realization that the transcriptional output
directed by a NR from a given gene may be reflected in the position of equilibrium between histone acetylation and deacetylation. Coactivators and corepressors may possess such activities or recruit proteins that
do to exact their function.
Recent work investigating the progesterone receptor (PR) has shown that
when bound by ligand, it exists in stable ternary complexes with SRC-1
and TIF-2 (20). Such complexes may exist with other NRs, enhancing the
potential for diverse interaction upon ligand binding. In addition,
many coactivators have been shown to interact with one another, such as
CBP/PCAF (21), SRC-1/CBP (22), SRC-1/p300 (23), and SRC-1/PCAF (11).
Also, multiple NR-interacting domains have been identified in certain
coactivators such as SRC-1 (24). The multiplicity of such protein
interactions therefore suggests that maximal transcriptional activation
is likely to require the targeted coupling of hyperacetylated histones with stabilized pre-initiation complexes and that this is likely to be
mediated by multimeric, coactivating, HAT-containing protein complexes.
The observed complexity of gene expression may be reflected in the
composition of these higher-order, activating complexes.
In this study, we sought to identify AR-interacting proteins in an
attempt to dissect the mechanism by which the AR exacts transcriptional
control. Due to the multitude of potential interacting proteins, we
utilized the yeast two-hybrid system (25). This led to the
identification of the Tat-interacting protein (Tip60) as an
AR-interacting protein. Tip60 was originally reported as a human
coactivator for the human immunodeficiency virus type I-encoded TAT
protein (26), and more recent work has shown that it is a member of a
family of related genes involved in transcriptional regulation (27).
Subsequently, we demonstrated that Tip60 acts as a
ligand-dependent coactivator for the AR, PR, and ER
(estrogen receptor). The level of enhancement observed is comparable to that of SRC-1, p300, and CBP in our system, and, taken together, these
results suggest that Tip60 is potentially an important member of the NR
coactivator family.
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EXPERIMENTAL PROCEDURES |
Yeast Two-hybrid Screening
Residues 559-918 of the hAR (GenBank accession number m23263)
containing the DNA and ligand binding domains were amplified by PCR
from pGEM3Z-AR (a gift from S. Liao, University of Chicago, Chicago, IL) using primers GGACTCTAGAATGCCCATTGACTATTACTT and TGTTTCTAGAGCTTCACTGGGTGTGGAA and subcloned into pT7-7 via
XbaI sites. This fragment was released by
NdeI-BamHI restriction of pT7-7hAR559-918 and
subcloned downstream of the GAL4 DNA binding domain (DBD) in pAS2-1
(CLONTECH). pAS2-1hAR559-918 was transformed into
the yeast strain PJ69-4A (a gift from P. James, University of
Wisconsin). PJ69-4A is an improved two-hybrid strain with an additional
nutritional reporter gene that serves to reduce false positives (28).
The subsequent tryptophan-positive strain was transformed with 50 µg
of a human brain cDNA:GAL4 activation domain fusion library
constructed in pACT2 (CLONTECH). 200,000 transformed cells were plated onto media lacking tryptophan, leucine,
and adenine, supplemented with 1 µM dihydrotesterone
(DHT). Adenine-positive, LacZ-positive, histidine-positive colonies
were isolated, and plasmid DNA was prepared and transformed into
Escherichia coli according to the protocol provided by
CLONTECH. pACT2 plasmids containing cDNA were
identified by colony PCR using primers specific for the LEU2
gene contained in pACT2. Specificity of interaction for the hAR was
determined by examining the liquid LacZ activity (see below) of
cDNA clones in the presence of the GAL4DBD:hAR559-918 fusion
protein versus the activity observed in the presence of the
GAL4DBD alone. After sequencing and examination of the
GenBankTM database, we determined that one interacting
clone was encoded by a 1.7-kb cDNA fragment with a 96% identity to
Tip60 (GenBank accession number u40989), which maintained the reported
open reading frame for Tip60 (25). Sequence alignment revealed that the
identified clone began 70 residues downstream of the NH2
terminus. Subsequent two-hybrid screens in our laboratory have
identified full-length Tip60 cDNAs as AR-interacting clones,
suggesting that the 70 residues absent from our construct did not
significantly interfere with the interaction. All Tip60 constructs used
in this work are derived from the original 1.7-kb clone.
