Molecular Cloning of xSRC-3, a Novel Transcription Coactivator from Xenopus, That Is Related to AIB1, p/CIP, and TIF2
Han-Jong Kim1,
Soo-Kyung Lee1,
Soon-Young Na,
Hueng-Sik Choi and
Jae Woon Lee
College of Pharmacy (H.-J.K., S.-K.L., J.W.L.) Department of
Biology (S.-Y.N.) Hormone Research Center (H.-S.C., J.W.L.)
Chonnam National University Kwangju, South Korea 500757
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ABSTRACT
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Nuclear receptors regulate transcription by
binding to specific DNA response elements of target genes. Herein, we
report the molecular cloning and characterization of a novel
Xenopus cDNA encoding a transcription coactivator xSRC-3 by
using retinoid X receptor (RXR) as a bait in the yeast two-hybrid
screening. It belongs to a growing coactivator family that includes a
steroid receptor coactivator amplified in breast cancer (AIB1),
p300/CREB-binding protein (CBP)-interacting protein (p/CIP), and
transcriptional intermediate factor 2 (TIF2). It also interacts with a
series of nuclear receptors including retinoic acid receptor (RAR),
thyroid hormone receptor (TR), and orphan nuclear receptors
[hepatocyte nuclear receptor 4 (HNF4) and constitutive androstane
receptor (CAR)]. However, it does not interact with small heterodimer
partner (SHP), an orphan nuclear receptor known to antagonize
ligand-dependent transactivation of other nuclear receptors. In
CV-1 cells, cotransfection of xSRC-3 differentially stimulates
ligand-induced transactivation of RXR, TR, and RAR in a dose-dependent
manner. Interestingly, xSRC-3 is highly expressed in adult liver and
early stages of oocyte development, suggesting that studies of xSRC-3
may lead to better understanding of the roles nuclear receptors play in
oocyte development as well as liver-specific gene expression.
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INTRODUCTION
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The nuclear receptor superfamily is a group of transcriptional
regulatory proteins linked by conserved structure and function (1). The
superfamily includes receptors for a variety of small hydrophobic
ligands such as steroids, T3, and retinoids, as well as a
large number of related proteins that do not have known ligands,
referred to as orphan nuclear receptors (2). The receptor proteins are
direct regulators of transcription that function by binding to specific
DNA sequences named hormone response elements (HREs) in promoters of
target genes. While some nuclear receptors apparently bind HREs only as
homodimers, retinoic acid receptors (RARs), thyroid hormone receptors
(TRs), vitamin D receptor, peroxisomal proliferator-activated receptors
(PPARs), and several orphan nuclear receptors bind their specific
response elements with high affinity as heterodimers with retinoid X
receptors (RXRs) (3, 4, 5, 6, 7, 8). Based on this high-affinity binding, such
heterodimers have been considered to be the functionally active forms
of these receptors in vivo. These heterodimers display
distinct HRE specificities to mediate the hormonal responsiveness of
target gene transcription, in that distinct HREs are comprised of
direct repeats (DRs) of a common half-site with variable spacing
between repeats playing a critical role in mediating specificity (2, 9). Accordingly, RARs activate preferentially through DRs spaced by two
or five nucleotides, whereas vitamin D receptor and TR activate through
DRs spaced by three and four nucleotides, respectively. RXR-PPAR
heterodimers as well as RXR homodimers activate preferentially through
DRs spaced by one nucleotide (referred to as DR1). In addition to DRs,
response elements composed of palindromes as well as inverted
palindromes, referred to as everted repeats, have been shown to mediate
transcriptional activation by RXR-RAR and RXR-TR heterodimeric
complexes (9). Such DNA-binding flexibility stands in contrast to the
steroid hormone receptors, which bind exclusively as homodimers to
inverted repeats spaced by three nucleotides (10).
