J. Biol. Chem.-2009-Xie-jbc.M109.034165

Document Sample
J. Biol. Chem.-2009-Xie-jbc.M109.034165 Powered By Docstoc
JBC Papers in Press. Published on August 18, 2009 as Manuscript M109.034165
  The latest version is at

         Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit
                    Nuclear Receptor ERRγ Transactivation

Yuan-Bin Xie1, Jeong-Hoh Park1, Don-Kyu Kim1, Jung Hwan Hwang2, Sangmi Oh3,
Seung Bum Park3, Minho Shong2, In-Kyu Lee4, Hueng-Sik Choi1*

 Hormone Research Center, School of Biological Sciences and Technology, Chonnam
National University, Gwangju, 500-757, Republic of Korea; 2Department of Internal
Medicine, Chungnam National University School of Medicine, Daejeon 301-721, Republic of
Korea; 3Department of Chemistry, College of Natural Science, Seoul National University,
Seoul 151-747, Republic of Korea; 4Department of Internal Medicine, Kyungpook National
University School of Medicine, Daegu 700-721, Republic of Korea.

           Running title: SMILE represses nuclear receptor ERRγ transactivation

*Correspondence: Hueng-Sik Choi, Hormone Research Center, School of Biological

                                                                                          Downloaded from by guest, on August 6, 2010
Sciences & Technology, Chonnam National University, Gwangju, 500-757, Republic of
Korea. Tel: +82-62-530-0503, Fax: +82-62-530-0506, E-mail:

SMILE (SMall heterodimer partner             SIRT1. Furthermore, ERRγ inverse
Interacting LEucine zipper protein) has      agonist     GSK5182        enhances    the
been identified as a corepressor of the      interaction of SMILE with ERRγ and
glucocorticoid receptor (GR), constitutive   SMILE-mediated repression. Knockdown
androstane     receptor      (CAR),    and   of SMILE or SIRT1 blocks the repressive
hepatocyte nuclear factor 4 alpha            effect of GSK5182. Moreover, chromatin
(HNF4α). Here, we show that SMILE            immunoprecipitation assays revealed that
also represses estrogen receptor-related     GSK5182 augments the association of
receptor γ (ERRγ) transactivation.           SMILE and SIRT1 on the promoter of
Knockdown of SMILE gene expression           ERRγ target pyruvate dehydrogenase
increases ERRγ activity. SMILE directly      kinase 4 (PDK4). GSK5182 and
interacts with ERRγ in vitro and in vivo.    adenoviral overexpression of SMILE
Domain mapping analysis showed that          cooperate to repress ERRγ-induced
SMILE binds to the AF2 domain of ERRγ.       PDK4 gene expression and this repression
SMILE represses ERRγ transactivation         is released by overexpression of a
partially through competition with           catalytically-defective SIRT1 mutant.
coactivators    PGC-1α,        PGC-1β, and   Finally, we demonstrated that ERRγ
GRIP1. Interestingly, the repression of      regulates SMILE gene expression, which
SMILE on ERRγ is released by SIRT1           in turn inhibits ERRγ. Overall, these
                 SMILE as a novel
inhibitors, a catalytically inactive SIRT1   findings implicate
mutant, and SIRT1 siRNA, but not by          corepressor of ERRγ and recruitment of
HDAC inhibitor. In vivo glutathione          SIRT1 as a novel repressive mechanism
S-transferase         pull-down        and   for SMILE and ERRγ inverse agonist.
co-immunoprecipitation assays validated
that SMILE physically interacts with             Estrogen-related receptor (ERRα, ERRβ,


Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

and ERRγ) are constitutively active nuclear   can homodimerize like other bZIP proteins
receptors (NRs) that contain high levels of   (15, 17). SMILE has been implicated in
sequence identity to estrogen receptors (ERs) herpes simplex virus (HSV) infection cycle
(1). All the ERR family members bind either   and related cellular processes through its
as a monomer or a homodimer or                association with HSV-related host-cell
heterodimeric complexes composed of two       factor (HCF) and CREB3 (17, 18). SMILE
distinct ERR isoforms to the consensus        has also been proposed as a coactivator of
sequence TCAAGGTCA, referred to as            activating      transcription      factor     4
ERR response element (ERRE) and as            (ATF4/CREB2) (19). Recently, we have
homodimers to the consensus estrogen          reported that SMILE functions as a
responsive element (1-3). Together with       coregulator of ER signaling and a
ERRα and ERRβ, ERRγ regulates a number        corepressor of the glucocorticoid receptor
of genes involved in energy homeostasis,      (GR), constitutive androstane receptor
cell proliferation and cancer metabolism (3,  (CAR), and hepatocyte nuclear factor 4
4). Targets of ERRγ known to date are         alpha (HNF4α) (16, 20). However, the
peroxisome proliferator-activated receptor γ  detailed roles of SMILE on other NRs still

                                                                                                Downloaded from by guest, on August 6, 2010
coactivator-1 alpha (PGC-1α), pyruvate        need to be clarified.
dehydrogenase kinase isoform 4 (PDK4),            Silent information regulator 2 proteins
RARα, cyclin-dependent kinase inhibitors      (Sirtuins) are class III histone protein
p21 (WAF1/CIP1) and p27 (KIP1) (4-7).         deacetylases (HDACs) and consist of 7
The ability of the ERRγ to regulate target    members named SIRT1 to SIRT7 in
genes transcription relies on its interaction mammals (21). Through deacetylating target
with coactivators and corepressors. The       proteins, Sirtuins play important roles in
coactivators      glucocorticoid     receptor cellular processes such as gene expression,
interacting protein 1 (GRIP1), PGC-1α and     apoptosis, metabolism and aging (21). Of 7
corepressors small heterodimer partner        Sirtuins, SIRT1 has been extensively studied.
(SHP), DAX-1 and receptor interacting         It has been reported that SIRT1 deacetylates
protein 140 (RIP140 or NRIP1) have been       and thereby deactivates the p53 and poly
reported to modulate ERRγ activity (5, 8-11). (ADP) ribose polymerase-1 (PARP1)
In addition, 4-hydroxytamoxifen (4-OHT)       protein, resulting in promoted cell survival
and its derivative GSK5182 act as inverse     (22, 23). In addition, SIRT1 regulates
agonists for ERRγ (12-14). However, the       glucose or lipid metabolism through its
deactivation mechanisms by these inverse      deacetylation activity on over two-dozen
agonists remain unclear.                      known      substrates,     including FOXO
    Small heterodimer partner interacting     transcriptional factors (24, 25) PPARα (26),
leucine zipper protein (SMILE), including     PPARγ (27) and PGC-1α (28). It has also
two alternative translation derived isoforms, been demonstrated that SIRT1 regulates
SMILE-L (CREBZF; long form of SMILE)          cholesterol        metabolism           through
                  activation of LXR
and SMILE-S (Zhangfei; short form of          deacetylation and
SMILE), has been classified as a member of    proteins (29).
the     CREB/ATF        family    of    basic     In this report, we have shown that
region-leucine zipper (bZIP) transcription    SMILE negatively regulates ERRγ through
factors (15, 16). However, SMILE cannot       direct interaction. We have demonstrated
bind to DNA as homodimers, although it        that coactivator competition and recruitment


of catalytically active SIRT1 are required        fragments of the ORFs into the EcoRI/XhoI
for the repression of ERRγ by SMILE.              sites of pcDNA3-Flag. pcDNA3-Flag-ERRγ
Moreover, ERRγ specific inverse agonist           was generated via subcloning the full ORF
GSK5182 enhances the interaction of               of ERRγ into the EcoRV/XhoI sites of
SMILE and ERRγ. siRNA SMILE and                   pcDNA3-Flag vector. pcDNA3-myc-SIRT1
siRNA SIRT1 experiments have revealed             was constructed via inserting the ORF of
that SMILE-SIRT association is required for       SIRT1       into    pcDNA3-myc       vector.
the inhibition of ERRγ by GSK5182. In             pcDNA3-myc-SIRT1H363Y was generated
addition,    we    have    observed    that       via PCR-mediated site-directed mutagenesis.
ERRγ induces SMILE gene expression in             pSUPER-siSIRT1 was constructed by
HepG2 cells by directly binding to SMILE          inserting     a    64-bp    double-stranded
promoter and that SMILE inhibits                  oligonucleotide                  containing
ERRγ transactivation of its own promoter.         5’-GAAGTTGACCTCCTCATTGT-3’                of
Overall, our observations suggest that            the human SIRT1 cDNA sequence into the
SMILE acts as a novel corepressor of              pSUPER vector between BglII and Xho I
ERRγ and that ERRγ belongs to a new               sites. To generate -1131bp-SMILE-Luc, the

