Steroid receptor coactivator-1 (SRC-1)
specifically bound to the transcription factor AP-1 subunits c-Jun and
c-Fos, as demonstrated by the yeast two-hybrid tests and glutathione
S-transferase pull down assays. The c-Jun and c-Fos binding
sites were localized to the C-terminal subregion of SRC-1 (amino acids
1101-1441) that encompasses the previously described histone
acetyltransferase and receptor-binding domains. In mammalian cells,
SRC-1, similar to the previous results with CBP-p300 (Arias, J.,
Alberts, A. S., Brindle, P., Claret, F. X., Smeal, T., Karin,
M., Feramisco, J., and Montminy, M. (1994) Nature 370, 226-229; Bannister, A. J., and Kouzarides, T. (1995) EMBO
J. 14, 4758-4762), potentiated the AP-1-mediated
transactivations in a dose-dependent manner and derepressed the
mutual inhibitions between nuclear receptors and AP-1. Furthermore,
coexpression of p300 further enhanced this SRC-1-potentiated level of
transactivations. Thus, we concluded that at least two distinct
coactivator molecules may cooperate to regulate
AP-1-dependent transactivations and mediate transrepression between AP-1 and nuclear receptors in vivo.
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INTRODUCTION |
The activation protein-1
(AP-1)1 transcription factors
are immediate early response genes involved in a diverse set of
transcriptional regulatory processes (1). The AP-1 complex consists of
a heterodimer of a Fos family member and a Jun family member. This
complex binds the consensus DNA sequence (TGAGTCA) (termed AP-1) sites
found in a variety of promoters (2, 3). The Fos family contains four
proteins (c-Fos, Fos-B, Fra-1, and Fra-2) (4-6), whereas the Jun
family is composed of three (c-Jun, Jun-B, and Jun-D) (7-10). Fos and
Jun are members of the bZIP family of sequence-specific dimeric
DNA-binding proteins (11). The C-terminal half of the bZIP domain is
amphipathic, containing a heptad repeat of leucines that is critical
for the dimerization of bZIP proteins (12, 13). The N-terminal half of
the long bipartite helix is the basic region that is critical for
sequence-specific DNA binding (14-16).
Transcription coactivators bridge transcription factors and the
components of the basal transcriptional apparatus (17). Functionally
conserved proteins CREB-binding protein (CBP) and p300 have been shown
to be essential for the activation of transcription by a large number
of regulated transcription factors, including nuclear receptors
(18-21), CREB (22-24), NF
B (25, 26), basic helix-loop-helix
factors (27), STATs (28, 29), and AP-1 (30, 31). In particular, the
nuclear receptor superfamily is a group of ligand-dependent
transcriptional regulatory proteins that function by binding to
specific DNA sequences named hormone response elements in promoters of
target genes (reviewed in Ref. 32). Transcriptional regulation by
nuclear receptors depends primarily upon a ligand-dependent
activation function, AF-2, located in the C terminus and predicted to
undergo an allosteric change upon ligand binding (32). Consistent with
this, CBP and p300 have been found to interact directly with nuclear
receptors in a ligand- and AF-2-dependent manner (18-21).
In addition, a series of factors that exhibit ligand- and
AF-2-dependent binding to nuclear receptors have been
identified both biochemically and by expression cloning. Among these, a
group of highly related proteins have been shown to form a complex with
CBP and p300 and enhance transcriptional activation by several nuclear
receptors, i.e. steroid receptor coactivator-1 (SRC-1) (20,
33), xSRC-3 (34), AIB1 (35), TIF2 (36), RAC3 (37), ACTR (38), TRAM-1 (39), and p/CIP (40). Interestingly, SRC-1 (41) and its homologue ACTR
(38), along with CBP and p300 (42, 43), were recently shown to contain
potent histone acetyltransferase activities themselves and associate
with yet another histone acetyltransferase protein p/CAF (44). In
contrast, it was shown that SMRT (45) and N-CoR (46), nuclear receptor
corepressors, form complexes with Sin3 and histone deacetylase proteins
(47, 48). From these results, it was suggested that chromatin
remodeling by cofactors may contribute, through histone
acetylation-deacetylation, to transcription factor-mediated
transcriptional regulation.