Transcriptional activation of nuclear receptors involves at least two
separate processes: derepression and activation (2). Repression is
mediated in part by interaction of unliganded receptors with
corepressors such as N-CoR (nuclear corepressor receptor) (11) and SMRT
(12). However, ligand binding triggers dissociation of these
corepressors and concomitant recruitment of coactivators. These
putative receptor-interacting coactivators include RIP-140 and RIP160
(13, 14), estrogen receptor (ER)-associated proteins ERAP-140 and
ERAP-160 (15), TIF1 (transcription intermediary factor 1) (16), TRIP1
(17), ARA70 (18), CBP (CREB-binding protein)/p300 (19, 20, 21), SRC-1
(steroid receptor coactivator 1) (19, 22), AIB1 (23), TIF2
(transcriptional intermediate factor 2) (24), RAC3 (25), ACTR (26),
TRAM-1 (27), and p/CIP (p300/CBP-interacting protein) (28). In
particular, the last seven proteins are highly related to each other
and can enhance transcritpional activation by several nuclear receptors
(19, 22, 23, 24, 25, 26, 27, 28). Functional analysis of nuclear receptors has shown that
there are two major activation domains. The N-terminal domain (AF-1)
contains a ligand-independent activation function, whereas the
extreme C-terminal region of the ligand-binding domain (AF-2) exhibits
ligand-dependent transactivation (1). The AF-2 region is conserved
among nuclear receptors, and deletion or point mutations in this region
impair transcriptional activation without changing ligand and DNA
binding affinities (29, 30, 31). Recent x-ray crystallographic studies of
the ligand-binding domain of nuclear receptors revealed that the ligand
binding induces a major conformational change in the AF-2 region
(32, 33, 34), suggesting that this region may play a critical role in
mediating transactivation by a ligand-dependent interaction with
coactivators. These coactivators are postulated to function to transmit
the signal of ligand-induced conformational change to the basal
transcription machinery. As expected, many coactivators fail to
interact with AF-2 mutants of nuclear receptors (13, 16, 24). Targeted
chromatin structure change has been postulated to associate with the
regulation of gene expression by nuclear receptors (35, 36, 37). In
particular, recent biochemical and genetic studies support the notion
that hyperacetylation of core histones is characteristic to gene
activation, while histone deacetylation is involved with
transcriptional repression (38). SRC-1 (39) and its homolog ACTR (26),
along with CBP/p300 (40, 41), were recently shown to contain potent
histone acetyltransferase activities themselves and associate with
histone acetyltransferase protein P/CAF (42). CBP/p300 also forms a
complex with SRC-1 (43). These results suggest that nuclear receptors
target at least three different and self-interacting histone
acetyltransferase activities (SRC-1 or related proteins, CBP/p300, and
P/CAF) to promoters (26). In contrast, it was shown that SMRT and
N-CoR, nuclear receptor corepressors, form complexes with Sin3 and
histone deacetylase proteins (44, 45). From these results, it was
suggested that chromatin remodeling by cofactors contributes, through
histone acetylation-deacetylation, to receptor-mediated transcriptional
regulation.
To understand the processes of ligand-dependent transactivation by
nuclear receptors, we screened a Xenopus oocyte cDNA library
for RXR-interacting proteins by using the yeast two-hybrid system (46).
We describe here the isolation and characterization of a novel
Xenopus transcription coactivator xSRC-3. It is a novel
member of a growing coactivator family that includes SRC-1 (19, 22),
AIB1 (23), p/CIP (28), and TIF2 (24). Studies of xSRC-3 will provide
new insights into the molecular mechanisms of transcriptional
regulation by nuclear receptors, particularly in oocyte development and
liver-specific gene expression, two target tissues where xSRC-3 is
highly expressed.
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RESULTS
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Isolation and Expression of xSRC-3
To isolate cDNAs encoding proteins that specifically interact with
RXR, we exploited the Gal4-based yeast two-hybrid system that has been
previously described (46). We screened a Xenopus oocyte cDNA
library (Clontech, Palo Alto, CA) by using a bait containing the ligand
binding domain (LBD) of human RXR
. Two independent isolates encoded
a novel coactivator molecule that we named xSRC-3. A full-length xSRC-3
cDNA, isolated from rescreening of the identical cDNA library, contains
an open reading frame of 1391 amino acids (Fig. 1A
). As indicated in Fig. 1B
, xSRC-3 is
highly related to AIB1 (23), p/CIP (28), and TIF2 (24) (overall, 77%,
71%, and 53% identity, respectively). These proteins share a basic
helix-loop-helix (bHLH)/PAS domain in the N-terminal, a nuclear
receptor-binding domain in the central, and a glutamine (Q) rich
sequence in the C-terminal region. In particular, xSRC-3 and its family
members have three specific motifs sharing a consensus sequence of
LXXLL (where L is leucine and X is any amino acid) within the central
receptor-binding domains. These motifs and their neighboring residues
are highly charged and well conserved among xSRC-3 and its family
members (22, 23, 24, 25, 26, 27, 28) (Fig. 1C
), and they were recently shown to mediate
protein-protein interactions between liganded nuclear receptors and
RIP-140, SRC-1, p/CIP, and CBP (28, 47).