                                                                                                 Downloaded from by guest, on August 6, 2010
autoregulatory loop that governs SMILE            SMILE promoter region spanning -1131 bp
gene expression.                                  to -15bp was PCR-amplified from human
                                                  genomic DNA and cloned into pGL3-basic
   EXPERIMENTAL PROCEDURES                        vector (Promega) between the SacI and
                                                  XhoI sites. -879bp- and -448bp-SMILE-Luc
    Plasmid and DNA construction - The            were constructed by inserting the PCR
plasmids of pCMV-β-gal, pcDNA3-ERRα,              fragments into the SacI/XhoI sites of
-ERRβ, -ERRγ, -ERRγ△AF2,                          pGL3-basic vector. The mutant reporters of
pSG5-HA-ERRγ , pGEX4T-1-ERRγ and                  SMILE-mtERRE1-Luc                       and
sft4-Luc were described elsewhere (9, 10).        SMILE-mtERRE2-Luc were subcloned via
(HNF4)8-tk-Luc,      pcDNA3-HA-HNF4α,             site-directed       mutagenesis        from
-PGC-1α,                 pSG5-HA-GRIP1,           -1131bp-SMILE-Luc.        The      mutated
pcDNA3-SMILE,                -Flag-SMLE,          sequences were shown in Figure 7D. All
-SMILE-83Leu, -SMILE-1Phe, pGEX4T-1,              plasmids were confirmed via sequencing
pGEX4T-1-SMILE, pEGFP-SMILE, pEBG,                analysis.
constructs, SMILE-L(239-267)V, pSUPER,          Chemicals and antibodies - SIRT1
pSUPER-siSHP,        -siSMILE-I,        and inhibitors Nicotinamide, Sortinol were from
-siSMILE-II were described previously (2).  Calbiochem, EX527 was purchased from
pcDNA3-Flag -SIRT6, -SIRT7 were kindly      TOCRIS, ERRγ inverse agonist GSK5182
provided by Dr. Eric Verdin (30).           was synthesized according the method
pcDNA3-HA-PGC-1β, -Flag-FOXO1 and           described in (14), and other chemicals were
the reporter PDK4-Luc were kind gifts from  from Sigma. Antibodies used in this work
Drs. Dieter Kressler (31),       Akiyoshi   were as follows: anti-Flag M2 (Stratagene,
Fukamizu (32) and Robert A. Harris (33),    #200472-21), anti-HA (12CA5, Roche
respectively.                               Molecular Biochemicals) , anti-SMILE
     pcDNA3-Flag-ERRα, -ERRβ were           (Abcam, #ab28700), anti-GST (Santa Cruz,
constructed by inserting the full PCR       sc-33614), anti-PGC-1α (H300, Santa Cruz,


sc-13067), anti-SIRT1 [Cell Signaling             were performed as previously described (20).
Technology           (CST),         #2493],       Briefly, ERR-α, -β, and -γ were labeled with
anti-acetyl-histone H3 (Lys9) (CST, #9671),       [35S]methionine using a TNT in vitro
anti-acetylated lysine (CST, #9441),              translation      kit     (Promega),       and
anti-Myc (CST, #2276), anti-tubulin (CST,         HA-PGC-1α, -PGC-1β and -GRIP1 were
#2146), and anti-ERRγ (R&D, pph6812-00)           labeled with cold methionine, according to
antibodies. The primary antibodies were           the manufacturer’s instructions. GST alone
used at dilution of 1:1000 in Western blot        and GST-fused SMILE (GST-SMILE)
analysis, at dilutioin of 1:200 in                proteins were prepared as previously
immunoprecipitation.                              described (16). The GST proteins were
                                                  prebound with glutathione-Sepharose beads
    Cell culture, transient transfection assay    (Amersham Biosciences) and then incubated
and luciferase assay - HEK293T (293T,             with            in           vitro-translated
human embryonic kidney), HepG2 (human             [ S]methionine-labeled ERR-α, -β, or -γ,
hepatoma), and HeLa (cervical cancer) cells       together    with      or    without      cold
were obtained from the American Type              methionine-labeled     PGC-1α, -PGC-1β or

                                                                                                  Downloaded from by guest, on August 6, 2010
Culture Collection (ATCC) and cultured            -GRIP1 in the binding buffer for 2-3 h at 4
according to the manufacturer's instructions.     ℃. The beads were washed three times with
Transient transfection was performed using        the binding buffer, analyzed by SDS-PAGE
Superfect transfection reagent (Qiagen) in        gel and visualized by a phosphorimage
293T cells and Lipofectamine 2000 reagent         analyzer (BAS-1500, Japan).
(Invitrogen) in HepG2 cells. 293T and
HepG2 cells were cotransfected with the            Co-Immunoprecipitation (Co-IP) and
reporter plasmids (HNF4)8-Luc, sft4-Luc or     Western blot analysis - Co-IP and western
PDK4-Luc coupled with various expression       blot analysis were performed as described
vectors.     The      plasmid     of     CMV   previously (20). In Western blot analysis of
(cytomegalovirus)-β-galactosidase         was  immunoprecipitated proteins, conventional
cotransfected as an internal control and the   HRP-conjugated anti-rabbit IgG was
total DNA employed in each transfection        replaced with rabbit IgG TrueBlot
was adjusted via the addition of an            (eBioscience, #18-8816) to eliminate signal
appropriate quantity of pcDNA3 vector.         interference by the immunoglobulin heavy
Approximately 36 h post-transfection, the      and light chains.
cells were treated with or without chemicals
as indicated in the figure legends for 12 h,       In vivo GST pull-down assay - In vivo
and then, cells were harvested, and the        GST      pull-down    experiments       were
luciferase activity was measured and           performed as previously described (20). In
normalized against β-galactosidase activity    brief, HepG2 cells were transfected with the
as previously described (16, 20). Fold         indicated plasmids using Lipofectamine
                  hours after
activity was calculated considering the        2000 reagent.
activity of reporter gene alone as 1.          transfection, the whole-cell extracts were
                                               prepared with 200 µl of lysis buffer [20 mM
    In vitro glutathione S-transferase (GST)   HEPES (pH 7.9), 10 mM EDTA, 0.1 M KCl,
pull-down assay and competition assay - In     and 0.3 M NaCl] containing 0.1% Nonidet
vitro GST pull-down and competition assays     P-40, and protease inhibitors. Then equal


amounts of total protein were used for in           and quantitative real-time PCR (qPCR)
vivo GST pull-down assays followed by               analysis - Total RNA was isolated using the
Western blot analysis.                              TRIzol reagent (Invitrogen) according to the
                                                    manufacturer’s instructions. The mRNAs of
    Confocal microscopy - The confocal              SMILE, SHP, PDK4, ERRγ, and SIRT1
microscopy assays were carried out as               were analyzed by RT-PCR or qPCR as
described previously (20). In brief, the HeLa       indicated. DNA samples from total RNA
cells grown on gelatin-coated coverslips            reverse transcription or from chromatin
were transfected with indicated plasmids            immunoprecipitation (ChIP) assays served
using Effectene transfection reagent (Qiagen)       as the templates for qPCR experiments,
according the manufacturer’s instructions.          which were performed with QuantiTect
Twenty four hours after transfection, the           SYBR GreenER PCR Kit (Qiagen) and the
cells were fixed with 2% formaldehyde               Roter-Gene 6000 real-time PCR system
followed by immunostaining. To detect               (Australia) in triplicate. Median cycle
HA-tagged ERRγ and the nucleus, the cells           threshold values were determined and used
were     incubated     with     dye     Alexa       for analysis. ChIP signals were presented as

