![siSMILE increases GR-, CAR- and HNF4α transactivation. (A) Effect of siRNAs for SMILE or SHP on the expression of SMILE and SHP. HepG2 cells were transfected with pSUPER siSMILE-I (siSM#1), siSMILE-II (siSM#2), siSHP or pSUPER [control (con)], and after 72 h the total RNA was isolated. The mRNA levels of SMILE and SHP were measured via RT–PCR analysis, with β-actin shown as a control. The data shown are representative of at least three independent experiments. (B–D) SMILE siRNA induces GR-, CAR- and HNF4α-mediated transactivation in HepG2 cells. HepG2 cells were transfected with pSUPER, pSUPER siSMILE-I (siSM#1), or pSUPER siSMILE-II (siSM#2). After 24 h, the cells were cotransfected with expression vector for GR (B), CAR (C) or HNF4α (D), and MMTV-Luc (B), (NR1)5-Luc (C) or (HNF4)8-Luc (D) luciferase reporter vectors, together with pCMV-β-gal as internal control. Twenty-four hours after transfection, the cells in (B) were treated for 24 h with or without 100 nM Dex prior to the measurement of luciferase activity. The mean and standard deviation (n = 3) of a representative experiment are shown. *P < 0.05; **P < 0.01, using Student's t-test.](https://www.researchgate.net/profile/Balachandar-Nedumaran/publication/24415354/figure/fig2/AS:216400317685774@1428605264573/siSMILE-increases-GR-CAR-and-HNF4a-transactivation-A-Effect-of-siRNAs-for-SMILE-or_Q320.jpg)
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4100–4115 Nucleic Acids Research, 2009, Vol. 37, No. 12 Published online 08 May 2009
doi:10.1093/nar/gkp333
Molecular characterization of SMILE as a novel
corepressor of nuclear receptors
Yuan-Bin Xie, Balachandar Nedumaran and Hueng-Sik Choi*
Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University,
Gwangju, 500-757, Republic of Korea
Received March 25, 2009; Revised and Accepted April 20, 2009
ABSTRACT
SMILE (small heterodimer partner interacting
leucine zipper protein) has been identified as a cor-
egulator in ER signaling. In this study, we have
examined the effects of SMILE on other NRs
(nuclear receptors). SMILE inhibits GR, CAR and
HNF4a-mediated transactivation. Knockdown of
SMILE gene expression increases the transactiva-
tion of the NRs. SMILE interacts with GR, CAR and
HNF4ain vitro and in vivo. SMILE and these NRs
colocalize in the nucleus. SMILE binds to the
ligand-binding domain or AF2 domain of the NRs.
Competitions between SMILE and the coactivators
GRIP1 or PGC-1ahave been demonstrated in vitro
and in vivo. Furthermore, an intrinsic repressive
activity of SMILE is observed in Gal4-fusion
system, and the intrinsic repressive domain
is mapped to the C-terminus of SMILE, spanning
residues 203–354. Moreover, SMILE interacts with
specific HDACs (histone deacetylases) and SMILE-
mediated repression is released by HDAC inhibitor
trichostatin A, in a NR-specific manner. Finally, ChIP
(chromatin immunoprecipitation) assays reveal that
SMILE associates with the NRs on the target gene
promoters. Adenoviral overexpression of SMILE
represses GR-, CAR- and HNF4a-mediated target
gene expression. Overall, these results suggest
that SMILE functions as a novel corepressor of
NRs via competition with coactivators and the
recruitment of HDACs.
INTRODUCTION
Small heterodimer partner interacting leucine zipper pro-
tein (SMILE) belongs to basic region leucine zipper
(bZIP) family (1,2). SMILE gene produces two isoforms,
SMILE-L (long isoform of SMILE, also known as
CREBZF) and SMILE-S (short isoform of SMILE,
previously known as Zhangfei), from alternative usage
of initiation codons (2). Although SMILE has the ability
to homodimerize like other bZIP proteins, it cannot bind
to DNA as a homodimer (1–3). SMILE has been identi-
fied as an interacting partner of herpes simplex virus-
related host-cell factor (HCF) and inhibits the replication
of the herpes simplex virus (1,3). SMILE has also been
reported as a coactivator of ATF4 and as a corepressor
of CREB3, another cellular HCF-binding transcription
factor (4,5). Recently, we have reported that SMILE
acts as a coregulator of estrogen receptor (ER) signaling
(2), but its role in other nuclear receptors (NRs) signaling
remains unknown.
NRs are transcription factors that modulate the expres-
sion of genes involved in embryonic development, main-
tenance of differentiated cellular phenotypes, metabolism
and cell death [see references (6–8) for reviews]. Members
of the NR superfamily include the conventional endocrine
receptors, the adopted orphan receptors, for which ligands
have been identified in recent years, and the orphan recep-
tors, ligands of which have not yet been identified (8).
Glucocorticoid receptor (GR) is a member of steroid
receptor family and mediates the effect of glucocorticoids
in a variety of cellular processes, including homeostasis,
cell growth, development, stress response and inflamma-
tion (8). GR regulates the transcription of target genes
either by binding to specific glucocorticoid response ele-
ments (GREs) within the target genes or by interacting
with other DNA-bound transcription factors. The inactive
GR resides in the cytoplasm bound to heat-shock protein.
It dissociates from heat-shock protein upon ligand binding
and enters the nucleus where it functions as a transcription
factor (9). GR plays an important role in various meta-
bolic pathways by regulating the expression of genes such
as phosphoenolpyruvate carboxykinase (PEPCK), glu-
cose-6-phosphatase (G6Pase) and insulin-like growth
factor-binding protein 1 (IGFBP1) (10,11).
Constitutive androstane receptor (CAR) is an adopted
orphan NR which functions as heterodimers with the reti-
noid X receptor (RXR) (8). CAR evidences constitutive
activity, and is expressed primarily in the liver, where it
*To whom correspondence should be addressed. Tel: +82 62 530 0503; Fax: +82 62 530 0506. Email: hsc@chonnam.ac.kr
ß2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
regulates many Phase I and Phase II biotransforming
enzymes, including Cyp2b6, Sult2a1, SultN and Ugt1a1
(12,13). This xenobiotic receptor can also regulate the
expression of membrane transporter proteins such as
organic anion transporting peptide 2 (Oatp2) and multi-
drug resistance-associated proteins. CAR can be modu-
lated by structurally diverse chemicals such as 1,4-bis-2
[-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phe-
nobarbital (12–14). Hepatocyte nuclear factor 4 (HNF4) is
an orphan nuclear receptor which is highly expressed in
the liver, kidney, and pancreatic b-cells. HNF4 contains
two subtypes in mammals, namely HNF4aand HNF4g,
and binds to the DR-1 element of target gene promoters as
homodimers (6,7). HNF4aplays critical roles not only in
the specification of the hepatic phenotype during liver
development but also in the transcriptional regulation of
genes involved in glucose, cholesterol, fatty acids and
xenobiotic metabolism (7, 15), including PEPCK, choles-
terol 7 alpha-hydroxylase (CYP7A1) and liver carnitine
palmitoyl transferase CPT (L-CPT) (16–18). Mutations
in the HNF4agene have been associated with maturity-
onset diabetes of the young (MODY) (7).
NR-mediated transcriptional effects are regulated by
NR coregulators, including coactivators and corepressor
(19). Coregulators modulate the transcription of NR
target genes through taking part in chromatin remodeling
or interacting with basal transcriptional machinery to
influence the main steps in transcriptional initiation (20).
In the presence of NR ligands, the SWI/SNF chromatin
remodeling complex, the histone acetyltransferase (HAT)
activity containing complexes CBP/p160/P/CAF, and the
TRAP/DRIP/ARC complex are sequentially recruited to
gene promoters to activate gene transcription (21–24).
Coactivators of the p160 family, including SRC1/
NCoA1 and TIF-2/GRIP1, interact with the ligand-bind-
ing domain (LBD)/activation function 2 (AF2) domain of
receptors through an LXXLL motif or NR boxes (25).