In light of the fact that SRC-1 is capable of forming a complex with
CBP and p300 that in turn coactivate AP-1 (30, 31), we tested whether
SRC-1 itself participates in the AP-1-mediated transactivations as
well. Herein, we show that 1) SRC-1 specifically binds to the AP-1
components c-Jun and c-Fos, 2) SRC-1 coactivates the AP-1-mediated
transactivations, 3) p300 synergizes with SRC-1 in this coactivation,
and 4) SRC-1 relieves the transrepression between nuclear receptors and
AP-1. These results suggest that at least two distinct coactivator
molecules (i.e. SRC-1 and p300) may cooperate to regulate
AP-1-dependent transactivations and mediate transrepression
between AP-1 and nuclear receptors in vivo.
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EXPERIMENTAL PROCEDURES |
Plasmids--
LexA, B42, T7, or GST vectors to express fragments
of SRC-1 (SRC-A through SRC-E as depicted in Fig. 1) were as described previously (49). Polymerase chain reaction-amplified fragments of c-Jun
and c-Fos were subcloned into EcoRI-SalI
restriction sites of the LexA fusion vector pEG202PL (50) and
EcoRI-XhoI restriction sites of the B42 fusion
vector pJG4-5 (50), the GST fusion vector pGEX4T (Amersham Pharmacia
Biotech) or the CMV/T7 vector pcDNA3 (Invitrogen, San Diego, CA).
The expression vectors for p300 (kind gift from Dr. David M. Livingston, Dana Farber Cancer Institute, Boston, MA) and SRC-1 (kind
gift from Dr. Ming Tsai, Baylor College of Medicine, Houston, TX),
along with the transfection indicator construct pRSV-
-gal, the
AP-1-responsive reporter construct (TRE)4-TK-Luc, and the
T3-responsive reporter construct TREpal-TK-Luc, were as described
previously (23, 41, 51, 52).
Yeast Two-hybrid Test--
For the yeast two-hybrid tests,
plasmids encoding LexA fusions and B42 fusions were cotransformed into
Saccharomyces cerevisiae EGY48 strain containing the
LacZ reporter plasmid, SH/18-34 (50). Plate and liquid
assays of
-gal expression were carried out as described (50, 52,
53). Similar results were obtained in more than two similar
experiments.
GST Pull Down Assays--
The GST fusions or GST alone was
expressed in Escherichia coli, bound to
glutathione-Sepahrose-4B beads (Amersham Pharmacia Biotech), and
incubated with labeled proteins expressed by in vitro
translation by using the TNT-coupled transcription-translation system,
with conditions as described by the manufacturer (Promega, Madison,
WI). Specifically bound proteins were eluted from beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0)
and analyzed by SDS-polyacrylamide gel electrophoresis and
autoradiography as described (52).
Cell Culture and Transfections--
CV1 cells were grown in
24-well plates with medium supplemented with 10% fetal calf serum for
24 h and transfected with 100 ng of LacZ expression
vector pRSV-
-gal and 100 ng of a reporter gene
(TRE)4-TK-Luc or TREpal-TK-Luc, along with increasing
amount of expression vectors for SRC-1 or p300. Total amounts of
expression vectors were kept constant by adding decreasing amounts of
pcDNA3 to transfections containing increasing amounts of the SRC-1
or p300 vector. After 12 h, cells were washed and refed with
Dulbecco's modified Eagle's medium containing 10% fetal calf serum.
Cells were harvested 24 h later, luciferase activity was assayed
as described (54), and the results were normalized to the
LacZ expression. Similar results were obtained in more than
two similar experiments.
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RESULTS AND DISCUSSION |
Interactions of SRC-1 and c-Jun and c-Fos--
We have recently
found that c-Jun and c-Fos interacts with a full-length xSRC-3 (34), a
Xenopus homologue of the nuclear receptor coactivator SRC-1
(20, 33), but not with a partial xSRC-3 that lacks the C-terminal
region encompassing the previously defined histone acetyltransferase-
and receptor-binding domains.2
Similarly, a full-length SRC-1 readily
interacted with c-Jun and c-Fos in yeast (Table
I). To localize the interaction domain, we examined LexA and B42 proteins fused to a series of SRC-1 fragments we recently described (49) (Fig. 1).