Analysis of Tip60-AR Interaction Using the Yeast Two-hybrid
System
pAS2-1hAR constructs were co-transformed with pACT2Tip60 or
pACT2 into PJ69-4A according to the manufacturer's guidelines (CLONTECH). Subsequent Trp-positive, Leu-positive
colonies were inoculated in triplicate into selective media and grown
at 30 °C overnight in the presence or absence of 1 µM
DHT. Samples were diluted to an A600 of 0.2 and
re-grown to an A600 of 0.6-0.8. Samples were
divided into three 1-ml aliquots. Cells were recovered by
centrifugation at 14,000 rpm for 5 min, washed once with buffer Z (0.1 M sodium phosphate, pH 7.0, 10 mM KCl, and 10 mM MgSO4) and resuspended in 800 µl of buffer
Z containing 21 µl of
-mercaptoethanol. 10 µl of 0.1% SDS were
added, followed by 50 µl of chloroform. Samples were vortexed for 1 min and placed at 30 °C, and 200 µl of
o-nitrophenyl-
-D-galactopyranoside (4 mg/ml in buffer Z) were added. Reactions were timed and terminated upon
observing an obvious yellow color or after 1 h via the addition of
500 µl of 1 M Na2CO3. A420 of the samples was determined, and activity
was calculated as follows: (A420 × 1000)/(A600 × time). All assays were performed in triplicate and repeated at least three times. hAR fusion proteins for these experiments were constructed as follows; hAR559-918 was
constructed as described above. Additional constructs were amplified by PCR using the following primer combinations: hAR1-150 (p1:TCAAGGATCCAAGTGCAGTTAGGGCTGGGA - p3:ACGTGGATCCGGCAGCTGAGTCATCCTCGT), hAR1-300
(p1-p5:CCTAGGATCCCGCCTTCTAGCCCTTTGGTG), hAR559-624
(p6:TATTGGATCCCACCCCAGAAGACCTGCCTG-p9:TCCGGGATCCCAGAGTCATCCCTGCTTCAT), and hAR624-918 (p8:GCCCGGATCCTGAAGAAACTTGGTAATCTG-
p10:TAGGGGATCCAATGCTTCACTGGGTGTGGA). PCR products were cloned
into the BamHI site of pAS2-1. All fusion constructs were
sequenced to confirm maintenance of the open reading frame, and
expression was confirmed by Western blotting using anti-AR antibodies (Novocastra).
In Vitro Interaction of AR and Tip60
Templates were prepared using a Geneclean kit (Anachem) and
resuspended in RNase-free distilled H2O. An in
vitro coupled transcription and translation kit (T7-TNT; Promega)
was used according to the manufacturer's instructions. After
completion of the 90-min reaction, samples were combined equally on
ice, as indicated. 1 ml of immunoprecipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.2 mM Na3VO4, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, 25 µg/ml leupeptin, 25 µg/ml aprotinin, and 25 µg/ml pepstatin) was added to each sample, mixed, and incubated on
ice for 30 min. 20 µl of protein G-Sepharose (PGS) pre-washed three
times in immunoprecipitation buffer were added to each sample and
incubated for an additional 4 h at 4 °C with rotation to remove
any proteins that interacted nonspecifically with PGS. PGS was removed
by centrifugation at 14,000 rpm for 3 min. The supernatant was
incubated with 2.5 µg of penta-His antibody (Qiagen) overnight at
4 °C with rotation. 20 µl of PGS were added to each sample and
incubated at 4 °C for an additional 60 min. PGS antibody conjugates
were recovered by centrifugation at 14,000 rpm for 3 min, resuspended
in 1 ml of buffer A (PBS, 0.2% Triton X-100, and 350 mM
NaCl), and re-spun. Samples were resuspended in 1 ml of buffer B (PBS
and 0.2% Triton X-100), re-spun, and resuspended in SDS sample buffer.