In the absence of NR ligands, on the other hand, many
NRs prevent gene transcription via recruitment of core-
pressors such as N-CoR and SMRT, which have been
proposed to antagonize the actions of coactivators and
to maintain a more repressed state in the chromatin struc-
ture. Histone deacetylases (HDACs)-dependent and
HDACs-independent mechanisms are involved in the
transrepression induced by N-CoR and SMRT (26).
In this study, we have identified that SMILE represses
the transcriptional activities of GR, CAR and HNF4a
through direct interaction. We have demonstrated that
SMILE represses the transactivities of the NRs via com-
petition with coactivators and the recruitment of HDACs
for its active repression. Overall, our findings suggest that
SMILE acts as a novel corepressor of NRs.
MATERIALS AND METHODS
Plasmid and DNA construction
The plasmids of pCMV-b-gal, pcDNA3mCAR, pcDNA3
mCARAF2 and pcDNA3-HA-mPPARg, -PGC-1a,
pSG5HA-GRIP1, (NR1)X5-Luc, Gal4-tk-Luc and
PPRE-Luc were described elsewhere (27–29).
p(HNF4)8-tk-Luc and MMTV-Luc were kindly provided
by Drs Akiyoshi Fukamizu and Yoon-Kwang Lee, respec-
tively. pcDNA3-SMILE, pcDNA3-Flag-SMLE,
pcDNA3-SMILE-83Leu, pcDNA3-SMILE-1Phe,
pGEX4T-1, pGEX4T-1-SMILE, pEBG, pEGFP-
SMILE, pSuper, pSuper-siSHP, pSuper-siSMILE-I and
pSuper-siSMILE-II were described previously (2).
pcDNA3-Flag-mCAR, pcDNA3-HA-mCAR and
pcDNA3-HA-HNF4awere constructed by inserting the
full PCR fragments of the ORFs into the EcoRI/XhoI
sites of pcDNA3-Flag, or pcDNA3-HA vector.
pcDNA3-HA-mGR was generated by subcloning the full
ORF of mouse GR into the XhoI/XbaI sites of
pcDNA3-HA vector. Mouse GR deletion constructs,
including pcDAN3-mGR-N (1–531 aa) and pcDNA3-
mGR-LBD (532–783 aa), were subcloned via the insertion
of the PCR fragments of mouse GR into pcDNA3
between the BamHI and XhoI sites. The pcDNA3-HA-
HNF4aCD (1–370 aa) and pcDNA3-HA-HNF4aLBD
(1–174 aa) plasmids were constructed via subcloning the
EcoRI-XhoI cDNA fragments of rat HNF4ainto
pcDNA3-HA vector. The SMILE leucine zipper region
mutant SMILE-L (239–267)V was generated via PCR-
mediated site-directed mutagenesis, and the PCR products
were cloned into the EcoRI/XhoI sites of pcDNA3-Flag
and the BamHI/KpnI sites of pEBG vector. pEBG-
SMILE and pEBG-SMILE deletion constructs were con-
structed by inserting full length SMILE or appropriate
SMILE deletion fragments into pEBG vector between
BamHI and KpnI sites. All plasmids were confirmed via
sequencing analysis.
Gal4-DBD fusion constructs were generated using
the pCMX-Gal4N expression vector (30). To generate
Gal4-DBD-SMILE, EcoRI/XhoI digested full-length
SMILE fragments from pcDNA3-Flag-SMILE were
cloned into EcoRI/SalI-digested pCMX-Gal4N vector.
To construct the Gal4-DBD-SMILE deletion constructs,
SMILE cDNA deletion fragments were obtained from
pcDNA3-Flag-SMILE via PCR, and cloned into
pCMX-Gal4N vector between the EcoRI and SalI sites.
pSuper-siHDAC1, pSuper-siHDAC3 and pSuper-
siHDAC4 constructs were constructed by inserting a 64-
bp double-stranded oligonucleotide containing 50-aagcaga
tgcagagattcaac-30of the human HDAC1 cDNA sequence,
or 50-aagatgctgaaccatgcacct-30of human HDAC3 cDNA
sequence, or 50-aatgtacgacgccaaagat-30of human HDAC4
cDNA sequence into the pSUPER vector between BglII
and Xho I sites. All plasmids were confirmed via sequen-
cing analysis.
Cell culture, transient transfection assay and
luciferase assay
HEK293T (293T), HepG2 and HeLa cells were obtained
from the American-type culture collection (ATCC) and
cultured according to the manufacturer’s instructions.
Transient transfection was performed using Superfect
transfection reagent (Qiagen) as described previously
(2). Total amounts of DNA in each transfection were
maintained at the same levels using empty pcDNA3 vec-
tors. Luciferase assays were performed as described
Nucleic Acids Research, 2009, Vol. 37, No. 12 4101
previously (31). Fold activity was calculated considering
the activity of reporter gene alone as 1.
In vitro glutathione S-transferase (GST) pull-down assay
and competition assay
In vitro GST pull-down and competition assays were
performed as described previously (31,32).
Co-immunoprecipitation (Co-IP) and western blot analysis
Co-IP and western blot analysis were performed as
described previously (2,33). Antibodies used for Co-IPs
were anti-GR (Santa Cruz, sc-8992), anti-CAR (Santa
Cruz, sc-13065) and anti-HNF4aantibody (Santa Cruz,
sc-8987). Control Co-IPs were carried out using rabbit
IgG (Santa Cruz, sc-2027). In western blot assays, the fol-
lowing antibodies were used at dilution of 1:1000: anti-
Flag M2 (Stratagene, #200472-21), anti-HA (12CA5)
(Roche Molecular Biochemicals), anti-SMILE (Abcom,
#ab28700), anti-GST (Santa Cruz, sc-33614) and anti-
tubulin (Cell Signaling Technology, #2146) antibodies.
In western blot analysis of immunoprecipitated proteins,
conventional HRP conjugated anti-rabbit IgG was
replaced with rabbit IgG TrueBlot (eBioscience, #18-
8816) to eliminate signal interference by the immunoglo-
bulin heavy and light chains.
In vivo GST pull-down assay
In vivo GST pull-down experiments were performed
as described previously (2). In brief, 293T cells were trans-
fected in 60 mm dishes with the indicated plasmids. Forty-
eight hours after transfection, the whole-cell extracts were
prepared and equal amounts of total protein were used for
in vivo GST pull-down assays followed by western blot
analysis.
Confocal microscopy
The confocal microscopy assays were carried out as
described previously (2). In brief, the HeLa cells grown
on gelatin-coated coverslips were transfected with indi-
cated plasmids using Effectene transfection reagent
(Qiagen) according to the manufacturer’s instructions.
Twelve hours after transfection, the cells were treated
with or without 100 nM dexamethasone for 12 h followed
by cell fixation and immunostaining. To detect HA-fusion
proteins and nucleus, the cells were incubated with dye
Alexa 594-conjugated anti-HA monoclonal antibody
(1:500 dilution; Invitrogen) for 1 h at room temperature
(258C), washed three times in PBS, and incubated with
0.1 mg/ml of DAPI (Invitrogen) solution for 10 min.
After three times washing with PBS, the cells were sub-
jected to observation by confocal microscopy.
Preparation of recombinant adenovirus
The adenovirus-encoding human SMILE was described
previously (2). The adenovirus-encoding rat HNF4awas
constructed via the previously described method (2).
In brief, the cDNA-encoding rat HNF4awas cloned
into the KpnI/XbaI sites of the pAdTrack-CMV vector.
The recombination of the AdTrack-CMV-rHNF4a
(where rHNF4ais rat HNF4a) with adenoviral gene
carrier vector was performed by transformation to pre-
transformed adEasy-BJ21-competent cells.
RNA interference
Knockdown of SMILE and histone deacetylases
(HDACs) was performed using the pSuper vector system
(2). 293T cells were transfected with siRNA constructs
using Lipofectamine2000 (Invitrogen) according to the
manufacturer’s instructions. siRNA-treated cells were
subjected to reverse transcription-PCR (RT–PCR) or
the second transfection as indicated in the figure legends.