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Figure 1. Sequence of xSRC-3
A, Amino acid sequences of xSRC-3. Boxed sequencesindicate bHLH, and underlined sequences indicate
two PAS domains. Shaded sequences are the LXXLL motifs
(28 47 ), while italicized sequences denote
glutamine-rich domain. Two original isolates are indicated as
solid and broken arrows, respectively. B, Schematic
representation of xSRC-3 and its family members. Regions in AIB1,
p/CIP, and TIF2-with significant homology are aligned with xSRC-3, and
percentage of amino acids identical to xSRC-3 in the area of bHLH/PAS
and nuclear receptor (NR)-CBP-binding domains, along with the overall
similarity percentages, are as indicated. Functional domains including
bHLH/PAS, five LXXLL motifs included in the NR- and CBP-binding
domains, as well as a stretch of glutamines (poly Q) are as indicated.
C, Sequence alignment of the three LXXLL motifs within the
receptor-interacting domain. Sequences of AIB, p/CIP, TIF2, and SRC-1
are aligned with xSRC-3. Three point-mutants in these motifs (LXXLL to
LXXAL) that are described in Fig. 3 are as indicated (xSRC-3-LR1, LR2,
and LR3, respectively). Mutated amino acids are
boldface.
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Northern blot analysis of poly(A)+ RNAs from
Xenopus oocytes and adult tissues indicated that the
expression of xSRC-3 is highly regulated. As shown in Fig. 2A
, xSRC-3 was expressed only in liver
among adult tissues examined (
5.8 kb), indicating that xSRC-3 may
play specific roles in nuclear receptor-mediated gene expression in
liver. As a control, 28S RNA was probed using the identical blot and
found to be expressed in comparable amounts throughout all the tissues
(data not shown). In addition, xSRC-3 was highly expressed in only
early stages of oocyte development (reviewed in Ref. 48) (Fig. 2B
; at
least five distinct sizes from approximately 5.811.0 kb). The
expression was most prominent in stage I, dramatically decreased in
stage II, and then gradually disappeared in later stages. In contrast,
28S RNA was expressed in comparable amounts throughout all the stages
(data not shown). Consistent with the multiple mRNAs of xSRC-3, we also
isolated a number of potential alternative splicing variants of xSRC-3
from the Xenopus oocyte cDNA library (data not shown). Each
of these splicing isoforms may play distinct roles in the development
of oocytes.

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Figure 2. Northern Blot Analysis of xSRC-3
Northern analyses were performed with Xenopus tissues
(A) and oocytes from different developmental stages (B). Tissues and
oocyte developmental stages (48 ) examined are indicated as well as the
approximate sizes for each mRNA. RNA loadings are as shown.
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Characterization of xSRC-3-Receptor Interactions
To examine receptor interaction properties of xSRC-3, we
constructed a chimeric LexA protein fused to the central
receptor-binding domain of xSRC-3 (amino acids 421-1263)
(LexA/xSRC-3-R). As shown in Fig. 3
, LexA/xSRC-3-R was transcriptionally inert with a lacZ
reporter construct controlled by upstream LexA sites (operators) in
yeast cells. However, coexpression of B42 fusions to RAR, RXR-LBD,
CAR-LBD (49), and hepatocyte nuclear factor 4 (HNF4)-LBD (50) enhanced
the expression of the lacZ reporter, indicating that these
receptors interact with xSRC-3 (Fig. 3
, A, B, D, and E). Addition of
9-cis-RA had no significant effects on the enhanced
expression by B42/RAR and B42/RXR-LBD. Interestingly, coexpression of
B42/TR-LBD resulted in minimal expression of the lacZ
reporter in the absence of T3. However, addition of 1
µM T3 led to significant enhancement of the
lacZ reporter expression, indicating that the interaction of
xSRC-3 and TR is ligand-dependent (Fig. 3C
). In contrast, coexpression
of B42 fusion to SHP (51, 52), an orphan nuclear receptor known to
antagonize ligand-dependent transactivations of other nuclear receptors
(B42/SHP), was without any effects, indicating that xSRC-3 does not
bind to SHP (Fig. 3F
).