Samples were resolved on 10% polyacrylamide gels at 200 V for 45 min
with equal loading. The gel was fixed for 30 min (10% propanol and 10% acetic acid), soaked in Amplify (Amersham Pharmacia Biotech) for
30 min, dried under vacuum, and exposed to x-ray film for 4-24 h at
70 °C. Templates used were pT7-7hAR1-918 and full-length Sap-1a
in pASG4 (a gift from A. D. Sharrocks, Newcastle University). pRSET-C-Tip60 was constructed via excision of a BamHI
fragment from pACT2Tip60 utilizing a BamHI site in the 3'
untranslated region of Tip60 cDNA and cloned in-frame with the
hexaHis tag of pRSET-C (R&D Systems). Orientation was determined via
restriction analysis and confirmed by sequencing.
Transient Transfections of COS-1 and LNCaP Cells
COS-1 Cells--
Cells were cultured for at least 48 h
before transfection in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal calf serum that had
been stripped of steroids by treatment with dextran-coated charcoal.
Cells were routinely grown to 60-80% confluence, washed, removed, and
seeded at a density of 5 × 104 cells/ml in fresh
medium in 35-mm wells (Corning). After 20 h, cells were washed
once with serum-free medium and transfected using Lipofectin (Life
Technologies, Inc.) according to the manufacturer's recommendations.
The amounts of DNA used are described in the figure legends. After
7 h, cells were washed twice with the appropriate medium and
incubated in 2 ml of medium containing steroid or vehicle, as
indicated. Mibolerone is used in mammalian experiments with the AR
because it is a specific androgen analogue. After 48 h, cells were
recovered and assayed for luciferase activity according to the
manufacturer's instructions (Promega). Luciferase activity was
corrected for the corresponding
-galactosidase activity to give
relative activity.
-Galactosidase assays were typically performed in
a 96-well plate (Corning) as follows: 10 µl of sample extract were
incubated with 80 µl of buffer Z and 10 µl of
o-nitrophenyl-
-D-galactopyranoside (4 mg/ml)
at 30 °C for 2 h. The reaction was terminated via the addition
of 50 µl of 1 M Na2CO3.
A420 values were obtained using a MR5000 plate
reader (Dynatech), and activity was calculated as described above.
Transfections were performed in triplicate and repeated at least three times.
LNCaP Cells--
These cells were routinely subcultured in RPMI
1640 medium (Life Technologies, Inc.) supplemented with 10% fetal calf
serum. Cells were seeded at 1 × 105 cells/well in
35-mm wells at least 24 h before transfection. For studies
examining the ER and PR, cells were cultured in phenol red-free RPMI
1640 medium supplemented with 10% dextran-coated charcoal-fetal calf
serum. Cells were transfected using Lipofectin for a period of 8 h
and washed, and medium containing vehicle or steroid was added as
indicated. Cells were harvested at 72 h after transfection and
analyzed as described above. pCMV5Tip60 was constructed via cloning of
a BglII fragment from pACT2Tip60 into pCMV5. Expression was
confirmed using an anti-HA antibody (Santa Cruz Biotechnology).