RT–PCR analysis
Total RNA was isolated using the TRIzol reagent
(Invitrogen) according to the manufacturer’s
instructions. The mRNAs of SMILE, SHP, insulin-like
growth factor-binding protein 1 (IGFBP1), CYP2B6 and
cholesterol 7a-hydroxylase (CYP 7A1) were analyzed by
RT–PCR as previously described (2), and the mRNA
levels of b-actin served as an internal control for
RT–PCR. The RT–PCR primers are provided in Supple-
mentary Table 1.
Chromatin immunoprecipitation (ChIP) Assay
ChIP assay was performed as previously described (32).
In brief, HepG2 cells seeded into 35 mm culture dishes
were treated as indicated in the figure legends and then
the cells were fixed with 1% formaldehyde, washed with
ice-cold PBS, harvested and sonicated. The soluble chro-
matin was then subjected to immunoprecipitation using
anti-GR, anti-CAR, anti-HNF4a, anti-SMILE (Santa
Cruz, sc-49329), or acetyl-histone H3 (Lys9) antibody
(Cell Signaling Technology, #9671) followed by protein
A agarose/salmon sperm DNA (Upstate). DNA was
recovered via phenol/chloroform extraction and amplified
by PCR for 30–35 cycles using specific primer sets for the
indicated specific regions of IGFBP1, CYP2B6 and
CYP7A1 genes. The PCR primers for ChIP assay are pro-
vided in Supplementary Table 2.
Statistical analysis
Student’s t-test was performed using GraphPad Prism ver-
sion 3.0 for Windows and results were considered to be
statistically significant when P<0.05.
RESULTS
SMILE inhibits the transactivation of nuclear receptor GR,
CAR and HNF4a
Previously, we have reported that SMILE regulates
orphan NR small heterodimer partner (SHP)-repressed
ER transactivation through direct interaction with SHP
(2). To investigate whether SMILE interacts with other
NRs, yeast two-hybrid interaction assays were performed.
Of great interest, SMILE was determined to interact with
many NRs, including GR, TRa, CAR, SF-1, ERRa,
ERRb, ERRg, HNF4aand Nur77 (Supplementary
Table 3). For detailed study, GR, CAR and HNF4a
4102 Nucleic Acids Research, 2009, Vol. 37, No. 12
were selected as a representative of classical endocrine
receptors, adopted orphan receptors, and orphan recep-
tors (8), respectively.
We have previously demonstrated that wild-type
SMILE generates two isoforms, SMILE long form
(SMILE-L) and SMILE short form (SMILE-S), which
can be produced by the mutants SMILE-83Leu and
SMILE-1Phe, respectively (2). To determine whether
these isoforms can regulate the transactivation of GR,
CAR and HNF4a, transient transfection was performed
in 293T cells. Overexpression of wild-type SMILE
dose-dependently repressed dexamethasone-stimulated
GR transactivation (Figure 1A), as well as CAR and
HNF4atransactivation (Figure 1B and C). Furthermore,
overexpression of SMILE-L or SMILE-S through
the aforementioned SMILE mutants evidenced similar
inhibitory effects on the NRs (Figure 1A–C). However,
SMILE did not show any significant effect on
PPARg-mediated transactivation (Figure 1D). Western
blot analysis demonstrated that the overexpression
of wild-type SMILE, SMILE-L or SMILE-S alone did
not significantly change the protein expression of
HA-GR and HA-CAR (Figure 1E and F). Taken
together, these results suggest that both SMILE isoforms
down-regulate GR, CAR and HNF4atransactivation.
Since SMILE-L and SMILE-S show similar effects on
those NRs, and SMILE-L is the major isoform in tested
cell lines and tissues (2), we have focused on wild-
type SMILE-generated SMILE-L (SMILE) for further
investigations.
Figure 1. SMILE represses GR-, CAR- and HNF4atransactivation. Reporter assays (A–D) were performed as described in the Materials and
methods section. HEK293T (293T) cells were cotransfected with 0.1 mg of pcDNA3-HA-GR (A), pcDNA3-HA-CAR (B), pcDNA3-HA-HNF4a(C)
or, pcDNA3-HA-PPARg(D) and 0.1 mg of MMTV-Luc (A), (NR1)5-Luc (B), (HNF4)8-Luc (C) or, PPRE-Luc (D) luciferase reporter vectors, 0.1mg
of pCMV-b-gal as internal control, together with indicated amounts of plasmids expressing wild-type SMILE, SMILE-L (SMILE-83Leu) and
SMILE-S (SMILE-1Phe). Twenty-four hours after transfection, the cells were treated with or without 100 nM of dexamethasone (Dex) (A) or
rosiglitazone (Rosi) (D) as indicated for 24 h prior to the measurement luciferase activity. The effects of overexpressed SMILE on the protein levels of
HA-GR (E) and HA-CAR (F). 293T cells were cotransfected with various plasmids as indicated. Fifty microgram of cellular extracts from the
transient transfection assay were subjected to western blot analysis. The proteins of HA-GR, HA-CAR, SMILE and tubulin were detected as
described in the Materials and methods section. wt, wild-type.
Nucleic Acids Research, 2009, Vol. 37, No. 12 4103
Knockdown of SMILE gene expression up-regulates GR,
CAR and HNF4atransactivation
To determine whether endogenous SMILE negatively reg-
ulates GR, CAR and HNF4atransactivation, we investi-
gated these NRs-mediated transcriptional activities after
individually knocking down the expression of SMILE and
SHP in HepG2 cells. As shown in Figure 2A, siSMILE-II
(siSM-II) efficiently knocked down the mRNA levels of
SMILE, whereas siSMILE-I (siSM-I) did not show any
significant effect, and siSHP efficiently silenced the gene
expression of SHP, which is a well-known corepressor of
GR, CAR and HNF4a(2). In the reporter assay, knock-
down of SMILE gene expression through siSMILE-II
increased GR-, CAR- and HNF4a-mediated transactiva-
tion by 65–100%, which is similar to the effect of positive
control siSHP (Figure 2B–D). Collectively, these results
indicate that endogenous SMILE is a functional corepres-
sor of receptor GR, CAR and HNF4a.
SMILE interacts with GR, CAR and HNF4ain vitro
and in vivo
To determine whether SMILE inhibits GR, CAR and
HNF4atransactivation through protein–protein interac-
tion, in vitro and in vivo GST pull-down experiments were
performed. For the in vitro GST pull-down assays, bac-
teria-expressed GST only, or GST-SMILE proteins were
incubated with in vitro translated
35
S-labeled GR, CAR,
or HNF4a. We found that
35
S-labeled GR was able to
bind to GST-tagged SMILE in the presence of dexametha-
sone, but not in the absence of the ligand (Figure 3G,
upper panel), indicating that SMILE can interact with
GR in a ligand-dependent manner. Moreover,
35
S-labeled
CAR, and HNF4awere also observed to bind to GST-
SMILE (Figure 3G, lower panel). These results suggest
that SMILE can interact with GR, CAR and HNF4a
in vitro. For the in vivo GST pull-down assays, mammalian
expression vectors encoding either pEBG (GST) alone or
pEBG-SMILE (GST-SMILE) together with indicated
pcDNA3-HA-GR, pcDNA-Flag-mCAR, or pcDNA3-
HA-HNF4awere cotransfected into 293T cells. As
shown in Figure 3A, HA-GR was detected in the copreci-
pitate only in the presence of its ligand when coexpressed
with GST-SMILE but not with GST alone. The expres-
sion levels of GST, GST-SMILE and HA-GR were con-
firmed by western blot analysis (Figure 3A, middle and
bottom panels, respectively). These results demonstrate
that ectopically expressed SMILE interacts with exoge-
nous GR in a ligand-dependent manner in 293T cells.