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Figure 3. Interactions of xSRC-3 with a Subset of Nuclear
Receptors
Host cells, in which B-galactosidase expression is dependent on the
presence of a transcriptional activator with a LexA DNA-binding domain,
were transformed with plasmids expressing the indicated LexA and B42
chimeras. Wild type denotes LexA/xSRC-3-R, a LexA fusion to the central
receptor-binding domain of xSRC-3 (amino acids 421-1263). LR1, LR2, and
LR3 are identical to LexA/xSRC-3-R except point-mutations within the
three LXXLL motifs previously shown to bind nuclear receptors (28 47 ),
as indicated in Fig. 1C . Open bars indicate coexpression
of B42 alone. Hatched bars (no ligand) or black
bars (addition of 1 µM ligand) indicate
coexpression of indicated B42-receptor fusions. These cells were grown
in liquid culture containing galactose, since expression of the B42
chimeras is under the control of the galactose-inducible GAL1 promoter
(53 ). ß-Galactosidase readings were determined and corrected for cell
density and for time of development (A415 nm/A600
nm) x 1000/min. The result is the average of at least three
different experiments, and the SDs are less than 5%.
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The LXXLL motifs were recently shown to mediate
protein-protein interactions between liganded nuclear receptors and
p/CIP, RIP-140, SRC-1, and CBP (28, 47). Thus, we examined the effects
of specific mutations introduced into each of the three LXXLL motifs in
the central receptor interaction domain of xSRC-3. As indicated in Fig. 1C
, three mutants, in which the second leucine in the LXXLL motifs was
mutated to alanine, were constructed in the context of LexA/xSRC-3-R by
using PCR (LexA/xSRC-3-LR1, LR2, and LR3, respectively). In binding to
B42/RAR, LexA/xSRC-3-LR1 showed similar results with the wild type.
LexA/xSRC-3-LR2 and LR3 showed significantly impaired interactions in
the absence of ligand, while they showed approximately 2550% of the
wild-type interactions in the presence of 1 µM
9-cis-RA (Fig. 3A
). In binding to B42/RXR-LBD,
LexA/xSRC-3-LR1 showed significant decrease (
30% of the wild-type
interactions) in the absence of ligand. However, the ligand-induced
interaction was comparable to that of the wild type. The results with
LexA/xSRC-3-LR2 and LR3 were similar to those of B42/RAR (Fig. 3B
). All
the mutants showed a significant decrease in binding to B42/TR-LBD
(
5070% of the wild type) and B42/HNF4-LBD (
30% of the wild
type), indicating that all the motifs should be intact for full
interactions with TR or HNF4 (Fig. 3
, C and D). Surprisingly,
interactions with B42/CAR-LBD was not affected by any of the mutations,
indicating that CAR interacts with distinct domains of xSRC-3 other
than these LXXLL motifs (Fig. 3E
).
Interactions of xSRC-3 with the AF-2 Domain of TR
Recent x-ray crystallographic studies of the ligand
binding domain of nuclear receptors revealed that the ligand binding
induces a major conformational change in the AF-2 region (32, 33, 34),
suggesting that this region may play a critical role in mediating
transactivation by a ligand-dependent interaction with coactivators.
Interestingly, xSRC-3 interacts with TR in a ligand-dependent manner,
while it constitutively interacts with RAR and RXR (Fig. 3
). However,
point-mutations in the LXXLL receptor-interacting motifs revealed
cryptic ligand-dependent interactions with RAR and RXR (Fig. 3
, A
and B). These results suggest that the xSRC-3-receptor interaction
interface may contain, at least for a subset of receptors, the AF-2
domain. To test this idea, we constructed a chimeric B42 protein fused
to the central receptor- binding domain of xSRC-3 (amino acids
421-1263) (B42/xSRC-3-R). In yeast cells, B42/xSRC-3-R was coexpressed
with either LexA/TR-LBD or LexA/TR-LBD-F459P, a previously described
point-mutant in which the AF-2 domain is specifically disrupted (53).
As expected, LexA/TR-LBD-F459P did not show any interactions with
B42/xSRC-3-R, while LexA/TR-LBD efficiently interacted with
B42/xSRC-3-R (Fig. 4
). These results
confirm that xSRC-3 indeed interacts with the AF-2 domain of TR.

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Figure 4. Interactions of xSRC-3 with the AF-2 Domain of TR
Wild-type and F459P denote LexA/TR-LBD, a LexA fusion to the LBD of rat
TRß 1, and a similar LexA fusion protein with a point mutation in the
AF-2 domain of TR, respectively, as previously described (53 ).