                                                                                                   Downloaded from by guest, on August 6, 2010
594-conjugated      anti-HA       monoclonal        percentage of input signals. mRNA
antibody (1:500 dilution; Invitrogen) for 1 h       expression levels of the interested genes
at room temperature (25℃), washed three             were normalized to those of β-actin. The
times in PBS, and incubated with 0.1mg/ml           RT-PCR and qPCR primers are provided in
of DAPI (Invitrogen) solution for 10                Supplementary Table 1.
minutes. After three times washing with
PBS, the cells were subjected to observation      Chromatin Immunoprecipitation (ChIP)
by confocal microscopy.                       Assay - ChIP assay was performed as
                                              previously described (20). In brief, treated
    Preparation of recombinant adenovirus     HepG2 cells in 60-mm culture dishes were
- The adenovirus encoding human SMILE         fixed with 1% formaldehyde, washed with
or mouse ERRγ was described previously        ice-cold PBS, harvested and solicited. The
(10, 20). The adenovirus expressing           soluble chromatin was then subjected to
SIRT1H355A was a kind gift from Dr.           immunoprecipitation using anti-ERRγ,
Myung-Kwan Han (34).                          anti-SMILE (Santa Cruz, sc-49329),
                                              anti-SIRT1, anti PGC-1α, acetyl-histone H3
    RNA interference - Knockdown of           (Lys9) or anti-HA antibodies followed by
SMILE, SHP, and SIRT1 was performed           using protein A agarose/salmon sperm DNA
using the pSuper vector system (16, 20).      (Upstate). Unrelated immunoglobin G (IgG)
293T or HepG2 cells were transfected with     was used a negative control for
siRNA constructs using Lipofectamine2000      immunoprecipitation. Precipitated DNA was
(Invitrogen) according to the manufacturer’s  recovered via phenol/chloroform extraction
                  qPCR or RT-PCR for
instructions. siRNA treated cells were        and amplified by
subjected to reverse transcription-PCR        35-40 cycles using specific primer sets for
(RT-PCR), or the second transfection as       the indicated specific promoter regions of
indicated in the Figure legends section.      PDK4 and SMILE genes. The PCR primers
                                              for ChIP assays are provided in
    Reverse transcriptase PCR (RT-PCR)        Supplementary Table 2.


                                                two-hybrid interaction assays, including GR,
    Primer       Extension      Analysis      - TRα, CAR, SF-1, ERRα, ERRβ, ERRγ,
Transcription start site was determined by      HNF4α and Nur77 (20). To further confirm
rapid amplification of cDNA ends (RACE)         the interaction of SMILE with ERRα,
using SMART™ RACE amplification kit             ERRβ, and ERRγ, in vitro and in vivo GST
(BD Biosciences Clontech). Total RNA            pull-down experiments were performed. For
isolated from HepG2 cells was reverse           the in vitro GST pull-down assays,
transcribed using SMART II A oligo and          bacterial-expressed         GST      only,   or
5’-CDS primer A according to the                GST-SMILE proteins were incubated with
manufacturer’s      recommendations.        BD  in vitro translated 35S-labeled ERRα, ERRβ,
PowerScript       RT      exhibits     terminal or ERRγ. As shown in Figure 1A,
transferase activity by adding three to five      S-labeled ERRγ was observed to bind to
residues of predominately dC to the 3’ end      GST-fused full length SMILE, but
of the first-strand cDNA. BD PowerScript          S-labeled ERRα and ERRβ was not.
RT switches templates from RNA to BD            These results suggest that SMILE
SMART oligonucleotides, generating a            specifically interacts with ERRγ in vitro. For

                                                                                                  Downloaded from by guest, on August 6, 2010
complete cDNA copy of the original RNA          the in vivo GST pull-down assays,
with BD SMART sequences at the end. The         mammalian expression vectors encoding
dC-tailed cDNA was amplified by the BD          either pEBG (GST) alone or pEBG-SMILE
Advantage 2 PCR system using a                  (GST-SMILE)                together        with
gene-specific                            primer pcDNA3-Flag-ERRα, pcDNA3-Flag-ERRβ,
(5’-TGGACCCCAGGCAACCGGACTGG                     or pSG5-HA-ERRγ were cotransfected into
CA-3’) corresponding to 332-356bp of            HepG2 cells. As shown in Figure 1B,
human SMILE cDNA, and a nested                  HA-ERRγ was co-precipitated with
gene-specific                            primer GST-SMILE, but not with GST alone. The
(5’-TGTTCGCTGCCCTCTGACCTGACC-                   expression of GST, GST-SMILE and
3’) corresponding to 85-108bp of human          HA-ERRγ proteins was confirmed by
SMILE cDNA. The amplified PCR                   Western blot analysis (Figure 1B, middle
fragments were subcloned into pGEM              and bottom panels, respectively). However,
T-easy vector (Promega) for DNA                 neither Flag-ERRα nor Flag-ERRβ was
sequencing.                                     found      to    be     co-precipitated    with
                                                GST-SMILE (Supplementary Figure 1A).
    Statistical analysis - Student’s t-test was These results demonstrate that exogenous
performed using GraphPad Prism version          SMILE         specifically     interacts   with
3.0 for Windows and results were                ectopically expressed ERRγ. To further
considered to be statistically significant      investigate whether endogenous ERRγ and
when P < 0.05.                                  SMILE can interact with each other in vivo,
                                                co-immunoprecipitation experiments were
                                                performed. Endogenous ERRγ proteins from
                                                HepG2 cells, mouse liver, kidney, and heart
    SMILE physically interacts with nuclear     tissues were observed to be co-precipitated
receptor ERRγ both in vitro and in vivo -       with SMILE (Figure 1C). Taken together,
Our previous work showed that SMILE             these results validate that SMILE can
interacted with many NRs in yeast               specifically interact with ERRγ both in vitro


and in vivo.                                        not     significantly   changed      by    the
    To investigate whether SMILE and                overexpression of wild-type SMILE,
ERRγ are colocalized to the same                    SMILE-L or SMILE-S alone (Figure 2E).
subcellular     compartments,        confocal       Taken together, these results demonstrate
microscopic studies were carried out. HeLa          that both SMILE isoforms can negatively
cells were cotransfected with the                   regulate      ERRγ transactivation.      Since
mammalian         expression        plasmids        SMILE-L and SMILE-S show similar
pEGFP-SMILE        and    pSG5-HA-ERRγ,             effects on ERRγ, and SMILE-L is the major
stained with dye Alexa 594-conjugated               isoform in tested cell lines and tissues (16),
anti-HA antibody and DAPI, and analyzed             we      have      focused     on     wild-type
via confocal microscopy. As shown in                SMILE-generated SMILE-L (SMILE) for
Figure 1D, GFP-SMILE was predominantly              further investigations.
localized within the nucleus, and was also              To examine whether endogenous SMILE
weakly detected in the cytoplasm, which             is    involved      in    regulating    ERRγ,
was consistent with our previous study (16,         ERRγ-mediated transcriptional activities
20). ERRγ was also observed mainly in the           were evaluated after knocking down SMILE