Similarly, interactions of exogenous SMILE with CAR
(Figure 3B) and HNF4a(Figure 3C) were verified using
in vivo GST pull-down assays. To further examine whether
endogenous SMILE and these NRs can interact in vivo,
co-immunoprecipitation assays were performed.
Endogenous SMILE proteins were found to be co-preci-
pitated with GR in a ligand-dependent manner
(Figure 3D), while with CAR in a ligand-independent
manner (Figure 3E). In addition, endogenous SMILE
was co-precipitated with endogenous HNF4a
(Figure 3F), confirming the interaction between that endo-
genous SMILE and the NRs. Collectively, these results
indicate that SMILE can interact with GR, CAR and
HNF4aboth in vitro and in vivo.
To examine whether SMILE and its binding partners
(GR, CAR, or HNF4a) are colocalized to the same sub-
cellular compartments, confocal microscopic studies were
performed. HeLa cells were cotransfected with the expres-
sion plasmids pEGFP-SMILE along with pcDNA3-HA-
GR, or pcDNA3-HA-mCAR, or pcDNA3-HA-HNF4a,
stained with dye Alexa 594-conjugated anti-HA antibody
Figure 2. siSMILE increases GR-, CAR- and HNF4atransactivation.
(A) Effect of siRNAs for SMILE or SHP on the expression of SMILE
and SHP. HepG2 cells were transfected with pSUPER siSMILE-I
(siSM#1), siSMILE-II (siSM#2), siSHP or pSUPER [control (con)],
and after 72 h the total RNA was isolated. The mRNA levels of
SMILE and SHP were measured via RT–PCR analysis, with b-actin
shown as a control. The data shown are representative of at least three
independent experiments. (B–D) SMILE siRNA induces GR-, CAR-
and HNF4a-mediated transactivation in HepG2 cells. HepG2 cells
were transfected with pSUPER, pSUPER siSMILE-I (siSM#1), or
pSUPER siSMILE-II (siSM#2). After 24 h, the cells were cotransfected
with expression vector for GR (B), CAR (C) or HNF4a(D), and
MMTV-Luc (B), (NR1)5-Luc (C) or (HNF4)8-Luc (D) luciferase
reporter vectors, together with pCMV-b-gal as internal control.
Twenty-four hours after transfection, the cells in (B) were treated for
24 h with or without 100 nM Dex prior to the measurement of lucifer-
ase activity. The mean and standard deviation (n= 3) of a representa-
tive experiment are shown. P<0.05; P<0.01, using Student’s t-test.
4104 Nucleic Acids Research, 2009, Vol. 37, No. 12
Figure 3. Interactions and colocalizations of SMILE with NRs. (A–C)In vivo interactions of exogenous GR (A), CAR(B) and HNF4a(C) with
exogenous SMILE. 293T cells were cotransfected with expression vectors for HA-GR (A), Flag-mCAR (B), or HA-HNF4a(C) with pEBG-
SMILE (GST-SMILE) or pEBG alone (GST). The in vivo GST pull-down assays were performed in the presence or absence of the GR ligand Dex
(100 nM) as indicated (A). The complex formation (top panel in A–C, GST puri.) and the amount of HA-GR, Flag-mCAR or HA-HNF4aused
for the in vivo binding assay (bottom panel in A–C, Lysate) were determined via western blot using an anti-HA or anti-Flag antibody. The same
blot was stripped and reprobed with an anti-GST antibody (middle panel in A–C) to confirm the expression levels of the GST fusion protein
(GST-SMILE) and the GST control (GST). In vivo interactions of endogenous GR (D), CAR (E) and HNF4a(F) with endogenous SMILE. Co-
immunoprecipitation assays were performed using cell extract from HepG2 cells using indicated antibodies in the presence or absence of 100 nM
Dex or 250 nM of TCPOBOP. Endogenous SMILE was immunoprecipitated with GR, CAR and HNF4a(upper panels). The proteins in the cell
lysates (middle and lower panels) were analyzed with western blot analysis using indicated antibodies. (G) In vitro GST pull-down assays. Upper
panel,
35
S-radiolabeled GR protein was incubated with GST, or GST-SMILE fusion proteins in the presence of 100 nM Dex or vehicle (DMSO).
Lower panel:
35
S-radiolabeled HNF4a, or CAR proteins were incubated with GST, or GST-SMILE fusion proteins. The input lane represents 10%
of the total volume of in vitro-translated proteins used for binding assay. Protein interactions were detected via autoradiography. (H–J) Co-
localizations of SMILE with NRs. Hela cells grown on coverslips on 12-well plates were transfected with 0.1 mg of expression vectors encoding
GFP-SMILE and HA-GR (H), HA-CAR (I) or HA-HNF4a(J). Twelve hours after transfection, the cells (H) were treated with 100 nM Dex
for 12 h. For the immunofluorescence of fixed cells, the HA-fusion proteins were detected with dye Alexa 594-conjugated anti-HA
monoclonal antibody. The cell images were captured under 400 magnifications. The data shown are representative of at least three independent
experiments.
Nucleic Acids Research, 2009, Vol. 37, No. 12 4105
and DAPI, and analyzed via confocal microscopy. As
shown in Figure 3H, GFP-SMILE was predominantly
localized within the nucleus, and was also weakly detected
in the cytoplasm, which was consistent with the results of
our previous study (2). In the presence of ligand, GR was
detected predominantly in the nucleus (Figure 3H). CAR
(Figure 3I) and HNF4a(Figure 3J) were also detected
mainly in the nucleus. The merged images indicated that
SMILE and GR, CAR, or HNF4awere colocalized to the
nucleus (Figure 3H–J). Collectively, these data reveal that
SMILE interacts and colocalizes with receptor GR, CAR
and HNF4ain vivo.
Dimerization of SMILE is not required for its repressive
function
It was reported that SMILE can homodimerize through
the leucine zipper region like other bZIP proteins (1,34)
and this homodimerization is important for the function
of leucine zipper protein (35). To determine whether the
homodimerization is essential for the repressive function
of SMILE, site-directed mutational analysis and in vivo
GST pull-down assays were performed. We generated a
mutant SMILE [SMILE-L (239–267) V], in which five
consecutive leucine residues in the leucine zipper region
were mutated to valine (Figure 4A). Next, the possibility
of homodimer formation was investigated via in vivo
GST pull-down assays. As expected, Flag-SMILE was
shown to be coprecipitated with GST-SMILE. However,
Flag-SMILE-L (239–267)V was not coprecipitated with
GST-SMILE-L (239–267)V (Figure 4B, upper panel),
although the mutant proteins were expressed to compara-
ble levels as the wild-type SMILE (Figure 4B, middle and
lower panel). These results indicate that SMILE is capable
of forming homodimers, and the mutation of the five con-
secutive leucine residues in the leucine zipper region
destroyed the homodimerization. Next, the functional
effects of the mutation were assessed using reporter
assays. SMILE-L (239–267)V repressed GR- and CAR-
mediated transcriptional activity as profoundly as the
wild-type SMILE (Figure 4C and D). Although the
repression of SMILE-L (239–267)V on HNF4awas not
so strong as wild-type SMILE, SMILE-L (239–267)V still
significantly inhibited HNF4a(Figure 4E). Collectively,
these results indicate that SMILE homodimerization
is not essential for its repressive function.