Open bars indicate coexpression of B42 alone.
Hatched bars (no ligand) or black bars
(addition of 1 µM ligand) indicate coexpression of
B42/xSRC-3-R, a B42 fusion to the central receptor-binding domain of
xSRC-3 (amino acids 421-1263). ß-Galactosidase readings were
determined and corrected for cell density and for time of development
(A415 nm/A600 nm) x 1000/min. The result is
the average of at least three different experiments, and the
SDs are less than 5%.
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Autonomous Transactivation of xSRC-3 and Interaction with
p300
To examine whether xSRC-3 can directly stimulate
transcription when recruited to a specific promoter by linking with a
heterologous DNA-binding domain, we constructed a chimeric LexA protein
fused to the central receptor binding domain of xSRC-3 to the C
terminus (amino acids 421-1391) (LexA/xSRC-3-C). LexA alone did not
activate the LacZ reporter controlled by upstream
LexA-binding sites, while LexA/xSRC-3-C efficiently stimulated the
reporter gene expression (Fig. 5
). In
contrast, a similar LexA fusion protein LexA/xSRC-3-R, which lacks the
C-terminal 128 amino acids, was transcriptionally inert. These results
indicate that xSRC-3 contains an autonomous transactivation domain,
functional in yeast cells, at the C-terminal region including the
C-terminal 128 amino acids. Next, we examined whether the autonomous
transactivation function of xSRC-3 is related to interactions with
CBP/p300. A chimeric B42 protein fused to the SRC-1-binding domain of
p300 (20) (amino acids 20412157) was constructed (B42/p300-C).
Coexpression of B42/p300-C further enhanced the transcriptional
activities of LexA/xSRC-3-C, while it did not enhance the
transcriptional activities of LexA alone or LexA/xSRC-3-R, which lacks
the autonomous transactivation function (Fig. 5
). These results suggest
that xSRC-3 interacts with p300, and the autonomous transactivation may
require efficient p300/CBP bindings. In addition, the C-terminal 128
amino acids of xSRC-3 should be important in binding to CBP/p300, as
opposed to the previous results (24, 25, 26, 27, 28).

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Figure 5. Autonomous Transactivation of xSRC-3
(-) denotes LexA alone. While xSRC-3-C denotes LexA/xSRC-3-C, a LexA
fusion to the central receptor-binding domain to the C terminus of
xSRC-3 (amino acids 421-1391), xSRC-3-R denotes LexA/XSRC-3-R, a LexA
fusion to the central receptor-binding domain of xSRC-3 (amino acids
421-1263). Open bars indicate coexpression of B42 alone.
Black bars indicate coexpression of B42/p300-C, a B42
fusion to the SRC-1-binding domain of p300 (amino acids 20412157).
ß-Galactosidase readings were determined and corrected for cell
density and for time of development (A415 nm/A600
nm) x 1000/min. The result is the average of at least three
different experiments, and the SDs are less than 5%.
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Coactivation of Nuclear Receptors by xSRC-3 in CV-1 Cells
To examine the coactivator activity of xSRC-3, cotransfection
studies were performed in CV-1 cells. As shown in Fig. 6
, cotransfection with expression vectors
for xSRC-3 significantly increased TR-mediated induction of the
TREpal-TK-Luc (49) reporter gene activity by T3. Similarly,
xSRC-3 enhanced RXR-mediated induction of the TREpal-TK-Luc reporter
gene activity by 9-cis-RA. However, the enhancement of
RAR-mediated induction of the ß-RARE-TK-Luc (49) reporter gene
activity by 9-cis-RA was only modest (Fig. 6
, A, B and C).
In all the cases, the basal level of reporter expression was not
significantly affected by cotransfected xSRC-3. We have also performed
the cotransfection experiments in the presence of additional expression
vectors for rTRß1, hRAR
, and hRXR
(Fig. 6D
and data not shown).
In particular, when 10 ng hRXR
were cotransfected, xSRC-3
dramatically enhanced the 9-cis-RA-induced transactivation
of the TREpal-TK-Luc reporter gene activity, with cotransfection of 100
ng xSRC-3 increasing the fold-activation more than 8-fold (Fig. 6D
).
Furthermore, cotransfection of p300 showed additive effects with xSRC-3
(Fig. 6D
), consistent with the interaction of xSRC-3 and p300 (Fig. 5
).