                                                                                                     Downloaded from by guest, on August 6, 2010
nucleus. The merged images indicated that           gene expression through siRNA in 293T and
SMILE and ERRγ were colocalized to the              HepG2 cells. As shown in Figure 2F,
nucleus (Figure 1D). Collectively, these data       siSMILE-II (siSM-II) efficiently silenced
demonstrate that SMILE interacts and                the mRNA expression of SMILE, whereas
colocalizes with nuclear receptor ERRγ in           siSMILE-I (siSM-I) did not show any
vivo.                                               significant effect. As expected, loss of
                                                    SMILE in 293T and HepG2 cells resulted in
    SMILE inhibits nuclear receptor                 a 60-90% increase in ERRγ-mediated
ERRγ transactivation - We have previously           transcription of the reporter gene (Figure 2C,
reported that wild-type (wt) SMILE gene             2D). This effect is similar to that shown
generates two isoforms through alternative          with siSHP, which knockdown the gene
translation, SMILE long form (SMILE-L)              expression of SHP (Figure 2D, 2F), a
and SMILE short form (SMILE-S), which               reported corepressor of ERRγ (9). These
can be produced by the mutants                      results indicate that endogenous SMILE can
SMILE-83Leu           and        SMILE-1Phe,        repress ERRγ transactivation.
respectively (16, 20). To examine whether
these isoforms can regulate ERRγ-mediated            Interaction domain mapping of SMILE
transcriptional activity, transient transfection with ERRγ - Since the AF2 domain of ERRγ
experiments were performed in 293T and           has been reported to be involved in its
HepG2 cells. Overexpressed wild-type             interactions with corepressor DAX-1 and
SMILE inhibited ERRγ transactivation in a        SHP (9, 10), we tested whether it could also
dose-dependent manner in both cells (Figure      mediate SMILE-ERRγ association via in
2A, 2B). Furthermore, overexpression of          vitro GST pull-down assays using ERRγ
SMILE-L or SMILE-S through the                   AF2 domain deletion construct (Figure 3A).
aforementioned SMILE mutants exerted             As expected, SMILE could not interact with
similar repressive effects on ERRγ (Figure       ERRγΔAF2, indicating that the AF2 domain
2A, 2B). Western blot analysis showed that       of ERRγ is essential for the interaction with
the protein expression of Flag-ERRγ was          SMILE (Figure 3B). To identify the SMILE


domains required for ERRγ interaction, we          binding to the AF2 pocket of ERRγ in cells.
performed in vivo GST pull-down                    To confirm the direct competition between
experiments using a series of previously           SMILE and PGC-1α, we performed in vitro
described (20) mammalian GST-tagged                competition binding assays, using in vitro
SMILE mutants (Figure 3C). We observed             translated    PGC-1α and     ERRγ     with
that the mutant GST-SMILE-N2 (1-202 aa)            GST-fused SMILE. Specifically, SMILE
and GST-SMILE showed significant                    interacted with 35S-labeled ERRγ, and
association with ERRγ, whereas the mutants         PGC-1α inhibited the interaction of SMILE
GST-SMILE-N1 (1-112 aa), GST-SMILE-                with ERRγ dose-dependently (lower panel
△202 (203-354 aa), and GST-SMILE-△                 in Figure 4A). Interestingly, quite similar
268 (269-354 aa) did not (Figure 3D, upper         results were obtained when coactivators
panel). Moreover, all the GST SMILE                PGC-1β (Figure 4B) and GRIP1 (Figure 4C)
fusions and ERRγ proteins used in the              were used. Τaken together, these
assays were expressed at comparable levels         observations indicate that competing with
(Figure 3D, middle and lower panels),              coactivators PGC-1α, PGC-1β and GRIP1
indicating that the differences in the             for binding to ERRγ may be involved in the

                                                                                                 Downloaded from by guest, on August 6, 2010
interactions between the SMILE mutants             repression of SMILE on ERRγ.
and ERRγ are not due to the differences in
protein expression levels. Overall, these          LxxLL motifs in SMILE are not involved
results demonstrate that ERRγ interacts with  in the inhibition of SMILE on ERRγ - LxxLL
the region spanning residues 113-202 of       motif has been identified in numerous
SMILE.                                        proteins that interact with the AF-2 domain
                                              of NRs LBD region (35). Many studies have
    SMILE competes with coactivators          shown that the motif plays an important role
PGC-1α, PGC-1β, and GRIP1 for binding         in the regulation of nuclear receptor
to ERRγ - Since SMILE interacts with          signaling by coregualtors, such as the p160
ERRγ AF2 domain, which is also the            family of coactivators (SRC-1, 2 and 3),
binding surface of coactivators PGC-1α        CBP/p300,         PGC-1α,        corepressors
PGC-1β (35) and GRIP1 (8), we postulated      receptor-interacting protein-140 (RIP140),
that coactivator competition might be         and SHP (36-38). Therefore we wondered
involved in the repression of SMILE on        whether the LxxLL motifs in SMILE are
ERRγ. To test this hypothesis, expression     crucial for the repression of SMILE on
vectors for PGC-1α, SMILE and ERRγ            ERRγ. To address this issue, transient
were introduced into HepG2 cells along        transfection and reporter assays were
with sft4-Luc reporter as indicated in Figure performed using previously described (20)
4A. As expected, PGC-1α coexpression          five SMILE LxxLL motif mutants (Figure
further stimulated ERRγ transactivation and   4D). Surprisingly, all these mutants
overexpression of SMILE repressed this        inhibited ERRγ transactivation to the level
                  observed with wt SMILE
induction in a dose-dependent manner. In a    comparable to that
reciprocal experiment, overexpression of      (Figure 4F), although expression levels of
PGC-1α released the inhibitory effect of      all the SMILE mutants used were similar to
SMILE on ERRγ dose-dependently (upper         that of wt SMILE (Figure 4E). Moreover,
panel in Figure 4A). These results indicate   the results from in vivo GST pull-down
that SMILE and PGC-1α may compete for         assays showed that the interactions of wt


SMILE and the LxxLL motif mutants with         and combination of               SIRT1 and
ERRγ are comparable (Figure 4G).               SMILE repressed the ERRγ activity by
Collectively, these observations indicate that approximately 86%. However, the dominant
all the LxxLL motifs in SMILE are not          negative SIRT1 mutant SIRT1H363Y
essential for SMILE to negatively regulate     increased ERRγ transactivation by about
ERRγ and the LxxLL motifs may not be           34%, and released the repression of ERRγ
involved in the interaction of SMILE with      by SMILE from 50% to 23%. These data
ERRγ.                                          suggest that SIRT1 deacetylase activity is
                                               needed for the repression of SMILE on
    SMILE recruits SIRT1 to inhibit            ERRγ.
ERRγ transactivation - Previously we have          To examine whether ERRγ and SMILE
reported that the classical HDACs are          directly interact with SIRT1, in vitro GST
involved in the SMILE repression of GR         pull-down experiments were carried out. As
and HNF4α (20). Το examine whether those       shown in Figure 5E, in vitro translated
HDACs take part in the inhibitory effect of      S-SIRT bound to GST-SMILE, but not
SMILE on ERRγ as well, we used inhibitor       GST-ERRγ. To further investigate whether

                                                                                              Downloaded from by guest, on August 6, 2010
trichostatin A (TSA) to block classical        SMILE specifically interacts with SIRT1,
HDACs activity in reporter assays. In          Myc-SIRT1,                Myc-SIRT1H363Y,
agreement with our previous report (20),       Flag-SIRT6, or Flag-SIRT7 expression
TSA treatment released SMILE repression        vectors were introduced into HepG2 cells
on HNF4α in a dose-dependent manner            along with pEBG(GST) or pEBG-SMILE
(Figure 5A). By contrast, SMILE repression     (GST-SMILE) and in vivo GST pull-down
of ERRγ was unaltered by the TSA               assays were performed. Myc-SIRT1 and
treatment (Figure 5B).                         Myc-SIRT1H363Y was detected in the
    Next we investigated whether the           coprecipitate only when coexpressed with
inhibition of SMILE on ERRγ is sensitive to    the GST-SMILE but not with GST alone
SIRT1 specific pharmacological inhibitors,     (top panel in Figure 5F). The protein
such as EX527 (39), Sirtinol, and              expression levels of GST, GST-SMILE,
Nicotinamide (40). Interestingly, all three    Myc-SIRT1, and Myc-SIRT1H363Y were
SIRT1      inhibitors   further    stimulated  confirmed via Western blot analysis (middle
ERRγ-mediated sft4-Luc reporter activity       and bottom panel in Figure 5F). By contrast,
and released the repression of SMILE on        Flag-SIRT6 and Flag-SIRT7 were not
ERRγ transactivation significantly (Figure     detected in the coprecipitate (Supplementary
5C), indicating that the catalytic activity of Figure 1B). These results demonstrate that
SIRT1 is necessary for the repression of       exogenous SMILE specifically interacts
ERRγ by SMILE. To further confirm              with SIRT1 in mammalian cells and the
whether the deacetylase activity of SIRT1 is   mutation of H363Y does not affect the
required for SMILE repression, we              interaction of SIRT with SMILE. To further
compared the effect of wild-type SIRT1 and     validate the association of endogenous
a reported deacetylase-defective SIRT1         SMILE and SIRT1, Co-IP assays were
(H363Y) mutant (41) on SMILE repression.       carried out. As shown in Figure 5G,
As shown in Figure 5D, overexpression of       endogenous SIRT1 proteins were observed
wild-type SIRT1 or SMILE alone inhibited       to be co-precipitated with SMILE. Taken
ERRγ transactivation by approximately 50%,     together, these results indicate that SMILE