Interaction domain mapping of SMILE with GR, CAR
and HNF4a
To identify the interaction domain of SMILE with the
NRs, a series of SMILE deletion fragments (Figure 5A)
were cloned into in vivo GST vector and in vivo GST pull-
down assays were performed. We found that the mutant
Figure 4. Homodimerization of SMILE is not essential for the repressive function. (A) Structure of human SMILE. The basic region and leucine
zipper domains are shown. The leucine zipper mutant SMILE-L (239–267)V indicates the leucine residues between positions 239 and 267 were
mutated to valine, as indicated by the arrows. The numbers in the figure indicate the amino-acid residues. (B)In vivo homodimerization possibility
analysis of wt SMILE and SMILE-L (239–267)V. 293T cells were cotransfected with expression vectors for Flag-SMILE, Flag-SMILE-L (239–267)V
with pEBG-SMILE (GST-SMILE wt) or pEBG-SMILE-L (239–267)V or pEBG alone (GST) as indicated. The complex formation (top panel, GST
puri.) and the amount of Flag-SMILE, Flag-SMILE-L (239–267)V used for the in vivo-binding assay (bottom panel, Lysate) were determined by
western blot using anti-Flag antibody. The same blot was stripped and reprobed with an anti-GST antibody (middle panel) to verify the expression
levels of the GST fusion proteins (GST-SMILE) and the GST control (GST). (C–E) The effect of SMILE-L (239–267)V on GR, CAR and HNF4a-
mediated transactivation. HepG2 cells were cotransfected with expression vector for GR (C), CAR (D), or HNF4a(E) and MMTV-Luc (C),
(NR1)5-Luc (D), or (HNF4)8-Luc (E) luciferase reporter vectors, pCMV-b-gal as internal control, together with expression vectors for wt
SMILE or SMILE-L (239–267)V as indicated. Twenty-four hours after transfection, the cells in (C) were treated with 100 nM Dex for 24 h.
Forty-eight hours after tranfection, luciferase activity was measured. wt, wild type. The mean and standard deviation (n= 3) of a representative
experiment are shown.
4106 Nucleic Acids Research, 2009, Vol. 37, No. 12
GST-SMILE-N2 (1–202 aa) and GST-SMILE were asso-
ciated strongly with GR, CAR and HNF4a, whereas the
mutants GST-SMILE-N1 (1–112 aa), GST-SMILE-202
(203–354 aa) and GST-SMILE-268 (269–354 aa) were
not significantly associated with the NRs (upper panel in
Figure 5B–D). Moreover, all the GST SMILE fusion pro-
teins and GR, CAR, HNF4aproteins were expressed
properly (middle and lower panel in Figure 5B–D), indi-
cating that the differences in the interactions between
the SMILE mutants and the NRs are not the result of
differences in protein expression. Taken together, these
results indicate that the region spanning residues
113–202 of SMILE is responsible for the interactions
with the NRs.
To identify the region of GR, CAR and HNF4a
involved in the interactions with SMILE, in vitro GST
pull-down experiments were performed using various
GR (Figure 6A), mCAR (Figure 6C) and HNF4a-deletion
constructs (Figure 6E). GST-SMILE was observed to bind
to
35
S-labeled full length GR in the presence of ligand
(Figure 6B, upper panel), as well as
35
S-labeled GR-
LBD (532–783 aa) (Figure 6C, lower panel), but did
not bind to GR-N (1–531 aa) (Figure 6B, middle panel),
indicating the LBD of GR is important for the ligand-
dependent interaction between GR and SMILE. In addi-
tion, deletion of AF2 domain abolished the interaction of
CAR with SMILE (Figure 6D) indicating that the AF2
domain of CAR is essential for the interaction. In the case
of HNF4a, GST-SMILE was capable of interacting with
full length HNF4aand HNF4aCD (1–370 aa) (Figure 6F,
upper and middle panel), which harbor the complete LBD
domain, but was not able to interact with HNF4aLBD
(1–174 aa) (Figure 6F, lower panel), thereby indicating
that the LBD domain of HNF4ais required for the inter-
action between HNF4aand SMILE. Collectively, these
results suggest that the LBD/AF2 domain of GR, CAR
and HNF4aare essential for the interactions with SMILE.
SMILE competes with coactivators
It has been well established that a host of coactivators,
including PGC-1a, CBP/p300 and GRIP1, can interact
with the LBD/AF2 region of NRs to form LBD-coactiva-
tor complexes and positively regulate NR-mediated tran-
scription (25,36,37). The aforementioned results that
SMILE interacts with the LBD/AF2 region of GR,
HNF4aand CAR prompted us to determine whether
SMILE could compete with coactivators. In the presence
of ligand, overexpression of GRIP1 increased GR-stimu-
lated transcriptional activity (Figure 7A), which is consis-
tent with previous report (37), and overexpression of
SMILE reduced the coactivation in a dose-dependent
manner (Figure 7A). In a reciprocal experiment, the trans-
fection of increasing quantities of GRIP1 expression
vector induced a gradual release of SMILE repression
on GR (Figure 7A). Interestingly, SMILE overexpression
also reduced PGC-1a-enhanced HNF4a-, and CAR-
stimulated transactivation in a dose-dependent fashion,
and overexpression of PGC-1arecovered the inhibitory
effect of SMILE on HN4aand CAR (Figure 7B and C).
These results indicate that SMILE can compete with the
coactivators GRIP1 and PGC-1afunctionally in vivo.To
further confirm the competition between SMILE and
either GRIP1 or PGC-1a,in vitro GST pull-down assays
Figure 5. Interaction domain of SMILE. (A) Schematic representation of the structures of SMILE mutants. bZIP indicates the basic region leucine
zipper domain. The numbers in the figure indicate the amino acid residues. (B–D)In vivo interaction assays between wt SMILE or SMILE mutants
and GR (B), CAR (C) or HNF4a(D). 293T cells were cotransfected with expression vectors for HA-GR (B), Flag-CAR (C) or HA-HNF4a(D) with
pEBG alone (GST) or pEBG-SMILE (GST-SMILE) fusions as indicated. The in vivo GST pull-down assays in (B) was performed in the presence or
absence of GR ligand Dex (100 nM). The complex formation (top panel in B–D, GST puri.) and the amount of HA-GR, Flag-mCAR or HA-
HNF4aused for the in vivo-binding assay (bottom panel in B–D, Lysate) were determined by western blot using an anti-HA or anti-Flag antibody.
The same blot was stripped and reprobed with an anti-GST antibody (middle panel in B–D) to confirm the expression levels of the GST fusion
proteins (GST-SMILE fusions) and the GST control (GST). wt, wild type. The data shown are representative of at least three independent
experiments with similar results.
Nucleic Acids Research, 2009, Vol. 37, No. 12 4107
Figure 7. SMILE competes with coactivators GRIP1 and PGC-1a. Reporter assays in (A–C) were performed as described in the Materials and methods
section. The mean and standard deviation (n= 3) of a representative experiment are shown. HepG2 cells were cotransfected with 0.1 mg of indicated
reporter plasmids, MMTV-luc (A), (NR1)5-luc (B), or (HNF4)8-Luc (C), and 0.1 mg of pcDNA3-HA-GR (A), pcDNA3-HA-mCAR (B) or pcDNA3-
HA-HNF4a(C), together with the indicated quantities of pcDNA3-Flag-SMILE, pSG5-HA-GRIP1 (A) or pcDNA3-HA-PGC-1a(B and C). Twenty-
four hours after transfection, the cells were treated with or without GR ligand Dex (100 nM) for 24h prior to the measurement of luciferase activity. (D)
In vitro competition between SMILE and GRIP1 or PGC-1a.
35
S-radiolabeled GR (in the presence of 100 nM DEX), or CAR, or HNF4aproteins were
incubated with GST, or GST-SMILE fusion proteins, together with an increasing amounts of unlabeled in vitro translated GRIP1 (0, 3, 6 or 12 ml, upper
panel) or PGC-1a(0, 3, 6 or 12 ml, middle and lower panel) proteins. After pull-down, the beads were washed and the samples separated on a 12%
SDS–PAGE gel and the protein interactions were detected via autoradiography. The data shown represent at least three independent experiments.
Figure 6. SMILE interacts with LBD/AF2 domain of the NRs. (A, C and E) Schematic representation of the structures of the GR (A), CAR (C) and
HNF4a(E) mutants. AF1, activation function-1 domain; DBD, DNA-binding domain; LBD, ligand-binding domain; AF2, activation function-2
domain. (B)
35
S-radiolabeled GR proteins were incubated with GST, or GST-SMILE fusion proteins in the presence of ligand Dex (100 nM) or
vehicle (DMSO). (Dand F)
35
S-radiolabeled CAR (D) or HNF4aproteins (F) were incubated with GST, or GST-SMILE fusion proteins. The input
lane represents 10% of the total volume of in vitro-translated proteins used for the binding assay. Protein interactions were detected via autoradio-
graphy. The data shown represent at least three independent experiments with similar results.