With cotransfection of 60 ng RXR
AF2, a previously described deletion
mutant with a specific defect for the AF2 function (50), xSRC-3 was not
able to show any coactivation, consistent with the importance of the
AF2 domain to interact with xSRC-3 (Fig. 4
). We also cotransfected
different cell lines with similar results (data not shown). From these
results, we concluded that xSRC-3 is indeed a transcriptional
coactivator for nuclear receptors.

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Figure 6. Coactivation of Nuclear Receptors by xSRC-3
CV-1 cells were transfected with ß-galactosidase expression vector
and increasing amount of xSRC-3-expression vectors along with a
reporter gene TREpal-TK-LUC (A, B, and D) or ß-RARE-TK-LUC (C), as
indicated. Cells were unstimulated (open bars) or
stimulated (black bars) with 0.1 µM
ligand. The cotransfection experiments were also performed with an
additional 10 ng of hRXR or 60 ng of RXR AF2 (50 ) expression
vectors (D). Normalized luciferase expressions from triplicate samples
are presented relative to the ß-galactosidase expressions. The result
is the average of at least two different experiments, and the
SDs are less than 5%.
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DISCUSSION
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Transcriptional activation of nuclear receptors involves at least
two classes of cofactors: corepressors and coactivators (1).
Corepressors that associate with unliganded nuclear receptors mediate
repression, while coactivators are recruited upon ligand binding and
concomitant dissociation of the corepressors. In this report, we added
a novel member to a growing family of distinct coactivators that
include SRC-1 (19, 22), AIB1 (23), TIF2 (24), and p/CIP (28). We named
this factor xSRC-3, which was isolated by using RXR as a bait in the
yeast two-hybrid screening of Xenopus oocyte cDNA library.
These proteins share the N-terminal bHLH/PAS domain (>60% identity
within 350 amino acids), which does not appear to be required for
xSRC-3 and its family members to enhance receptor-mediated
transactivation. Even though its function is currently unknown, it may
contribute to some functional aspects. Interestingly, xSRC-3 and its
family members were shown to have rather conservative, a few
alternative splicing isoforms (Refs. 22, 23, 24, 25, 26, 27, 28 and data not shown). The
conservation of these splicing events suggests that distinct roles may
be played by each of these isoforms. It is an interesting hypothesis
that xSRC-3 and its family members might be involved with multiple
transactivator-mediated signalings, as was the case with CBP/p300
(19, 20, 21).
Currently, at least two mechanistic details have been proposed to
describe the function of these coactivators. First, they are postulated
to function to transmit the signal of ligand-induced conformational
change to the basal transcription machinery. Second, they have been
associated with targeted chromatin structure change by nuclear
receptors (35, 36, 37). Recent biochemical and genetic studies support the
notion that hyperacetylation of core histones is characteristic to gene
activation, and histone deacetylation is involved with transcriptional
repression (38). For instance, it was shown that SMRT (12) and N-CoR
(11), nuclear receptor corepressors, form complexes with Sin3 and
histone deacetylase proteins (44, 45). In contrast, SRC-1 (39) and its
homolog ACTR (26), along with CBP/p300 (40, 41), were recently shown to
contain potent histone acetyltransferase activities themselves and
associate with histone acetyltransferase protein P/CAF (42), while
CBP/p300 can form a complex with SRC-1 (43). These results suggest that
nuclear receptors target at least three different and self-interacting
histone acetyltransferase activities (SRC-1 or related proteins,
CBP/p300, and P/CAF) to promoters (26). In light of these results, it
will be interesting to test whether xSRC-3 itself contains histone
acetyltransferase activities.
The nuclear receptor and CBP-interaction domains within xSRC-3 and its
family members contain a number of a short sequence motif LXXLL (where
L is leucine and X is any amino acid) (28, 47). The third helical motif
in the receptor interaction domain of NCoA-1 was recently shown to be
absolutely required for the RAR function, but not for the ER function
(28). Consistent with this, the third helical motif of xSRC-3 seems to
be most important for interactions with RAR and RXR (Fig. 3
, A and B).
However, mutation of the second helical motif of xSRC-3 resulted in
reasonably strong interactions with RAR and RXR (Fig. 3
), while similar
mutation in NcoA-1 completely abolished interactions with RAR and ER
(28). These results suggest that these motifs could provide the
molecular basis of specificity in nuclear receptor-mediated
transcriptional responses by xSRC-3 and its family members.