Consistent with an idea that c-Jun and c-Fos interact with SRC-E,
coexpression of a B42 fusion to the full-length c-Jun and c-Fos further
stimulated the LexA/SRC-E-mediated LacZ expression, whereas
coexpression of B42 alone was without any effects (Table I). In
contrast, the LacZ expressions mediated by LexA fusions to
SRC-A, -B, -C, or -D were not further stimulated by coexpression of
B42/c-Jun or B42/c-Fos. Similar results were also obtained with B42
fusions to SRC-1 fragments and LexA fusions to c-Jun and c-Fos, in
which coexpression of the B42/SRC-E and LexA/c-Jun or LexA/c-Fos pair
efficiently stimulated the LacZ reporter expression (data
not shown).
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Table I
Interactions of SRC-1 with c-Jun and c-Fos in yeast
The indicated B42 and LexA plasmids were transformed into a yeast
strain containing an appropriate LacZ reporter gene. At
least six separate transformants from each transformation were
transferred to indicator plates containing 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside, and reproducible results were
obtained using colonies from a separate transformation. +++, strongly
blue colonies after 2 days of incubation and strong interaction; ++,
light blue colonies after 2 days of incubation and intermediate
interaction; +, light blue colonies after more than 2 days of
incubation and weak interaction; , white colonies and no interaction.
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Fig. 1.
Schematic representation of the SRC-1
constructs. The full-length human SRC-1 (20, 33) and a series of
five SRC-1 fragments are as depicted. The nuclear receptor-interacting
(Receptor), CBP-p300-interacting (p300), basic
helix-loop-helix/PAS (bHLH/PAS), serine-threonine-rich
(S/T-rich), and glutamine-rich domains (Q), along
with the recently identified histone acetyltransferase domain
(HAT) (41) and the NF B component p50-binding domain
(p50) (49), are as indicated. The amino acid numbers for
each construct are shown.
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To further characterize these interactions in vitro, GST
alone and GST fusions to c-Fos or c-Jun were expressed, purified, and
tested for interaction with in vitro translated luciferase and SRC-1. The radiolabeled SRC-1 interacted with GST/c-Jun and GST/c-Fos, but not with GST alone (Fig.
2A). In contrast, the radiolabeled luciferase did not bind any of the GST proteins, as
expected. In agreement with the yeast two-hybrid results (Table I),
only SRC-E, among various SRC-1 fragments, specifically interacted with
GST/c-Jun and GST/c-Fos but not with GST alone (Fig. 2B). These results, along with the yeast results, suggest that SRC-1 directly associates with c-Jun and c-Fos through a subregion of SRC-1
containing the histone acetyltransferase and receptor-binding domains
(i.e. SRC-E).

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Fig. 2.
Interactions of c-Fos and c-Jun with
SRC-1. Luciferase, SRC-1 (A) and a series of five SRC-1
fragments (B) were labeled with
[35S]methionine by in vitro translation and
incubated with glutathione beads containing GST alone, GST/c-Fos, or
GST/c-Jun as indicated. Beads were washed, and specifically bound
material was eluted with reduced glutathione and resolved by
SDS-polyacrylamide gel electrophoresis. Approximately 10-20% of total
input was typically retained.