4108 Nucleic Acids Research, 2009, Vol. 37, No. 12
were performed. In the presence of the ligand, increasing
amounts of the cold competitor, HA-GRIP1, reduced
the binding of
35
S-methionine-labeled GR protein to
GST-SMILE (Figure 7D, upper panel). Moreover,
increasing amounts of the cold competitor, HA-PGC-1a,
reduced the association of GST-SMILE with
35
S-HNF4a
and
35
S-CAR (Figure 7D, lower panel). Taken together,
these results indicate that coactivator competition is one
mechanism underlying for the repression of GR, HN4a
and CAR by SMIILE.
SMILE has intrinsic repressive activity
Many corepressors, including SHP (38,39), and RIP140
(40), were reported to inherently possess transcriptional
repressive activity. To determine whether SMILE also
has an intrinsic repressive function, the transcriptional
activities of a set of Gal4-SMILE deletion constructs
were investigated (Figure 8A). The reporter plasmid
Gal4-tk-Luc, and indicated expression vectors encoding
Gal4-DBD alone, Gal4-SMILE, or Gal4-SMILE dele-
tions were cotransfected into 293T cells. As indicated in
Figure 8B, Gal4-SMILE, Gal4-SMILE-112 (113–354
aa), -202 (203–354 aa) showed only 10% of Gal4-
DBD-stimulated reporter activity, and 268 (269–354
aa) showed only 15% of the activity by Gal4-DBD.
However, Gal4-SMILE-N1 (1–112 aa) and Gal4-
SMILE-N2 (1–202 aa) displayed 2–3-fold activity of
that by Gal4-DBD, and Gal4-SMILE-NC (113–202
aa) displayed a comparable effect to Gal4-DBD.
Moreover, all of the Gal4-fusions were expressed properly
(Figure 8C), indicating the distinct reporter activities sti-
mulated by the Gal4-SMILE fusions were not the conse-
quence of different protein levels. Taken together, these
results indicate that the SMILE N-terminus (1–112 aa)
has intrinsic activation activity, whereas the C-terminus
(203–354 aa) has intrinsic repression. As a whole,
SMILE showed repression activity, indicating the intrinsic
repression derived from the C-terminus predominates.
SMILE recruits HDACs in a NR-specific manner
It has been reported previously that the recruitment of
HDAC contributes to the intrinsic repressive function
of corepressors, including RIP140 (41), and SHP (42).
To determine whether SMILE could also recruit
HDACs, the effect of the HDAC-specific inhibitor trichos-
tatin A (TSA) on SMILE-mediated repression was exam-
ined. The results showed that TSA treatment partially but
significantly reversed the repression of GR and HNF4aby
SMILE (Figure 9A and B), whereas TSA treatment did
not significantly affect the repression of CAR (Figure 9C).
These results demonstrate that the recruitment of HDACs
is required for the inhibition of SMILE on GR and
HNF4a, but not required for the inhibition on CAR, indi-
cating that the recruitment of HDACs by SMILE might
be NR-specific.
To further determine the HDACs involved in the
repression of GR and HNF4aby SMILE, the potential
interactions between HDACs (HDAC1, HDAC2,
HDAC3, HDAC4, HDAC5 and HDAC6) and SMILE
were investigated via in vivo GST pull-down assays.
HDAC1 (Figure 9D), and HDAC3 (Figure 9E), as well
as HDAC4 (Figure 9F) were detected in the coprecipitate
only when coexpressed with the GST-SMILE but not with
GST alone. The expression levels of GST, GST-SMILE,
Flag-HDAC1, HA-HDAC3 and Flag-HDAC4 were con-
firmed via western blot analysis (middle and bottom panel
in Figure 9D–F). However, Flag-HDAC2, Flag-HDAC5
and Flag-HDAC6 were not detected in the coprecipitate
(data not shown). These results demonstrate that SMILE
specifically interacts with HDAC1, HDAC3 and HDAC4
in vivo, thereby indicating that the recruitment of HDAC1,
HDAC3 and HDAC4 may play a role in the SMILE-
mediated repression of GR and HNF4a.
To further investigate whether HDAC1, HDAC3 and
HDAC4 are also involved in the repressive effect of
SMILE on GR and HNF4a, reporter assays combined
with siRNA-mediated knockdown of the HDACs gene
expression were performed. As shown in Figure 9G,
Figure 8. Intrinsic repressive function of SMILE. (A) Schematic representation of wt SMILE and its deletion mutants fused in-frame to the yeast
Gal4 DBD-(1–147 amino acid). bZIP, basic region leucine zipper domain. (B) 293T cells were cotransfected with the reporter plasmid Gal4-tk-Luc
and the indicated expression vectors of Gal4-SMILE or Gal4-SMILE deletion mutants together with pCMV-b-gal vector. Forty-eight hours after
transfection, the luciferase activity was measured as described in the Materials and methods section. The normalized luciferase activity values are
shown as the percentage of the Gal4-tk-Luc reporter activity stimulated by Gal4-DBD. The mean and standard deviation (n= 3) of a representative
experiment are shown. (C) Western blot analysis of Gal4-SMILE and Gal4-SMILE deletion mutants. 293T cells were transfected with the indicated
Gal-SMILE chimeras. Whole cell extracts (50 mg) were analyzed via western blot using anti-Gal4 rabbit polyclonal antibody.
Nucleic Acids Research, 2009, Vol. 37, No. 12 4109
siHDAC3 or siHDAC4 alone induced a slight reduction
in the repression of GR by SMILE, but siHDAC1 exerted
no detectable effects. Moreover, the combination of
siHDAC3 and siHDAC4 additively and significantly atte-
nuated the repression. In the case of HNF4a, only the
combination of siHDAC1, siHDAC3 and siHDAC4 sig-
nificantly attenuated the repression of HNF4aby SMILE
(Figure 9H). In addition, all the siRNAs for HDAC1,
HDAC3, or HDAC4 were demonstrated to knockdown
the specific HDAC gene expression effectively (Figure 9I).
Taken together, these results indicate that HDAC3 and
HDAC4 contribute to the inhibition of GR by SMILE,
and HDAC1, HDAC3 and HDAC4 contribute to the
repression of HNF4aby SMILE.
Figure 9. SMILE recruits HDACs. (A–C) TSA releases SMILE-mediated repression on GR and HNF4a. HepG2 cells were cotransfected with 0.1 mg
of reporter plasmids, MMTV-luc (A), (HNF4)8-luc (B) or (NR1)5-Luc (C), and 0.1 mg of pcDNA3-HA-GR (A), pcDNA3-HA-HNF4a(B) or
pcDNA3-HA-mCAR (C), together with or without 0.2mg of pcDNA3-Flag-SMILE as indicated. Twenty-four hours after transfection, the cells were
treated for 12 h with or without 100 nM of Dex. Then the cells were treated with indicated concentration of TSA for 12 h in the absence or presence
of Dex (100 nM). In vivo interactions of HDAC1 (D), HDAC3 (E) and HDAC4 (F) with SMILE. 293T cells were cotransfected with expression
vectors for Flag-HDAC1, or HA-HDAC3 or Flag-HDAC4 with pEBG-SMILE (GST-SMILE) or pEBG alone (GST). The complex formation (top
panel in D–F, GST puri.) and the amount of Flag- or HA-tagged HDAC fusion proteins used for the in vivo-binding assay (bottom panel in D–F,
Lysate) were determined using anti-Flag or anti-HA antibody, respecitively. The same blot was stripped and reprobed with an anti-GST antibody
(middle panel in D–F) to confirm the expression levels of the GST fusion protein (GST-SMILE) and the GST control (GST). (Gand H) The effect of
HDAC siRNAs on the inhibition of GR- and HNF4a-mediated transactivation by SMILE. HepG2 cells were transfected with pSuper siHDAC1,
siHDAC3, or siHDAC4 as indicated and after 24 h, the cells were cotransfected with 0.1 mg of indicated reporter plasmids, MMTV-luc (G), or
(HNF4)8-Luc (H), and 0.1 mg of pcDNA3-HA-GR (G) or pcDNA3-HA-HNF4a(H), together with 0.1 mg of pcDNA3-Flag-SMILE and after 24 h,
the cells were treated with or without 100 nM of Dex for 24 h prior to the measurement of luciferase activity. The mean and standard deviation
(n= 3) of a representative experiment are shown. P<0.05, using Student’s t-test. (I) Effect of siRNAs on the expression of HDACs. HepG2 cells
were transfected with pSUPER siHDAC1, siHDAC3, siHDAC4 or pSUPER [control (con)], and after 72 h the total RNA was isolated. The mRNA
levels of HDACs were measured via RT–PCR analysis, with b-actin shown as a control. The results are representative of three experiments.