Interactions of xSRC-3 with RAR and RXR were relatively strong and, at
least for a partial xSRC-3 (LexA/xSRC-3-R), ligand-independent, while
mutations in the LXXLL motifs revealed cryptic ligand-dependent
interactions (Fig. 3
, A and B). Accordingly, it will be important to
test whether a full-length xSRC-3 shows more prominent ligand
dependency in interactions with RAR and RXR. The LXXLL motifs within
the receptor-binding domains of xSRC-3 and its family members seem to
be functionally redundant; i.e. a single mutation within the
three motifs only weakens the overall interactions without complete
abolishment (Ref. 28 and Fig. 3
). Strikingly, none of the interaction
mutants affected interactions with CAR (49), indicating that CAR should
recognize domains other than these motifs (Fig. 3E
). In light of these
CAR results, it is noteworthy that TRAM-1 (27) was recently shown to
bind subdomains of nuclear receptors including a helix 3 rather than
the AF-2 domain, in contrast to SRC-1/TIF2 (19, 22, 24). Accordingly,
it will be interesting to test whether xSRC-3 binds similar subdomains
of CAR (49). Most exciting was the finding that xSRC-3 constitutively
binds HNF4 (51) and CAR, but does not bind SHP (52, 53). These results
were consistent with the fact that HNF4 and CAR are constitutive
transactivators (49, 51), while SHP, an orphan nuclear receptor known
to antagonize ligand-dependent transactivations of other nuclear
receptors, contains an active repressor domain (53). Similarly, Rac3
(25) did not interact with COUP-TF (chicken ovalbumin upstream
promoter-transcription factor) (55), an orphan nuclear receptor that
also antagonizes other receptors.
Our results indicate that the C-terminal 128 amino acids of xSRC-3 are
essential in binding to p300 and the autonomous transactivation may
require efficient p300/CBP bindings (Fig. 6
). These results contradict
previous results (24, 25, 26, 28), in which this C-terminal region seemed
unnecessary for the CBP/p300 interactions. Additional, more thorough
domain mapping and mutagenesis experiments will be required to provide
further insights into the receptor-xSRC-3-p300/CBP interactions. We
clearly demonstrated that xSRC-3 enhances transcriptional activation by
RXR and TR in mammalian cells. However, its not clear why we observed
only a modest enhancement by RAR, while a much higher level of
enhancement was observed by its close relative ACTR (26), for instance.
This might be due to the fact that, in our experiments with CV-1 cells,
a more than 1,200-fold induction was initially obtained with RAR by
hormone treatment in the absence of cotransfected xSRC-3. In the
experiments with ACTR, only a 10-fold induction was observed with A549
lung carcinoma cells (26). Thus, a saturation effect might have limited
our ability to detect a higher level of enhancement. In addition,
cotransfection of an additional 10 ng RXR resulted in more than 8-fold
activation (compare the results between Fig. 6B
and Fig. 6D
),
suggesting that the ratio between receptor and xSRC-3 can be important.
Thus, it is possible that target receptors for xSRC-3 could be present
in adequate amounts relative to xSRC-3 in vivo.
In conclusion, we identified a novel coactivator xSRC-3 that is related
to a growing coactivator family that includes SRC-1 and TIF2.
Accordingly, xSRC-3 displays various properties of transcriptional
coactivator, including the capacity for ligand-dependent interactions
with the receptors, autonomous transactivation, and transcription
coactivation. However, xSRC-3 shows very distinct expression patterns
from other members. As such, xSRC-3 may lead us to better understand
tissue-specific control of nuclear receptor-mediated gene
expressions.
 |
MATERIALS AND METHODS
|
---|
Hormones, Yeast Cells, and Plasmids
T3 and 9-cis-RA were obtained from Sigma
Chemical Co. (St. Louis, MO). Y190 cells and a parental vector pGBT are
as described (Clontech, Palo Alto, CA). A chimeric protein consisting
of Gal4 DNA-binding domain fused to the ligand binding domain of human
RXR
was constructed by using PCR (Gal4-DBD/RXR-LBD). EGY48 cells,
the lexA-ß-galactosidase reporter construct, and the LexA- and
B42-parental vectors were as reported (52). LexA or B42 fusions to the
LBDs of CAR, HNF-4, RXR
, and rat TRß as well as B42 fusions to
full-length SHP and RAR
were as previously described (52, 54, 56).