inhibits    ERRγ transactivation       through   ERRγ-mediated transcriptional activity.
SMILE-mediated SIRT1 recruitment.                However, those SIRT1 activators failed to
     To further assess whether the               repress ERRγ after shutdown of SMILE
recruitment of SIRT1 by SMILE alters             gene expression by siSMILE-II (siSM#2)
ERRγ and SMILE acetylation status,               (Figure 5I, 5J). Taken together, these
expression vectors for Flag-FOXO1,               observations suggest that the activation of
Flag-ERRγ or Flag-SMILE, together with           SIRT1 by chemicals resulted in the
Myc-SIRT1 or Myc-SIRT1H363Y were                 inhibition of ERRγ transactivation and this
cotransfected      into      HepG2        cells. issue depends on the expression of SMILE.
Subsequently, the acetylation/deacetylation
levels of FOXO1, ERRγ and SMILE were                  SMILE is required for the ERRγ inverse
examined. As expected, overexpression of         agonist GSK5182-mediated transrepression
wild-type SIRT1 but not the catalytically-       - It has been reported that GSK5182 (Figure
inactive mutant SIRT1H363Y deacetylated          6F), a synthetic 4-hydroxytamoxifen
FOXO1 (Supplementary Figure 2A), a               (4-OHT) derivative, acts as an ERRγ inverse
well-known target of SIRT1 (24), and the         agonist through binding to ERRγ LBD

                                                                                                Downloaded from by guest, on August 6, 2010
acetylation of FOXO1 was blocked by              region (14). Since SMILE binds to the AF2
treatment of SIRT1 inhibitor EX527. By           domain of ERRγ LBD region, we
contrast,            no             significant  hypothesized that ERRγ inverse agonist
acetylation/deacetylation       signal     was   might recruit SMILE to repress ERRγ
observed      for    ERRγ      and     SMILE     transactivation. Therefore we tested whether
(Supplementary Figure 2B, 2C), indicating        SMILE and GSK5182 could cooperatively
that        the         phenomenon           of  inhibit ERRγ transactivation. As expected,
acetylation/deacetylation may not occur in       GSK5182 treatment and overexpression of
either SMILE or ERRγ protein.                    SMILE alone inhibited ERRγ-mediated
     To further determine the importance of      sft4-Luc reporter activity by approximately
SIRT1 recruitment in SMILE repression on         50-55% (Figure 6A, compare lanes 3 and 4
ERRγ, reporter assay experiments were            to lane 2). Moreover, SMILE enhanced the
performed after knocking down SIRT1 gene         transrepression of ERRγ by GSK5182 up to
expression using siRNA in HepG2 cells. As        about 88%. Next, we examined whether the
shown in Figure 5J, siSIRT1 efficiently          GSK5182-mediated           repression     of
knocked down the mRNA expression of              ERRγ depends on SMILE or SIRT1.
SIRT1. The silencing of SIRT1 significantly      Interestingly, knock down of SMILE or
increased ERRγ transactivation and released      SIRT1 gene expression decreased the
SMILE-mediated repression of ERRγ by             transrepression of ERRγ by GSK5182 to
approximately 60%, indicating SIRT1 is           approximately 17% or 13% (Figure 6A,
required for the full inhibition of SMILE on     compare lanes 7 to lane 6 & lane 9 to lane
ERRγ (Figure 5H). In a parallel experiment,      8), respectively. In the presence of SIRT1
                  Nicotinamide, GSK5182
we investigated the effect of SIRT1              inhibitor EX527 or
activators Resveratrol and Piceatannol on        did not show any significant repression on
ERRγ transactivation before and after            ERRγ (Figure 6A, compare lanes 10-11 to
siRNA-mediated gene silencing of SMILE.          lane 2). Furthermore, similar results were
Of great interest, Resveratrol and               obtained when PKD4-Luc reporter was used
Piceatannol treatment significantly inhibited    (Figure 6B). Taken together, these


observations indicate that ERRγ inverse      promoter and the recruitment of SMILE can
agonist GSK5182 can strengthen the           be increased by GSK5182, which leads to
repressive effect of SMILE on ERRγ and       the dissociation of PGC-1α.
the transrepression of ERRγ by GSK5182           Based on the results that the repression
relies on SMILE and SIRT1.                   of SMILE was sensitive to the inhibition of
    To determine whether GSK5182 affect      SIRT1 catalytic activity and SMILE
the interaction of ERRγ and SMILE,           interacted with SIRT1 (Figure 5C-5G), we
expression vector for Flag-ERRγ along with   speculated that the recruitment of SMILE to
pEBG(GST)           or       pEBG-SMILE      PDK4       promoter      might     lead    to
(GST-SMILE) were cotransfected into          promoter-complexed histone deacetylation.
HepG2 cells and then in vivo GST             To address this issue, ChIP assays were
pull-down assays were performed with the     performed      with     antibodies    against
whole-cell extracts in the presence or       acetylated lysine 9 of histone H3 (Ace-H3).
absence of GSK5182. GSK5812 treatment        As shown in Figure 6D, in the absence of
substantially increased the binding of       GSK5182, high acetylation level of the
Flag-ERRγ to GST-SMILE (Figure 6C,           histone H3 on the ERRγ binding region of

                                                                                             Downloaded from by guest, on August 6, 2010
upper panel). In addition, the protein       PDK4 promoter was observed (lower left
expression levels of Flag-ERRγ and           panel). However, the acetylation level
GST-SMLE were not significantly changed      significantly decreased by the treatment of
by GSK5812 (Figure 6C, middle and            GSK5182 (left in lower panel), which
bottom panels).                              coincides with the increased SMILE and
                                             SIRT1 association. Interestingly, the
    ERRγ inverse      agonist     GSK5182    decrease in the acetylation level of histone
enhances     overexpressed     SMILE     to  H3 was recovered by SIRT1 inhibitor
down-regulate the expression of ERRγ target  EX527, but not by HDAC inhibitor (TSA)
gene PDK4 - Next, we performed ChIP          treatment (lower left panel). Taken together,
assays to examine whether SMILE associate    these results indicate that the increased
with the ERRγ on the promoter of PDK4, a     recruitment of SMILE on PDK4 promoters
known target of ERRγ (42, 43). Specific      is associated with increased chromatin
primers that flanked an ERRE AGGTCA in       histone deacetylation.
PDK4 promoter were used for quantitative         Since aforementioned data suggest that
real-time PCR analysis (Figure 6D, upper     GSK5182 strengthens SMILE to repress
panel). We observed that low levels SMILE,   ERRγ-mediated transactivation and to form
SIRT1 and high levels PGC-1α were            complex with ERRγ on ERRγ target PDK4
associated on PDK4 promoter in the           promoter, we next examined the effect of
absence of GSK5182. Treatment of             GSK5182 and SMILE on PDK4 gene
GSK5182 increased the occupancy of           expression. As expected, overexpresison of
SMILE and SIRT1, but decreased the           ERRγ though adenovirus increased PDK4
                 approximately 10.5 folds
occupancy of PGC-1α (Figure 6D, lower        mRNA levels by
left panel). However, no significant         (Figure 6E, compare lane 3 to lane 1), and
recruitment was observed in the control      overexpressed SMILE and GSK5182 alone
region of PDK4 promoter (Figure 6D, lower    conspicuously repressed Ad-ERRγ-induced
right panel). These results indicate that    as well as the basal mRNA levels of PDK4
SMILE form complex with ERRγ on PDK4         in HepG2 cells (Figure 6E, compare lane