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Cotransfection of SRC-1 Stimulates AP-1-mediated
Transactivations--
To assess the functional consequences of these
interactions, SRC-1 was cotransfected into CV1 cells along with a
reporter construct (TRE)4-TK-Luc. This reporter construct,
previously characterized to efficiently mediate the
AP-1-dependent transactivations in various cell types,
consists of a minimal promoter from the thymidine kinase gene and four
upstream consensus AP-1 sites (51). Increasing amounts of cotransfected
SRC-1 enhanced the reporter gene expressions in an SRC-1
dose-dependent manner, with cotransfection of 100 ng of
SRC-1 increasing the activation approximately 4-fold (Fig. 3). Consistent with the reports that CBP
and p300 are transcription coactivators of AP-1 (30, 31), increasing
amounts of cotransfected p300 also had stimulatory effects on the
reporter gene expressions, with cotransfection of 200 ng of p300
increasing the activation approximately 8-fold. Consistent with an idea
that SRC-1 and p300 synergize to coactivate the AP-1-mediated
transactivations, coexpression of p300 and SRC-1 further increased the
reporter gene expressions above the levels observed with SRC-1 or p300
alone (Fig. 3). In HeLa and CV-1 cells, SRC-1 also coactivated the
TPA-induced level of transactivations (data not shown). Similar results
were also obtained with xSRC-3 (34), AIB1 (35), and p/CIP
(40).2 In contrast, cotransfection of SRC-1 did not affect
the LacZ reporter expression of the transfection indicator
construct pRSV-
-gal either in the presence or absence of TPA (data
not shown).

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Fig. 3.
Effects of SRC-1 and p300 cotransfection on
the AP-1-mediated transactivations. CV1 cells were transfected
with 50 ng of c-Fos expression vector and LacZ expression
vector and increasing amounts of SRC-1 or p300 expression vectors along
with a reporter gene (TRE)4-TK-Luc as indicated.
Cotransfection of 50 ng of c-Fos activated the reporter gene
expressions approximately 40-fold (data not shown). Normalized
luciferase expressions from triplicate samples are presented relative
to the LacZ expressions, and the standard deviations are
less than 5%.
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Cotransfection of SRC-1 Relieves Transrepression between Nuclear
Receptors and AP-1--
The mutual antagonism between receptor and
AP-1 signaling pathways (55) has been shown to be blocked by
overexpression of CBP and p300, which also interact with both receptors
and AP-1 (20, 56). SRC-1 shows similar effects. Thus, liganded retinoid X receptor efficiently repressed the TPA-dependent
transactivations of AP-1-responsive reporter construct, and c-Fos
inhibited the 9-cis-retinoic acid-dependent activation
of TREpal-TK-Luc reporter. These inhibitory effects (55) were largely
relieved upon addition of increasing amounts of SRC-1 (Fig.
4). Similar results were also obtained
with transrepressions by other nuclear receptors (data not shown).
Thus, competition for limiting amounts of SRC-1 could account for the
mutual inhibitions between receptors and AP-1.

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Fig. 4.
Effects of SRC-1 on the transrepression
between AP-1 and receptors. CV1 cells were transfected with
LacZ expression vector and retinoid X receptor, c-Fos or
SRC-1 expression vectors along with a reporter gene
(TRE)4-TK-Luc (A) or TREpal-TK-Luc
(B) as indicated. Black bars indicate the
presence of 1 µM 9-cis-retinoic acid (RA).
Normalized luciferase expressions from triplicate samples are presented
relative to the LacZ expressions, and the standard
deviations are less than 5%.
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In summary, we have shown that SRC-1 interacts with the AP-1 subunits
c-Jun and c-Fos and coactivates AP-1-mediated transactivations in
synergy with p300, which was also shown to be a coactivator of AP-1
(30, 31). This synergy is believed to reflect a cooperative recruitment
of two different coactivator molecules (i.e. SRC-1 and
CBP-p300) by c-Jun and c-Fos. It is possible that these two distinct
histone acetyltransferases (38, 41-43) either modify selective sites
on the histone tails or act in a concerted fashion to control different
aspects of transcriptional activation. It is also notable that SRC-1
was originally identified as a coactivator molecule for the nuclear
receptor superfamily (20, 33). However, the results presented in this
report, along with the recent reports in which SRC-1 and its homologue
p/CIP were shown to be coactivators for NF
B (49), CREB, and STAT-1
(57), suggest that SRC-1 may regulate many different transcription
factors. Thus, we propose to regroup SRC-1 into the class of proteins
that were termed integrators (i.e. CBP and p300) (18-21).
Finally, competition for a limiting amount of these molecules should be
involved with cross-talks between distinct signaling pathways such as
the well defined antagonisms between the nuclear receptor- and
AP-1-mediated transactivations (55).
We thank Dr. Ming Tsai for the SRC-1 clones
and Dr. David Livingston for the p300 mammalian expression vector.