LexA/xSRC-3-R, a chimeric LexA protein fused to the central
receptor-binding domain of xSRC-3 (amino acids 421-1263) and
LexA/xSRC-3-C (amino acids 421-1391), a LexA fusion to the central
receptor-binding domain to the C terminus of xSRC-3 were similarly
constructed. LexA/xSRC-3-LR1, LexA/xSRC-3-LR2, and LexA/xSRC-3-LR3 were
constructed, in the context of LexA/xSRC-3-R, by introducing alanines
into the second leucines of the three LXXLL motifs (28, 47) within the
receptor-binding domains of xSRC-3, respectively. B42/p300-C (amino
acids 20412157), a B42 fusion to the SRC-1 binding domain of p300
(20) was also constructed. Yeast expression vectors, YEP-RXR
,
YEP-RXRß, YEP-RXR
, YEP-RAR
, YEP-RARß, YEP-RAR
, and
YEP-TR
and YEP-TRß were as described (54). Mammalian expression
vectors for rTRß1, hRXR
, RXR
AF2 and hRAR
, the reporter
constructs TREpal-TK-LUC and ß-RARE-TK-LUC, and the transfection
indicator construct pRSV-ß-gal are as described (49, 50).
Yeast Two-Hybrid Screening
Gal4-DBD/RXR-LBD was used as a bait to screen a
Xenopus oocyte cDNA library in pGAD10 vector (Clontech) for
RXR-interacting proteins in the absence of ligand, as previously
described (46). The library plasmids from positive clones that
expressed both HIS3 and LacZ reporters were
rescued and retransformed into yeast cells, together with the original
bait and other constructs, for testing the specificity of
protein-protein interaction. Two independent isolates encoding xSRC-3
was selected for further analysis in this study.
Yeast ß-Galactosidase Assay
The cotransformation and quantitative liquid ß-galactosidase
assays in yeast were performed with the following changes as described
previously (54). The yeast culture was initially diluted to an
A600 nm of 0.05, and plated into 96-well culture dishes
with the various concentrations of hormone. The cultures were then
incubated in the dark at 30 C for 16 h. The A600 nm
was determined, and then cells were lysed and substrate was added and
A415 nm was read after 1030 min. The normalized
galactosidase values were determined as follows: (A415
nm/A600 nm) x 1000/min developed. For each
experiment, at least three independently derived colonies expressing
chimeric receptors were tested.
Northern Blot Analysis
Poly(A)+ RNAs were isolated from Xenopus
multiple tissues and Xenopus oocytes of different
developmental stages (48), electrophoresed on a formaldehyde-agarose
gel, transferred to membrane, hybridized with a random primed
32P-labeled DNA probes (encompassing amino acids 421-1263
of xSRC-3), and exposed on x-ray film, as described previously
(57).
Cell Culture and Transfection
CV-1 cells were grown in 24-well plates with medium supplemented
with 10% charcoal-stripped serum. After 24 h incubation, cells
were transfected with 100 ng ß-galactosidase expression vector
pRSV-ß-gal and 100 ng of a reporter gene TREpal-TK-LUC (49) or
ß-RARE-TK-LUC (49), either in the presence or absence of 10 ng of
rTRß1-, hRAR
-, or hRXR
-expression vectors, as previously
described (57). Total amounts of expression vectors were kept constant
by adding decreasing amounts of the CDM8 expression vector to
transfections containing increasing amounts of the TRß-, RAR
,- or
RXR
-vector. After 12 h, cells were washed and refed with DMEM
containing 10% charcoal-stripped FBS. After 12 h, cells were left
unstimulated or stimulated with 0.1 µM ligand. Cells were
harvested 24 h later, and luciferase activity was assayed as
described (57), and the results were normalized to the
ß-galactosidase expression. Similar results were obtained in more
than two similar experiments.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Tae-Sung Kim, Wongi Seol, Yoon Kwang Lee, and
David D. Moore for valuable advice, plasmids, and critical readings of
this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jae Woon Lee, Ph.D., College of Pharmacy, Hormone Research Center, Chonnam National University, 300 Yongbong-dong Puk-gu, Kwangju 500757, Korea. E-mail:
jlee{at}chonnam.chonnam.ac.kr
This research was supported by the academic research fund of Ministry
of Education, Republic of Korea (GE 9681/97143 to J.W.L) and Korean
Science and Engineering Foundation (HRC to H.S.C and J.W.L).
1 The first two authors contributed equally. 
Received for publication December 31, 1997.
Revision received March 31, 1998.
Accepted for publication April 2, 1998.
 |
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