4-5 to lane 3 & lane 2 to lane 1).              shown in Figure 7B, overexpression of
Interestingly, GSK5182 enhanced the             ERRγ significantly       activated      SMILE
inhibitory effect of SMILE on PKD4 gene         promoter activity in a dose-dependent
expression (Figure 6E, compare lane 8 to        manner, whereas ERRα and ERRβ did not
lane 4-5). Moreover, the treatment of SIRT1     show      any    significant    effect.   Our
inhibitor EX527 recovered SMILE- and            aforementioned data demonstrated that
GSK5182-repressed mRNA levels of PDK4           SMILE inhibits ERRγ transactivation via
by approximately 80%, and overexpression        coactivator competition; therefore, we next
of a deacetylase-defective SIRT1(H355A)         investigated whether SMILE could repress
mutant (34) via adenovirus showed a similar     ERRγ on its own promoter. As expected,
effect as EX527 (Figure 6E, compare lane        overexpression of SMILE repressed
9-10 to lane 8). In addition, those treatments  ERRγ-mediated and PGC-1α-enhanced
did not significantly affect the mRNA levels    SMILE promoter transcriptional activity
of PDK2, which is a non-ERRγ target (6).        (Figure7C).
Collectively, these observations demonstrate        Previously we have shown that ERRγ
that ERRγ inverse agonist GSK5182 and           recognizes the sequence T(N)AAGGTCA or

                                                                                                 Downloaded from by guest, on August 6, 2010
SMILE function cooperatively to reduce          AGGTCA (half sites), or TCAAGGTGG (9,
ERRγ-mediated PDK4 gene expression, and         10). Sequence analysis of SMILE promoter
this repression depends on SIRT1 activity.      showed that there are two putative
                                                ERRγ response elements (ERRE1 and
    Autoregulatory loop controlling SMILE       ERRE2) in the SMILE promoter (Figure 7D).
gene expression by ERRγ - Interestingly,        To further examine whether these elements
overexpression of ERRγ through adenovirus       were required for ERRγ-induced SMILE
vector increased SMILE mRNA levels              promoter transcriptional activity, a series of
(Figure 6E, compare lane 3 to lane 1),          mutants of the SMILE promoter were
indicating that SMILE might be a target of      generated (Figure 7D). As shown in Figure
ERRγ. Indeed,            adenovirus-mediated    7E, deletion of the promoter sequence up to
overexpression of ERRγ up-regulated both        nt    -879     decreased     ERRγ-dependent
mRNA and protein levels of SMILE                activation of the SMILE promoter by
dose-dependently (Figure 7A). We next           approximately 95%. Moreover, mutation of
examined whether ERR family members             ERRE2         (mtERRE2-Luc)         decreased
could regulate human SMILE promoter. To         ERRγ-stimulated SMILE promoter activity
address this issue, we first identified the     by approximately 95%, whereas mutation of
transcription start site in SMILE gene          ERRE1 (mtERRE1-Luc) had no significant
through primer extension analysis. The          effect. Taken together, these results indicate
apparent start site of transcription identified that ERRE2 is essential for ERRγ-mediated
by these studies locates 224 nucleotides        transactivation of SMILE promoter. To
upstream from the translation start codon       further examine whether ERRγ directly bind
                   SMILE promoter, ChIP
ATG (Supplementary Figure 4). Next, we          to the ERRE in
cloned an approximately 1-kilobase              assays were carried out using specific
fragment of the human SMILE promoter            primers to amplify the region spanning the
sequences into a luciferase reporter            ERRE1 and ERRE2. Expression vectors
construct     and      performed      transient encoding HA fusion protein of ERRγ
transfection experiments in HepG2 cells. As     (HA-ERRγ), or HA alone were transfected


into HepG2 cells. As shown in Figure 7F, a     play an important role in ERRγ signaling.
262bp fragment (corresponding to ERRE1              Our previous work has shown that
and ERRE2) was amplified from cells that       SMILE interacted with ERRα, ERRβ, and
expressed HA-ERRγ, but not HA alone. In        ERRγ in yeast two-hybrid assays (20).
addition, no significant ERRγ binding was      However, in this study, we observed that
observed in the control region of SMILE        SMILE specifically interacts with ERRγ in
promoter. These results demonstrate that       mammalian cells, as demonstrated through
ERRγ is specifically associated with the       in vivo GST pull-down and Co-IP assays
SMILE promoter in vivo.                        (Figure 1). This discrepancy of interaction
    Overall, these results suggest that ERRγ   pattern could be due to the difference
regulates SMILE gene expression via            between yeast and mammalian cell system.
directly binding to the ERRE in the SMILE      Our previous report has shown that the
promoter, and SMILE in turn inhibits           region spanning residues 113-202 of SMILE
ERRγ transactivation on the SMILE              interacts with LBD/AF2 domain of GR,
promoter, indicating the existence of an       CAR, and HNF4α (20). Similarly, we have
autoregulatory loop.                           observed that SMILE uses the same region

                                                                                              Downloaded from by guest, on August 6, 2010
                                               for binding to AF2 domain of ERRγ (Figure
               DISCUSSION                      3). Ιt is well known that AF2 domain of NRs
                                               is usually involved in its binding to
    The bZIP protein SMILE has been            coactivators (1), and that ERRγ binds to
reported to regulate the transactivation of    coactivators PGC-1α, PGC-1β, and GRIP1
several transcription factors, including ERs,  though its AF2 domain (34, 35). Therefore,
HCF, CREB3, and ATF4 (6, 17-19).               it is not surprising to find that SMILE
Recently, we have reported that SMILE acts     competes        with   PGC-1α, PGC-1β, and
as a novel corepressor of nuclear receptors    GRIP1 for binding to ERRγ (Figure
GR, CAR and HNF4α (20). In this study,         4A-4C). As numerous other coactivators
we identified ERRγ as a novel target of        and corepressors are known to interact with
SMILE repression. SMILE directly interacts     ERRγ (3), whether SMILE may also affect
with ERRγ in vitro and in vivo. SMILE          the interactions of ERRγ with those
inhibits    ERRγ transactivation       through coregulators still needs to be determined.
coactivator competition and recruitment of         Ιt has been well established that LXXLL
SIRT1, a class III HDAC. Moreover,             is a common motif found in NR
knockdown of the endogenous SMILE and          coregulators to interact with the LBD/AF2
SIRT1      gene      expression      increases domain of NRs (35, 36). However, the
ERRγ-mediated transcriptional activity. In     LXXLL motifs in SMILE are apparently not
addition, ERRγ specific inverse agonist        important for the SMILE-ERRγ association
GSK5182         increased       SMILE-ERRγ     and the repression of ERRγ by SMILE
association and enhanced the repression of     (Figure 4B-4E). This LXXLL-independent
SMILE on ERRγ-mediated transactivation         interaction between coregulator and
and PDK4 mRNA level. Given the                 LBD/AF2 domain of NRs has also been
coexpressions of SMILE and ERRγ in the         demonstrated in our previous report (20), in
liver, heart, skeletal muscle, kidney, and     the association between            ERα and
brain (16, 43, 44), these observations         proline-rich nuclear receptor coregulatory
indicate that the corepressor SMILE may        protein (PNRC), and in the case of


corepressor RIP140 (45, 46). In addition,    LXRα, and LXRβ (24-29). Ιn contrast, the
the bZIP region is known to be essential for regulation of SIRT1 on ERRγ is not through
the dimerization and functions of b-zip      a direct interaction between SIRT1 and
proteins (47). However, we found that bZIP   ERRγ, but through association with SMILE
region of SMILE is required for the          (Figure 5E-5G). Moreover, the phenomenon
homodimerization, but is not essential for   of acetylation/deacetylation does not occur
the repressive effect of SMILE on            on either SMILE or ERRγ protein
ERRγ (Supplementary Figure 3), which is      (supplementary Figure 2), indicating SIRT1
consistent with our previous observation     regulation of SMILE and ERRγ may not be
(20).                                        due to direct deacetylation of these two
    Competition with coactivators is a       proteins. Of note, the repression of SMILE
common repression mechanism among            on ERRγ activity is not only sensitive to the
certain corepressors, including SHP (9),     inhibition of SIRT1 catalytic activity by
DAX-1 (10), RIP140 (11), and the             pharmacological inhibitors (Figure 5C, 6A,
corepressor silencing mediator of retinoid   6B and 6E), but also sensitive to the
and thyroid receptors (SMRT) (48). Our       overexpression       of     two     different