hAR1-918 was cloned into the XbaI site of pCDNA3. The
following mammalian expression plasmids were gratefully received:
RSVhPRB and full-length hSRC-1a in pCR3.1 (M.-J. Tsai; Baylor College
of Medicine, Houston, TX), pHEGO (wtER; P. Chambon; Institute de
Genet/Biology Molec/Cell), pRc/RSVmCBP (R. H. Goodman, Oregon
Health Sciences University), and pCMV
-p300 (D. M. Livingston,
Dana-Farber Cancer Institute, Boston, MA). The reporter plasmid
pMTV-Luc (a gift from R. Vogel, Merck and Co., Inc.) was used for
experiments with the AR in COS-1 cells and for experiments with the PR
in LNCaP cells. For the ER in LNCaP cells, the reporter plasmid was
pVITEREluc (a gift from B. Westley, Newcastle University). In addition,
pPSAluc, a reporter construct derived from the PSA promoter, was used
for AR-based experiments in LNCaP cells. A 658-base pair fragment
containing two androgen-responsive elements was amplified by PCR from
the PSA promoter (GenBank accession numbers u37672 and x13940) using primers GTGGAGCTGGATTCTGGGT and CTCAGCTTGACAGTCAGGG. This product was
cloned into the TA vector (Invitrogen), released via
KpnI/XhoI digestion, and inserted into the
corresponding sites in the luciferase reporter plasmid pGL3basic
(Promega). For Fig. 6, pSGp53 expressing GAL4DBDp531-42
was obtained from A. D. Sharrocks (Newcastle University). The
reporter plasmid TK-GAL4UASLuc and the GAL4DBD-expressing plasmid
pSG424 were gifts from R. Clifton-Bleigh (Cambridge University). The
Tip60:GAL4DBD fusion construct was created by the insertion of the
BamHI fragment from pACT2Tip60 into the BamHI
site of pSG424 downstream of the GAL4DBD. Correct orientation was
determined by sequencing, and expression was confirmed via Western
analysis with an anti-GAL4DBD antibody
(CLONTECH).
 |
RESULTS |
Tip60 Interacts with the Ligand Binding Domain of the
AR--
Yeast two-hybrid screening, which was originally described by
Fields and Song (25), using the AR construct AR559-918 comprising the
DNA and ligand binding domains (DBD and LBD, respectively) as bait
identified Tip60 as an AR-interacting protein (see "Experimental Procedures"). In an attempt to define the specificity of the
interaction with the AR, we examined the association of Tip60 with
various GAL4DBD:AR constructs in the yeast two-hybrid system in the
presence of 1 µM DHT. AR constructs spanning the first
300 residues (AR1-150 and AR1-300) and the DBD of the AR (AR559-624)
failed to interact with the Tip60GAL4AD fusion protein (Fig.
1a), whereas both AR559-918 and AR624-918 interacted specifically with Tip60. Therefore, Tip60 appears to interact specifically with the LBD of the AR. The
interaction appeared to be increased significantly with the attachment
of the DBD to the LBD, although the DBD of the AR fails to interact by
itself. A potential explanation for this observation is that the LBD
may be prevented from folding into the optimal conformation for
interaction when positioned directly at the COOH terminus of the
GAL4DBD. The presence of the DBD may serve to distance the LBD from the
GAL4DBD in this scenario, or, alternatively, it may be that the AR DBD
is essential for directing or influencing the conformational change
upon ligand binding.

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Fig. 1.
Tip60 interacts specifically with the ligand
binding domain of the AR. a, the interaction of Tip60
with various regions of the AR was examined in the presence of 1 µM DHT using the yeast two-hybrid system and quantified
using a liquid LacZ assay. AR559-918 and AR624-918 interacted with
Tip60, whereas AR1-150, AR1-300, and AR559-624 did not.
b, the interaction of Tip60 with the LBD is not
ligand-dependent but is enhanced in the presence of 1 µM DHT; yeast two-hybrid analysis performed in the
absence and presence of 1 µM DHT showed a significant
increase in interaction in the presence of DHT. Data are expressed as
the mean of at least three separate experiments performed in
triplicate ± S.D.
|
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The interaction of AR and Tip60 is not ligand-dependent
(Fig. 1b) but is enhanced in the presence of ligand in the
yeast two-hybrid system. We examined the interaction of the AR regions
559-918 and 624-918 with Tip60 in the presence and absence of 1 µM DHT and found that the interaction was significantly
enhanced in the presence of DHT. Values obtained in the absence of DHT
for AR559-918 and AR624-918 were similar, indicating that the AR DBD
may have a role to play in altering the conformation of the AR upon
ligand binding in this system. The majority of interactions reported to
date, such as ARA70 with the AR (6), have been
ligand-dependent. It is therefore interesting that the
AR:Tip60 interaction is not ligand-dependent.