4110 Nucleic Acids Research, 2009, Vol. 37, No. 12
Adenovirus-mediated overexpression of SMILE
down-regulates the expression of GR, CAR and
HNF4atarget genes
Next, we performed ChIP assays to determine whether
SMILE can associate with the NRs on the promoter of
the IGFBP1, CYP2B6 and CYP7A1 genes, which are
known targets of GR, CAR and HNF4a, respectively
(11,12,17). As shown in Figure 10A, low levels of GR
and SMILE were associated on IGFBP1 promoter in the
absence of dexamethasone (upper panel, lanes 5 and 9).
We observed an increased occupancy of GR after 1 h of
dexamethasone treatment (upper panel of Figure 10A,
compare lanes 5 and 6), whereas the occupancy of
SMILE significantly increased after 2 h of dexamethasone
treatment (upper panel of Figure 10A, compare lanes 9
and 11). On the promoter of CYP2B6, the occupancy of
CAR did not significantly changed upon the treatment of
CAR agonist TCPOBOP (upper panel of Figure 10B,
compare lanes 6–7 to lane 5), whereas the association of
SMILE was increased after 12 h TCPOBOP treatment
(upper panel of Figure 10B, compare lanes 9 and 11).
On the promoter of CYP7A1, the occupancy of HNF4a
was significantly increased after adenovirus (Ad)-mediated
overexpression of HNF4a(upper panel of Figure 10C,
compare lanes 6–7 to lane 5), whereas the occupancy of
Figure 10. SMILE down-regulates the transcription of IGFBP1, CYP2B6 and CYP7A1 gene. (A–C) The recruitment of SMILE on IGFBP-1,
CYP2B6 and CYP7A1 promoters is associated with histone deacetlyation. (A and B) HepG2 cells were stimulated with or without 100 nM of Dex
(A) or 250 nM of TCPOBOP (B) in the presence or absence of 300mM TSA as indicated. (C) HepG2 cells were infected with or without adenovirus-
HNF4a(Ad-HNF4a) in the presence or absence of 300 mM TSA as indicated. Cell lysates from the treated HepG2 cells were then collected for Chip
assays. Chromotian fragments were prepared and immumoprecipitated with the indicated specific antibodies. DNA fragments covering a GRE on
IGFBP1 promoter (A, upper panel), or CAR-binding site on CYP2B6 promoter (B, upper panel), BARE-I and BARE-II element on CYP7A1
promoter (C, upper panel) were PCR-amplified as described in the Materials and methods section. The lower panels in A–C indicate the amplifi-
cation of the control regions. (D–F) Shown are RT–PCR carried out using PCR primers for SMILE, IGFBP1, CYP2B6, CYP7A1, HNF4aand
b-actin, using total RNA prepared from HepG2 cells infected with indicated adenovirus vector (Ad-null, Ad-SMILE and Ad-HNF4a) (100 pfu/cell).
After 24 h of infection, the cells were stimulated with vehicle (DMSO) or 100 nM of Dex (D) for 2 h or 250 nM of TCPOBOP (E) for 24 h before total
RNA was isolated. Data shown are representative of three experiments.
Nucleic Acids Research, 2009, Vol. 37, No. 12 4111
SMILE increased after 24 h of Ad-HNF4ainfection
(upper panel of Figure 10C, compare lanes 9 and 11).
However, no recruitment was observed in the nonregula-
tory regions of target gene promoters (Figure 10, lower
panels of A–C, see lanes 5–12). These results indicate that
SMILE dynamically forms complex with GR, CAR, or
HNF4aon their target gene promoters.
Since SMILE interacted with HDACs (Figure 9), we
assume that the recruitment of SMILE to the target gene
promoters may lead to histone deacetylation. To test this
hypothesis, ChIP assays were performed using antibodies
against acetylated lysine 9 of histone H3. One hour dexa-
methasone treatment increased the acetylation of histone
H3 on the GR-binding region of IGFBP1 promoter,
whereas theacetylation decreased to basal level after 2 h
treatment of dexamethasone, which coincides with the
timing of increased SMILE association. Interestingly,
the decline of the acetylated histone H3 was recovered
by HDAC inhibitor (TSA) treatment (upper panel of
Figure 10A, see lanes 9–16). Moreover, 2 h TCPOBOP
treatment resulted in increased acetylation of histone H3
on the CAR-binding region of CYP2B6 promoter and the
acetylation diminished after 12 h TCPOBOP treatment.
Although this deacetylation of histone H3 occured in line
with the recruitment of SMILE, it did not change upon
TSA treatment (upper panel of Figure 10B, see lanes
9–16). In addition, acetylated histone H3 on the HNF4a-
binding region of CYP7A1 promoter increased 12 h after
Ad-HNF4ainfection and reduced to basal level 24 h after
Ad-HNF4ainfection, which also coincides with the
recruitment of SMILE. Similar to the case of IGFBP1
promoter, the decrease in acetylated histone H3 on
CYP7A1 promoter was prevented by the treatment of
TSA (upper panel of Figure 10C, see lanes 9–16).
Collectively, these results demonstrate that the recruitment
of SMILE on these target gene promoters is associated
with chromatin histone deacetylation.
As the aforementioned data show that SMILE is able to
inhibit the transactivation of GR, CAR and HNF4a, and
these three NRs form complex with SMILE on IGFBP1,
CYP2B6 and CYP7A1 promoters, respectively, we specu-
lated that SMILE may repress IGFBP1, CYP2B6 and
CYP7A1 gene expression. As expected, the overexpression
of SMILE in HepG2 cells using adenovirus vector mark-
edly reduced dexamethasone-induced as well as the basal
mRNA levels of IGFBP1 (Figure 10D, compare lane 4 to
lane 3 and lane 2 to lane 1). Moreover, SMILE overex-
pression blocked CAR agonist TCPOBOP-mediated
increase in CYP2B6 mRNA levels (Figure 10E, compare
lane 4 to lane 3). In addition, SMILE overexpression also
inhibited the basal and Ad-HNF4a-mediated increase in
CYP7A1 mRNA levels (Figure 10F, compare lane 2 to 1
and lane 4 to 3). Taken together, these results reveal that
SMILE is capable of down-regulating GR, CAR and
HNF4atarget gene expression.
DISCUSSION
Previous results have demonstrated that the bZIP protein
SMILE plays an important role in repressing the
replication of the herpes simplex virus (1,3) and serves
as a coregulator in ER signaling (2). The results presented
in this study extend the role of SMILE in NR signaling.
SMILE inhibited GR-, HNF4a- and CAR-mediated tran-
scriptional activity through direct binding to the LBD/
AF2 domain of the NRs. Moreover, the knockdown of
SMILE gene expression increased the GR, HNF4aand
CAR transactivation. Furthermore, the overexpression
of SMILE via adenovirus vector inhibited the transcrip-
tion of the NRs’ target genes, including IGFBP-1,
CYP2B6 and CYP7A1. In addition, SMILE also inhibited
the transactivation by receptor LXR, FXR, Nur77 and
ERRgthrough direct interactions (data not shown).