                                                                                             Downloaded from by guest, on August 6, 2010
study indicates that SMILE also competes     deacetylase-defective mutants of SIRT1
with coactivators PGC-1α, PGC-1β, and        (H363Y and H355A) (Figure 5D and 6E),
GRIP1 to repress ERRγ transactivation        indicating that the deacetylase activity of
(Figure 4A-4C). Ηowever, overexpression      SIRT1 is required for the repression of
of these coactivators only partially release ERRγ by SMILE. Moreover, we have
the repression by SMILE (Figure 4A-4C),      demonstrated that the recruitment of SIRT1
indicating that coactivator competition is   by SMILE on ERRγ target PDK4 promoter
not completely responsible for the           is correlated with the promoter-complexed
transrepression. Of interest, coactivator    histone 3 deacetylation in ChIP assays, and
competition was also involved in the         this deacetylation is sensitive to SIRT1
repression of SMILE on GR, CAR, and          inhibitor EX527, but not to classical HDAC
HNF4α (20). In contrast with our previous    inhibitor TSA (Figure 6D). These results
observation that certain Class I and II      further support the importance of SIRT1
HDACs are required for the full repression   deacetylase activity in SMILE-stimulated
of SMILE on GR and HNF4α (20), the class     repression on ERRγ and implicate SIRT1 as
III HDAC SIRT1 took the place of classical   a histone H3 deacetylase in mammalian
HDACs to contribute to the inhibition of     cells. Of interest, SIRT1 has been reported
SMILE on ERRγ, as demonstrated by the        to play a similar role in chicken ovalbumin
findings     that   the   repression    was  upstream promoter transcription factor
significantly released by SIRT1 inhibitors,  (COUP-TF)-interacting protein 2-mediated
and siSIRT1, but not by the specific         transcriptional repression (49). However,
classical HDACs inhibitor TSA (Figure 5B,    SMILE is also a corepressor of GR,
5C and 5H).     (20), whether SIRT1
                                             HNF4α, and CAR
    SIRT1 has been reported to regulate a    plays a similar role in the repression of
large number of transcription factors        SMILE on those NRs needs to be further
through direct deacetylation of target       clarified.
proteins, including FOXO transcriptional         In the current model of transcriptional
factors,      PPARα,      PPARγ, PGC-1α,     regulation by NRs, agonist-bound receptors


recruit coactivators such as PGC-1α and       SMILE is involved in this insulin-mediated
histone acetyltransferase complex, leading    repression awaits further exploration. Since
to expression of target genes. The            PGC-1α, RARα, cyclin-dependent kinase
antagonist-bound         receptors       bind inhibitors p21 and p27 are known targets of
corepressors such as N-CoR/SMRT and           ERRγ (4, 5, 7), it would be interesting to
histone deacetylase complexes, leading to     further examine whether SMILE represses
silencing of target genes (1). For instance,  their expression through inhibiting ERRγ. Ιn
promoter-bound OHT-complexed ERα is           addition, as our observations were obtained
associated with NCoR-HDAC3 complexes,         from cell culture studies, in the future, it
resulting in the suppression of ER-mediated   would be necessary to investigate whether
pS2 and c-myc gene transcription (50)         ERRγ is more functionally active in SIRT1
Keeping in line with this model, our study    or SMILE knockout mice.
demonstrated       that    inverse    agonist     Previously, we have reported that
GSK5182-bound            ERRγ        recruits ERRγ regulates SHP and DAX-1 gene
SMILE-SIRT1 corepressor complex to            promoters (9, 10). Our current study
PDK4 gene promoter, resulting in the          indicates that SMILE promoter is also a

                                                                                               Downloaded from by guest, on August 6, 2010
dissociation of coactivator PGC-1α and        target of ERRγ. Αlthough there are two
repressed gene expression of PDK4 (Figure     potential ERRE in the human SMILE
6). These results shed light on a mechanism   promoter, the data from mutation analysis
for the repression of ERRγ by GSK5182.        suggest that ERRE2 is responsible for
    In the oxidation of glucose to            ERRγ-mediated transactivation of SMILE
acetyl-CoA, the pyruvate dehydrogenase        promoter (Figure 7). It has been reported
complex (PDC) is a key enzyme catalyzing      that ERRs (ERRα, ERRβ, and ERRγ) bind
the conversion of pyruvate to acetyl-CoA      to the same DNA response elements (1-3);
(51). PDK2 and PDK4, highly expressed         however,       in    this     work,      only
PDK isoforms in the liver, heart, and         ERRγ significantly activated the SMILE
skeletal muscle, negatively regulate PDC      promoter (Figure 7). This indicates that
activity via phosphorylation (52). It has     some other factors except DNA-binding
been reported that the decrease in PDC        may affect the transactivation. As
activity in diabetes is a consequence of      demonstrated in Figure 7, ERRγ regulated
increased PDK activity (51). In light of our  the transcription of SMILE, which in turn
results that SMILE and ERRγ inverse           repressed ERRγ transactivation. These
agonist        GSK5182          cooperatively results suggest the existence of an
down-regulate ERRγ-mediated PDK gene          autoregulatory loop. Of great interest,
expression in a SIRT1 activity-dependent      similar autoregulatory loops also exist in the
manner (Figure 6E), it is very likely that    regulation      of   SHP,     DAX-1         by
SMILE and SIRT1 function synergistically      ERRγ (9, 10). Certainly, the physiological
in the regulation of glucose oxidation.       role of ERRγ regulation of SMILE gene
                  be further investigated.
However, it should be pointed out that        expression needs
further investigations are required to find
out the upstream signaling of SMILE.              In a summary, as depicted in Figure 7G,
Previously, it has been reported that insulin we proposed that ERRγ activates the SMILE
inhibits the induction of the PDK4 gene by    promoter, whereas SMILE in turn inhibits
both ERRγ and PGC-1α (6). Whether             ERRγ. The binding of the inverse agonist


GSK5182 to ERRγ recruits corepressor           observations provide new insight into
SMILE-SIRT complex, which leads to the         understanding the repressive mechanism of
dissociation of coactivator PGC-1α and         SMILE, SIRT1, and ERRγ inverse agonist.
silencing of ERRγ target gene PDK4. Our

                                                                                           Downloaded from by guest, on August 6, 2010




1.    Giguère, V. (1999) Endocr. Rev. 20, 689-725.
2.    Pettersson, K., Svensson, K., Mattsson, R., Carlsson, B., Ohlsson, R., and
      Berkenstam, A. (1996) Mech. Dev. 54, 211–223.
3.    Giguère, V. (2008) Endocr. Rev. 29, 677-696.
4.    Yu, S., Wang, X., Ng, C.F., Chen, S., and Chan, F.L. (2007) Cancer Res. 67,
5.    Wang, L., Liu, J., Saha, P., Huang, J., Chan, L., Spiegelman, B., and Moore, D.D.
      (2005) Cell Metabolism, 2, 227-238.
6.    Zhang, Y., Ma, K., Sadana, P., Chowdhury, F., Gaillard, S., Wang, F., McDonnell,
      D.P., Unterman, T.G., Elam, M.B., and Park, E.A. (2006) J. Biol. Chem. 281,
7.    Dufour, C.R., Wilson, B.J., Huss, J.M., Kelly, D.P., Alaynick, W.A., Downes, M.,
      Evans, R.M., Blanchette, M., and Giguère, V. (2007) Cell Metabolism, 5, 345-356.
8.    Hong, H., Yang, L., Stallcup, M.R. (1999) J. Biol. Chem. 274, 22618-22626.

