From the Department of Biology, the
§ College of Pharmacy, the ¶ Hormone Research
Center, and the
Department of Microbiology, Chonnam
National University, Kwangju 500-757, Korea
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ABSTRACT |
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Steroid receptor coactivator-1 (SRC-1)
specifically bound to the transcription factor NFB subunit p50 but
not to p65 as demonstrated by the yeast two hybrid tests and
glutathione S-transferase pull down assays. The p50-binding
site was localized to a subregion of SRC-1 (amino acids 759-1141) that
encompasses the previously described CBP-p300-binding domain. In
mammalian cells, SRC-1 potentiated the NF
B-mediated transactivations
in a dose-dependent manner. Coexpression of p300 further
enhanced this SRC-1-potentiated level of transactivations, consistent
with the recent findings in which CBP and p300 were shown to be
transcription coactivators of the p65 subunit (Perkins, N. D.,
Felzien, L. K., Betts, J. C., Leung, K., Beach, D. H.,
and Nabel, G. J. (1997) Science 275, 523-527; Gerritsen, M. E., Williams, A. J., Neish, A. S., Moore,
S., Shi, Y., and Collins, T. (1997) Proc. Acad. Natl. Sci.
U. S. A. 94, 2927-2932). These results suggest that at least
two distinct coactivator molecules may cooperate to regulate the
NF
B-dependent transactivations in vivo and
SRC-1, originally identified as a coactivator for the nuclear
receptors, may constitute a more widely used coactivation complex.
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INTRODUCTION |
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The transcription factor nuclear factor B
(NF
B)1 is important for
the inducible expression of a wide variety of cellular and viral genes
(reviewed in Refs. 1 and 2). NF
B is composed of homo- and
heterodimeric complexes of members of the Rel (NF
B) family of
polypeptides. In vertebrates, this family comprises p50, p65 (RelA),
c-Rel, p52, and RelB. These proteins share a 300-amino acid region,
known as the Rel homology domain, which binds to DNA and mediates homo-
and heterodimerization. This domain also is target of the I
B
inhibitors, which include I
B
, I
B
, I
B
, Bcl-3, p105,
and p100 (3). In the majority of cells, NF
B exists in an inactive
form in the cytoplasm, bound to the inhibitory I
B proteins.
Treatment of cells with various inducers results in the degradation of
I
B proteins. The bound NF
B is released and translocates to the
nucleus, where it activates appropriate target genes. I
B
is
degraded in response to all of the known inducers of NF
B, whereas
I
B
is degraded only when cells are stimulated with inducers such
as lipopolysaccharide (LPS) and interleukin-1, which cause persistent
activation of NF
B (4). Following degradation of the initial pool of
I
B
in response to LPS or interleukin-1, newly synthesized
I
B
accumulates in the nucleus as an unphosphorylated protein that
forms a stable complex with NF
B and prevents it from binding to
newly synthesized I
B
(5, 6). Bcl-3 is an unusual I
B protein in
that it can not only inhibit nuclear NF
B complexes but can bind to
p50 and p52 dimers on DNA and provide the complexes with
transactivating activity (7, 8).
Transcription coactivators bridge transcription factors and the
components of the basal transcriptional apparatus (reviewed in Ref. 9).
Functionally conserved proteins 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 (10-13),
CREB (14-16), AP-1 (17, 18), bHLH factors (19), STATs (20, 21), and,
most recently, the NFB component p65 (22, 23). 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. 24). 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 (24). Consistent with
this, CBP and p300 have been found to interact directly with nuclear
receptors in a ligand- and AF-2-dependent manner (10-13).
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) (12,
25), AIB1 (26), TIF2 (27), RAC3 (28), ACTR (29), TRAM-1 (30), p/CIP
(31), and XICO (32). Interestingly, SRC-1 (33) and its homologue ACTR
(29), along with CBP and p300 (34, 35), were recently shown to contain
potent histone acetyltransferase activities themselves and associate
with yet another histone acetyltransferase protein p/CAF (36). In
contrast, it was shown that SMRT (37) and N-CoR (38), nuclear receptor
corepressors, form complexes with Sin3 and histone deacetylase proteins
(39, 40). 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 the NFB component p65 (22, 23),
we tested whether SRC-1 itself participates in the NF
B-mediated
transactivations as well. Herein, we show that 1) SRC-1 specifically
binds to the NF
B component p50 but not to p65, 2) SRC-1 coactivates
the NF
B-mediated transactivations, and 3) p300 synergized with SRC-1
in this coactivation. These results suggest that at least two distinct
transcription coactivator molecules with histone acetyltransferase
activities (i.e. SRC-1 and CBP-p300) may regulate the
NF
B-mediated transactivations in vivo, and SRC-1,
originally identified as a coactivator for the nuclear receptors, may
regulate many different transcription factors.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
LexA, B42, T7, or GST vectors to express p50 and
p65 were as described previously (41). Polymerase chain
reaction-amplified fragments of SRC-1 (A-E as depicted in
Fig. 1) were subcloned into EcoRI-SalI
restriction sites of the LexA fusion vector pEG202PL (42),
EcoRI-XhoI restriction sites of the B42 fusion
vector pJG4-5 (42), or EcoRI-XhoI restriction
sites of the T7 vector pBS-SK (Stratagene, San Diego, CA). The GST
fusion vectors encoding CBP-N (amino acids 1-450) and CBP-C (amino
acids 1891-2441) (kind gifts from Dr. Chris Glass, University of
California at San Diego) were as described (12). 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 and the NF
B-responsive reporter
construct (
B)4-IL-2-Luc, were as described previously
(15, 33, 43).