Significantly, PCAF, a human homologue of the yeast Gcn5 (21), has been
shown to bind to the AR and ER in a ligand-independent manner and to
function as a coactivator for nuclear receptors (29). The interaction
reported in the yeast two-hybrid system between PCAF and the AR is
similar to the interaction observed between Tip60 and the AR in that it
is enhanced in the presence of ligand (29). Thus, Tip60 and PCAF may
represent a class of coactivators that are complexed with the
aporeceptor. It is possible that the ligand-independent Tip60:AR
complex could hold the AR in a state of readiness, thus facilitating a
rapid activation response upon ligand binding, enabling the directed recruitment of other cofactors to AR-regulated genes.
Tip60 Interacts with the AR in Vitro--
To confirm the
interaction data obtained from the two-hybrid system, we sought to
determine whether Tip60 could interact with the AR in vitro.
Using proteins transcribed and translated in vitro in the
presence of [35S]methionine, we attempted to
co-immunoprecipitate the AR with Tip60 using an anti-His antibody
recognizing His-tagged Tip60. The transcription factor Sap-1a (Elk4)
(30), an Ets family-related protein, was included as a control for this
experiment. Before immunoprecipitation, labeled HexaHisTip60 was
incubated with rabbit reticulocyte lysate extracts containing Sap-1a or
AR, as indicated (Fig. 2, lanes
2 and 4, respectively). Lanes 1 and
3 show the control samples, in which immunoprecipitations
were carried out on extracts containing Sap-1a or AR in the absence of
Tip60 to determine the levels immunoprecipitated that are not
specifically associated with Tip60. Confirmation of the two-hybrid
results was obtained via the observation that full-length AR
co-immunoprecipitated with Tip60 (lane 8),
whereas very low amounts of nonspecific AR were visible in the absence
of Tip60 (lane 7). This interaction was not affected by the
presence of ligand (data not shown), again a feature previously
described for PCAF (29). Importantly, Sap-1a did not interact with
Tip60 in this experiment, confirming the specificity of the
immunoprecipitation procedure because no additional Sap-1a is recovered
in the presence of Tip60 (lane 6) versus the absence of Tip60 (lane 5). We also attempted to demonstrate
the interaction using anti-AR. In this system, the only anti-AR that works consistently in our hands for the purposes of immunoprecipitation is sc-805 (Santa Cruz Biotechnology). However, this proved
unsuccessful. Subsequent investigation leads us to believe that the
antibody directed against the ligand binding domain and Tip60 compete
for AR binding. This phenomenon has been reported for other studies in
which antibody against an AR-interacting protein, BAG-1L, failed to
co-immunoprecipitate the AR (31). However, our experiment, in
combination with the two-hybrid data, provides sufficient evidence for
the AR:Tip60 interaction.

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Fig. 2.
Tip60 interacts specifically with the AR
in vitro. 35S-labeled proteins were
mixed after in vitro translation and before
immunoprecipitation (Pre-IP) as indicated in lanes
1-4. Samples were incubated with anti-His antibody at 4 °C
overnight, which recognized the His-tagged Tip60. Antibody complexes
were recovered with PGS, washed, and subjected to SDS-polyacrylamide
gel electrophoresis. Resultant gels were soaked in Amplify (Amersham
Pharmacia Biotech) and exposed to x-ray film (lanes 5-8 , Post-IP). Significant amounts of AR co-immunoprecipitate
with Tip60 (lane 8) versus immunoprecipitation in
the absence of Tip60 (lane 7). Incubation of Tip60 with
Sap-1a (lane 6) did not alter the amount of Sap-1a recovered
(lane 5), demonstrating that the Tip60:AR interaction is
specific.