These findings indicate that SMILE may be an important
modulator of NR signaling.
We have investigated the roles of potential functional
domains of SMILE for its repressive function, including
the leucine zipper motif (1), the HCF-binding motif
(HBM) (1,3,5) and the LXXLL motifs (NR boxes)
(25,43). The leucine zipper region is known to be essential
for the dimerization and functions of b-zip proteins (44).
For instance, the leucine zipper of cyclic AMP response
element-binding (CREB) protein is required for the dimer-
ization and transcriptional activation (35). By way of con-
trast, our findings support the notion that the bZIP region
of SMILE is required for the homodimerization, but is not
essential for the repressive effect of SMILE on GR and
CAR (Figure 4). It has been reported that Jun dimeriza-
tion protein 2 (JDP-2) functions as a progesterone recep-
tor (PR) coactivator through direct interaction via the
DBD of PR and the bZIP region of JDP-2 (45).
However, the domain-mapping results have demonstrated
that the bZIP region of SMILE is not involved in the
interactions with GR, CAR and HNF4a(Figure 5).
Although HBM-mediated association of SMILE with
HCF is required for SMILE to repress CREB3 (5), our
reporter assay results have shown that wild-type SMILE
and HBM-defective SMILE mutant (Y306A), which
was demonstrated not able to interact with HCF (1),
have similar inhibitory effect on GR, CAR and HNF4a
(Supplementary Figure 1), indicating the repression of the
NRs by SMILE is independent of HBM.
LXXLL motif is commonly found in NR coregultors
and has been reported to be important for coregulators
function through interaction with the LBD/AF2 domain
of NRs (25,43). The results of domain-mapping analysis
manifests that SMILE binds to the LBD/AF2 domain of
GR, CAR and HNF4athrough the region spanning resi-
dues 113–202, which contain a LXXLL motif.
Surprisingly, we found that the repressive effects of
SMILE on GR, CAR and HNF4awere not significantly
changed by single mutation or combinatorial mutation of
four LXXLL motifs (Supplementary Figure 2), indicating
that LXXLL motifs are not essential for the interactions
and repressive effects of SMILE in the cases of GR, CAR
and HNF4a. Interestingly, this LXXLL-independent
interaction was also observed between proline-rich nuclear
receptor coregulatory protein (PNRC) and LBD of ERa
(46). In addition of using LXXLL motifs to interact
with NRs, corepressor RIP140 also uses its C-terminus,
which contains no LXXLL motifs, to interact with LBD
4112 Nucleic Acids Research, 2009, Vol. 37, No. 12
of NRs (40). However, it remains to be determined
whether the LXXLL motifs are also dispensible for the
repressive effect of SMILE on other NRs, such as
Nur77, LXR and FXR.
We have recently reported that SMILE functions as a
coregulator in ER signaling in association with SHP. The
regulation of ER by SMILE depends on the existence of
SHP in breast cancer MCF-7 cells (2). In contrast, the
results of our siRNA knockdown experiments indicate
that SHP is not involved in the SMILE-mediated repres-
sion of GR, CAR and HNF4a(data not shown). In our
previous study, SMILE regulates the inhibition of ER
by SHP in a cell-type specific manner (2). However, the
repression of GR, CAR and HNF4aby SMILE is not
cell-type specific, since similar repressive effects were
observed in 293T, HepG2 and HeLa cells (data not
shown).
Our results suggest that multiple mechanisms are
involved in SMILE-mediated repression. One such mech-
anism could be competition with coactivators such as
GRIP and PGC-1a, which is a common mechanism
among certain NR corepressors, including SHP (31),
DAX-1 (29), RIP140 (43) and the ligand-dependent cor-
epressor (LCoR) (47). Interestingly, besides coactivator
competition, SMILE has an intrinsic repressive function,
like the corepressors SHP (42) and RIP140 (41).
Moreover, we found that SMILE specifically interacts
with HDAC1, HDAC3 and HDAC4. The inhibition of
HDAC activity using the HDAC inhibitor TSA, or the
knockdown of the HDACs gene expression through
siRNA partially released the repression of GR and
HNF4aby SMILE. In contrast, TSA showed little effect
on the repression of CAR by SMILE, indicating HDAC-
dependent and -independent mechanism of repression.
Consistently, our ChIP assay results also evidenced that
TSA was able to prevent SMILE-associated deacetylation
of histone H3 on GR and HNF4atarget gene promoters,
but not on CAR target gene promoter. Of note, the TSA-
sensitive and -insensitive actions of SMILE are similar to
several other corepessors, including RIP140 (41) and
LCoR (47). In addition, HDAC1, HDAC3 and HDAC4
are required for the repression of HNF4aby SMILE,
whereas HDAC1 is not essential for the repression of
GR, indicating that SMILE associations with HDACs
exhibits promoter specificity. Similar phenomenon
has been reported with the corepressors NCoR and
SMRT (26).
It is worth noting that the inhibition of DNA binding is
one of the common repression mechanisms utilized by cer-
tain corepressors. For instance, this mechanism underlies
the inhibition of TR and GR by tumor suppressor p53
(48,49), and the inhibition of hepatic nuclear factor-3
(HNF3) family by the corepressor SHP (30). However,
our results indicate that the inhibition of DNA binding
is not involved in the repression of GR, CAR, and
HNF4aby SMILE, as the recruitment of SMILE exerted
no detectable effect on the binding of the NRs to
the promoters of IGFBP1, CYP2B6 and CYP7A1
(Figure 10A–C). Whether this mechanism is involved in
the inhibitory effect of SMILE on other NRs, including
Nur77, LXR and FXR, still needs to be clarified.
GR, CAR and HNF4aare crucial for liver function,
including the regulation and processing of glucose,
lipids, amino acids and drug metabolism, as well as bile
acid homeostasis (14,15,50). Therefore, the repression of
their transcriptional activity by SMILE indicates that
SMILE may function as a negative coregulator in the
aforementioned physiological processes. It has been
reported that as integrators of various biological pro-
cesses, several transcriptional coregulators are regulated
by distinct nutritional and hormonal signals (51). For
example, activation of cAMP signaling by fasting induces
the coactivator PGC-1aexpression in hepatocytes,
whereas the activation of insulin-signaling pathway by
refeeding exhibits quite opposite effect (51). Increased
bile acid levels switch on the feedback pathway of bile
acid synthesis through induction of the corepressor SHP
(52). Therefore, it would be necessary to study the regu-
lation of SMILE gene expression by diverse physiological
settings and intracellular signaling pathways, which is cur-
rently under investigation. Moreover, to better understand
the function of SMILE in those aforementioned physio-
logical processes, the SMILE knockout and transgenic
animal model will be useful. In addition, the identification
of more SMILE-interacting proteins and the elucidation
of SMILE crystal structure will be helpful to illuminate
the detailed mechanism of SMILE-mediated repression.
In summary, we have identified that SMILE represses
GR-, CAR- and HNF4a-mediated transactivation
through direct interaction. At least two mechanisms are
involved in SMILE-mediated repression of the NRs, com-
petition with coactivators, and active repression through
the recruitment of HDACs. Taken together, these obser-
vations indicate that SMILE is novel corepressor and may
play an important role in NR signaling.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Drs Richard W. Hanson, Keesook Lee,
Changsoo Kim, Hee-Sae Park and Eungseok Kim for
helpful discussions and comments on the project. We
would like to acknowledge Dipanjan Chanda and
Dr Seok-Yong Choi for critical reading of the manuscript.
FUNDING
National Research Laboratory grant (ROA-2005-000-
10047-0) and the Korea Research Foundation grant
(KRF-2006-005-J03003). Funding for open access
charge: Brain Korea 21 programme.
Conflict of interest statement. None declared.
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