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 (42). Plate and liquid
assays of -gal expression were carried out as described (42,
44-46). 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-Sepharose-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 (47).
Cell Culture and Transfections--
L929 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
(
B)4-IL-2-Luc (43), along with increasing amounts 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
(48), 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 |
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Interactions of SRC-1 and p50-- We have recently found that XICO (32), a Xenopus homologue of the nuclear receptor coactivator SRC-1 (12, 25), interacts with p50 but not with p65.2 The smallest clone among the original isolates of XICO that readily interacted with p50 consisted of only the central nuclear receptor-binding and CBP-p300-binding domains, suggesting that the interaction interface should be included within these two domains.2 These interactions were further characterized in the yeast two-hybrid tests (42), in which formations of p50-p50 homodimer and p50-p65 heterodimer were readily detected as previously reported (41) (Table I). A series of SRC-1 fragments were subcloned into B42 and LexA vectors as depicted in Fig. 1. Among the LexA fusions, only SRC-D (SRC-1 amino acids 759-1141) encompassing the previously described CBP-p300-binding domain (12, 25-32) was found to confer autonomous transactivation function to a LacZ reporter construct controlled by upstream LexA-binding sites (42) (Table I). This is consistent with the previous findings in which the CBP-p300-binding domain was shown to be essential for autonomous transactivation functions (12, 25-32). Consistent with an idea that p50 interacts with SRC-D, coexpression of a B42 fusion to the full-length p50 further stimulated the LexA/SRC-D-mediated LacZ expression, whereas coexpression of a B42 fusion to the full-length p65 was without any effects (Table I). In contrast, the LacZ expressions mediated by LexA fusions to SRC-A, -B, -C, or -E were not stimulated by coexpression of B42/p50 or B42/p65. Similar results were also obtained with B42 fusions to SRC-1 fragments and LexA fusions to p50 and p65, in which coexpression of the B42/SRC-D and LexA/p50 pair efficiently stimulated the LacZ reporter expression (data not shown).
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Cotransfection of SRC-1 Stimulates the NFB-mediated
Transactivations--
To assess the functional consequences of these
interactions, SRC-1 was cotransfected into L929 cells along with the
reporter construct (
B)4-IL-2-Luc. This reporter
construct, previously characterized to efficiently mediate the
NF
B-dependent transactivations in various cell types,
consists of a minimal promoter from the interleukin-2 gene and four
upstream
B sites from the IL-6 gene (43). Increasing amounts of
cotransfected SRC-1 enhanced the reporter gene expressions in an SRC-1
dose-dependent manner, with cotransfection of 800 ng of
SRC-1 increasing the fold activation approximately 3-fold (Fig.
3). Consistent with the reports that CBP
and p300 are transcription coactivators of the NF
B component p65
(22, 23), increasing amounts of cotransfected p300 had stimulatory
effects on the reporter gene expressions, with cotransfection of 100 ng
of p300 increasing the fold activation approximately 2-fold. Consistent
with an idea that SRC-1 and p300 synergize to coactivate the
NF
B-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 various cells, SRC-1
also coactivated the LPS- or tumor necrosis factor
-induced level of
transactivations (data not shown). 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
the absence of LPS or tumor necrosis factor
(data not shown).
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ACKNOWLEDGEMENTS |
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We thank Dr. Ming Tsai for the SRC-1 clones,
Dr. Chris Glass for the GST-CBP clones, Dr. Tae Ho Lee for the
NFB-responsive reporter construct, and Dr. David Livingston for the
p300 mammalian expression vector.
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FOOTNOTES |
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* This work was supported by Grant 96-0401-08-01-3 from the Korea Science and Engineering Foundation (to J. W. L.), grants from the Hormone Research Center (to J. W. L, S.-Y. I., and H.-S. C.), and Grant BSRI 97-4426 (to S.-Y. I.) and Grant GE 96-81/97-143 (to J. W. L.) from the Academic Research Fund of the Ministry of Education, Republic of Korea.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. Tel.: 82-62-530-2934; Fax: 82-62-530-2949; E-mail: jlee{at}chonnam.chonnam.ac.kr.
1
The abbreviations used are: NFB, nuclear
factor
B; LPS, lipopolysaccharide; SRC-1, steroid receptor
coactivator-1; GST, glutathione S-transferase;
-gal,
-galactosidase; IL, interleukin; CBP, CREB-binding protein.
2 S.-K. Lee and J. W. Lee, unpublished observations.
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REFERENCES |
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