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Tip60 Enhances AR-mediated Transactivation--
As stated earlier,
Tip60 was previously identified as a coactivator for the human
immunodeficiency virus type I-encoded TAT protein. This fact led us to
believe that it could act in a similar manner for the AR. We
investigated the effect of increasing levels of Tip60 upon
AR-dependent transactivation in transiently transfected LNCaP and COS-1 cells (Fig. 3). In LNCaP
cells, pPSAluc, a PSA-derived reporter plasmid containing two
androgen-responsive elements, was utilized. Co-transfection of the
indicated plasmids demonstrated a ligand-dependent
enhancement of AR-mediated transactivation by Tip60 (Fig.
3a). In these experiments, Tip60 expression produced a
5-fold enhancement of AR transactivation without any effect on basal
transcription. We also investigated the effect of Tip60 on AR-mediated
transactivation in COS-1 cells using a reporter plasmid derived from
the murine mammary tumor virus promoter pMTVluc (Fig. 3b).
Expression of Tip60 in this context resulted in a
ligand-dependent, 3-4-fold enhancement of transcriptional
activation without any effect on basal transcription levels. These
results demonstrate that Tip60 is a ligand-dependent
coactivator for the AR. This effect was observed using two different
cell lines and reporter constructs, which provides confidence in the
coactivating properties of Tip60.

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Fig. 3.
Tip60 enhances AR-mediated transactivation in
LNCaP and COS-1 cells. a, effect of increasing amounts
of pCMV5Tip60, as indicated, upon LNCaP cells transfected with 0.5 µg
of pPSAluc and 0.2 µg of pCMV- gal per 35-mm well. pSK was added to
a total of 1 µg of DNA per transfected well. After transfection,
medium containing 10 nM mibolerone (Mib) or
vehicle was added. Mibolerone is a specific androgen analogue. Cells
were harvested after 72 h, assayed for luciferase activity, and
corrected for gal activity to give relative activity. b,
effect of increasing amounts of pCMV5Tip60 on COS-1 cells transfected
with 0.2 µg of pCDNA3-AR1-918, 0.5 µg of pMTVluc,
and 0.2 µg of pCMV- gal per 35-mm well. pCMV5 was added to 1.1 µg/transfection. After transfection, medium containing 10 nM mibolerone (Mib) or vehicle was added. Cells
were harvested 48 h later, and activity was calculated as
described above. Data are expressed as the mean of at least three
separate experiments performed in triplicate ± S.D.
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Tip60 Enhances PR- and ER-dependent
Transactivation--
Having outlined the role of Tip60 in AR-regulated
promoters, we wished to investigate the role of Tip60 in
transcriptional coactivation with other nuclear receptors. pMTVluc was
used to study the effect of Tip60 on PR-dependent
transactivation. Co-transfection of LNCaP cells with pMTVluc, PR, and
Tip60 in the presence of progesterone led to a 4-fold increase in
PR-mediated transactivation (Fig.
4a) without any effects on
basal levels of transcription. This Tip60-mediated increase was
comparable to levels observed with the AR (Fig. 3a) and was
also observed with the PR in COS-1 cells (data not shown). We also
investigated the effect of Tip60 on ER-dependent
transactivation (Fig. 4b). Using pVITEREluc, a reporter
vector derived from the vitellogenin gene, a 3-fold increase in
ER-dependent transactivation was noted. Levels of
enhancement were not as high as those observed with the PR; however,
this may reflect the promoter constructs studied.

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Fig. 4.
Tip60 enhances ER- and
PR-dependent transactivation. a, effect of
increasing amounts of Tip60 on PR-dependent
transactivation. pCMV5-Tip60 was added as indicated to LNCaP cells
transfected with 0.5 µg of pPSAluc and 0.2 µg of pCMV- gal per
35-mm well. pSK was added to a total of 1 µg of DNA/transfected well.
After transfection, medium containing 10 nM progesterone
(P) or vehicle was added. Cells were harvested 72 h
later, and relative activity was determined as described earlier.
b, effect of increasing amounts of Tip60 on
ER-dependent transactivation. pCMV5-Tip60 was added as
indicated to cells transfected as described in a, with the
exception that medium contained 1 nM estradiol or vehicle
after transfection. Cells were harvested 72 h later, and relative
activity was determined as described earlier. Data are expressed as the
mean of at least three separate experiments performed in
triplicate ± S.D.
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Tip60 Coactivates to Levels Observed with SRC-1, CBP, and
p300--
Having established that Tip60 is a nuclear receptor
coactivator, we attempted to make a comparison of the relative extents of coactivation exacted by SRC-1, CBP, p300, and Tip60 upon the AR.
SRC-1, CBP, and p300 are established as important coactivators (reviewed in Refs. 4 and 5), and due to the variability generally
observed in reporter assays, LNCaP cells were transiently transfected
with differing coactivators to confirm the effects observed with Tip60,
as indicated in Fig. 5. In all instances, mean activity is enhanced at least 2-fold. It is clear from these results that Tip60 enhances transcription and therefore coactivates to
levels comparable with SRC-1, CBP, and p300. Hence, this experiment suggests that Tip60 could be an equally important AR coactivator and
could be potentially as influential upon transactivation by nuclear
receptors as SRC-1, CBP, and p300.

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Fig. 5.
Tip60 enhances AR-dependent
transactivation to levels comparable with SRC-1, CBP, and p300.
Effect of 100 ng of plasmids expressing SRC-1, CBP, p300, or Tip60, as
indicated, upon LNCaP cells transfected with 0.5 µg of pPSAluc and
0.2 µg of pCMV- gal per 35-mm well. pSK was added to a total of 1 µg of DNA/transfected well. After transfection, medium containing 10 nM mibolerone (Mib) or vehicle was added.
Relative activity was calculated as described previously. Data are
expressed as the mean of at least three separate experiments performed
in triplicate ± S.D.
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|
Tip60 Contains No Intrinsic Transcriptional Activity--
In an
attempt to define the mechanism by which Tip60 acts as a coactivator,
we sought to determine the existence, if any, of a transcriptional
activation function within Tip60. The presence of an activation
function has been demonstrated for SRC-1, whereas PCAF was shown to
possess no activation function (24). The determination of this property
is essential for the characterization of a coactivator. Although Tip60
contains an atypical zinc finger region (26), no DNA binding ability
has been demonstrated. Thus, we fused Tip60 to a GAL4DBD and examined
its effect upon a GAL4-responsive reporter plasmid (Fig.
6). The observed activity was compared
with the activity of a p53 fragment containing an identified activation domain, p531-42, fused to a GAL4DBD, and the GAL4DBD
alone. It can clearly be seen that Tip60 appears to contain no
intrinsic activation function, producing no additional activity when
compared with GAL4DBD alone, thus distinguishing it from SRC-1. This
feature groups Tip60 with PCAF with respect to coactivator
properties.

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Fig. 6.
Tip60 contains no intrinsic
transcriptional activity. Effect of 200 ng of plasmids expressing
GAL4DBD alone, GAL4DBDp531-42 or GAL4DBDTip60, as
indicated, co-transfected with 0.2 µg of GAL4-responsive reporter
plasmid into COS-1 cells. Cells were recovered after 48 h, and
relative activity was calculated as described earlier. Data are
expressed as the mean of at least three separate experiments performed
in triplicate ± S.D.
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|
 |
DISCUSSION |
In this study, we have identified Tip60 as a nuclear receptor
coactivator. Tip60 is a member of the MYST/SAS family of genes that is
implicated in transcriptional control and conserved between species
(32). This family includes the yeast SAS2 and SAS3 genes implicated in
transcriptional silencing (32), yeast gene product ESA1 with HAT
activity that is essential for growth (33), and the
Drosophila MOF gene required for X-linked dosage
compensation (27). A significant member of the family is the human MOZ
protein, which exhibits strong homology to Tip60, originally identified via a common translocation found in acute myeloid leukemia. Other related human cDNA sequences contain the MYST domain but have no
defined function at present (34). Thus, it is apparent that Tip60 is a
member of a family of proteins that are likely to be important in the
normal and abnormal control of cellular growth.
The MYST/SAS family genes contain HAT-like domains, which are conserved
between members, including Tip60 (32). However, no importance can yet
be attached to this domain in Tip60 with respect to its role in
coactivation. Tip60 has been shown to acetylate free histones but
failed to acetylate nucleosomal histones (35), thus raising concerns
about the physiological substrates, if any, for this domain. If it is
not required for histone acetylation, there may be other non-histone
substrates, for example p53 is acetylated by p300, resulting in a
20-fold increase in affinity for a p53 binding site (36). Thus, an
important future task is to determine the significance, if any, of the
HAT-like domain within Tip60.
An interesting feature contained within the amino acid sequence of
Tip60 is the presence of a LXXLL motif in the COOH region of
the protein (amino acids 458-462, GenBank accession number u40989).
This LXXLL region is present in other NR coactivators such
as GRIP1, CBP, and SRC-1 and has been shown to mediate
hormone-dependent binding to nuclear receptors in some
detail, with the deletion of this region abolishing the interaction
between a NR and coactivator in some instances (37-39). Hence, peptide
regions containing single or multiple LXXLL signatures are
often designated as NR boxes. The presence of this NR box in Tip60 may
be responsible for its ability to act as a coactivator for several NRs.
Other unidentified motifs may account for the ligand-independent
binding observed between Tip60 and the AR.
The lack of intrinsic transcriptional activity within Tip60 raises
mechanistic questions. This feature is similar to PCAF (24), which is
known to interact with several other coactivators and is therefore
thought to recruit additional necessary factors once it is bound to a
NR. PCAF has also recently been shown to enhance transcription when
tethered to a promoter in the presence of an enhancer element and its
cognate transcription factor (40), but not in its absence, suggesting
that it may well recruit factors necessary for the action of a
particular transcription factor. It is interesting to hypothesize that
Tip60 can act in a similar mode, and we are currently investigating
this possibility.
Tip60 and the recently identified nuclear receptor coactivator NCoA-62
(41) both display broad specificity, like most coactivators (5). CBP
interacts with many other transcription factors (reviewed in Ref. 42),
and it is becoming increasingly evident that SRC-1 is a signal
integrator not unlike CBP and p300. For example, it has recently been
shown that SRC-1 can mediate AP-1-dependent transcription
by directly interacting with c-Jun and c-Fos (43). The combination of
SRC-1 and p300 at the AP-1 sites studied resulted in a synergistic
response, highlighting cooperation between coactivators. Also, SRC-1
has been demonstrated to act as a coactivator for nuclear factor
B
and is able to bind specifically to the p50 subunit of nuclear factor
B (44). However, as these features become more evident, the lack of
specificity demonstrated by coactivators provokes questions regarding
the manner in which distinct responses are elicited by nuclear
receptors. The phosphorylation status of the coactivators is not well
studied, although TIF1
was recently shown to be hyperphosphorylated
in the presence of the ER upon estradiol treatment (45). TIF1
was
also shown to possess the ability to phosphorylate TFIIE
,
TAFII28, and TAFII55. It would therefore seem
that specific transcriptional control is unlikely to be achieved by
coactivators exclusive to a particular NR. This is more likely to be
achieved by post-translational modification of transcription factors by
coactivators and of coactivators themselves by other cellular factors.
This will serve to alter the dynamic state of flux between distinct
activating complexes that share many cofactors in order to achieve specificity.
In conclusion, we have demonstrated that Tip60 is a
ligand-dependent coactivator for the AR and that it
interacts directly via the LBD of the AR. The demonstration of this
function has been achieved using transiently transfected reporter
constructs. It would perhaps be more pertinent to examine the effects
of Tip60 on AR-responsive genes, such as ornithine decarboxylase (46) and prostate specific antigen (47), in their normal chromosomal context
to truly assess its significance. However, it is clear that Tip60, in
our system, appears to be as important as SRC-1 and CBP in
AR-dependent transactivation and is thus worthy of further
investigation into its mechanism of action.