                                                                                               Downloaded from by guest, on August 6, 2010
9.    Sanyal, S., Kim, J.Y., Kim, H.J., Takeda, J., Lee, Y.K., Moore, D.D., and Choi, H.S.
      (2002) J. Biol. Chem. 277, 1739-1748.
10.   Park, Y.Y., Ahn, S.W., Kim, H.J., Kim, J.M., Lee, I.K., Kang, H. and Choi, H.S.
      (2005) Nucleic Acids Res. 33, 6756-6768.
11.   Castet, A., Herledan, A., Bonnet, S., Jalaguier, S., Vanacker, J.M., and Cavaillès, V.
      (2006) Mol. Endocrinol. 20, 1035-1047.
12.   Tremblay, G.B., Bergeron, D., and Giguere, V. (2001) Endocrinology. 142,
13.   Coward, P., Lee, D., Hull, M.V., and Lehmann, J.M. (2001) Proc. Natl Acad. Sci.
      USA. 98, 8880–8884.
14.   Chao, E.Y., Collins, J.L., Gaillard, S., Miller, A.B., Wang, L., Orband-Miller, L.A.,
      Nolte, R.T., McDonnell, D.P., Willson, T.M., and Zuercher, W.J., (2006) Bioorg.
      Med. Chem. Lett. 16, 821-824.
15.   Lu, R. and Misra, V. (2000) Nucleic Acids Res., 28, 2446–2454.
16.   Xie, Y.B., Lee, O.H., Nedumaran, B., Seong, H.A., Lee, K.M., Ha, H., Lee, I.K., Yun,
      Y. and Choi, H.S. (2008) Biochem. J., 416, 463-473.
17.   Akhova, O., Bainbridge, M. and Misra, V. (2005) J. Virol., 79, 14708–14718.
18.   Misra, V., Rapin, N., Akhova, O., Bainbridge, M., and Korchinski, P. (2005) J. Biol.
      Chem. 280, 15257-15266. Zhangfei is a potent and specific inhibitor of the host cell
      factor-binding transcription factor Luman.
19.   Hogan, M.R., Cockram, G.P., and Lu, R. (2006) FEBS Lett. 580, 58-62.
20.   Xie, Y.B., Nedumaran, B., Choi, H.S. (2009) Nucleic Acids Res. May 8. [Epub ahead
      of print].
21.   Blander, G., and Guarente, L. (2004) Annu. Rev. Biochem.73, 417-435.
22.   Luo, J., Nikolaev, A.Y., Imai, S., Chen, D., Su, F., Shiloh, A., Guarente, L. and Gu,
      W. (2001) Cell 107, 137–148.
23.   Rajamohan, S.B., Pillai, V.B., Gupta, M., Sundaresan, N.R., Konstatin, B., Samant,
      S., Hottiger, M.O., and Gupta, M.P. (2009) Mol. Cell. Biol. [Epub ahead of print]


24.   Brunet, A., Sweeney, L.B., Sturgill, J.F., Chua, K.F., Greer, P.L., Lin, Y., Tran, H.,
      Ross, S.E., Mostoslavsky, R., and Cohen, H.Y. et al., (2004), Science 303,
25.   Motta, M.C., Divecha, N., Lemieux, M., Kamel, C., Chen, D., Gu, W., Bultsma, Y.,
      McBurney, M., and Guarente, L. (2004) Cell 116, 551–563.
26.   Purushotham, A., Schug, T.T., Xu, Q., Surapureddi, S., Guo, X., and Li, X. (2009)
      Cell Metab. 9, 327-38.
27.   Picard, F., Kurtev, M., Chung, N., Topark-Ngarm, A., Senawong, T., Machado, De.
      Oliveira, R., Leid, M., McBurney, M.W., and Guarente, L. (2004) Nature, 429,
28.   Nemoto, S., Fergusson, M.M., and Finkel, T. (2005) J. Biol. Chem. 280,
29.   Li, X., Zhang, S., Blander, G., Tse, J.G., Krieger, M., and Guarente L. (2007) Mol.
      Cell. 28, 91-106.
30.   North, B.J., Marshall, B.L., Borra, M.T., Denu, J.M., Verdin, E. (2003) Mol. Cell. 11,

                                                                                               Downloaded from by guest, on August 6, 2010
31.   Kressler, D., Schreiber, S.N., Knutti, D., Kralli A. (2002) J. Biol. Chem. 277,
32.   Yamagata, K., Daitoku, H., Shimamoto, Y., Matsuzaki, H., Hirota, K., Ishida, J.,
      Fukamizu, A. (2004) J. Biol. Chem. 279, 23158-23165.
33.   Kwon, H.S., Huang, B., Unterman, T.G., and Harris, R.A. (2004) Diabetes. 53,
34.   Han, M.K., Song, E.K., Guo, Y., Ou, X., Mantel, C., Broxmeyer, H.E. (2008) Cell
      Stem Cell. 2, 241-251.
35.   Hentschke, M., Süsens, U., Borgmeyer, U. (2002) Biochem. Biophys. Res. Commun.
      299, 872-879.
36.   Savkur, R.S., and Burris, T.P. (2004) J, Pept. Res. 63, 207-12. Review.
37.   Heery, D.M., Hoare, S., Hussain, S., Parker, M.G., and Sheppard, H. (2001) J. Bio.l
      Chem., 276, 6695-6702.
38.   Chanda, D., Park, J.H., and Choi, H.S. Endocr J. 55, 253-268, review.
39.   Solomon, J.M., Pasupuleti, R., Xu, L., McDonagh, T., Curtis, R., DiStefano, P.S., and
      Huber, L.J. (2006) Mol Cell Biol. 26, 28-38.
40.   Pfister, J.A., Ma, C., Morrison, B.E., D'Mello, S.R. (2008) PLoS ONE. 3, e4090.
41.   Dai, Y., Ngo, D., Forman, L.W., Qin, D.C., Jacob, J., Faller, D.V. (2007) Mol.
      Endocrinol. 21, 1807-1821.
42.   Wang, J., Fang, F., Huang, Z., Wang, Y., and Wong, C. (2009) FEBS Lett. 583,
43.   Zhang, Z, and Teng, C.T. (2007) Mol. Cell. Endocrinol .264, 128-141.
      Yang, X., Downes, M., Yu, R.T., Bookout, A.L., He, W., Straume, M., Mangelsdorf,
      D.J., Evans, R.M. (2006) Cell, 126, 801-810.
45.   Zhou, D., Ye, J.J., Li, Y., Lui, K., and Chen, S. (2006) Nucleic Acids Res. 34,
46.   Lee, C.H., and Wei, L.N. (1999) J. Biol. Chem., 274, 31320-31326.
47.   Deppmann, C.D., Alvania, R.S., and Taparowsky, E.J. (2006) Mol. Biol. Evol., 23,


48.   Ruse, M.D. Jr, Privalsky, M.L., and Sladek. F.M. (2002) Mol. Cell. Biol. 22,
49.   Senawong, T., Peterson, V.J., Avram, D., Shepherd, D.M., Frye, R.A., Minucci, S.,
      and Leid, M. (2003) J. Biol. Chem. 278, 43041-43050.
50.   Liu, X.F., and Bagchi, M.K. (2004) J. Biol. Chem. 279, 15050-15058.
51.   Harris, R. A., Bowker-Kinley, M. M., Huang, B., and Wu, P. (2002) Adv. Enzyme
      Regul. 42, 249–259.
52.   Kwon, H. S., and Harris, R. A. (2004) Adv. Enzyme Regul. 44, 109–121.

                                                                                          Downloaded from by guest, on August 6, 2010




We appreciate Drs. Seok-Yong Choi, Balachandar Nedumaran and Dipanjan Chanda for
critical reading of the manuscript. This work was supported by the National Research
Laboratory grant (ROA-2005-000-10047-0) and the Korea Research Foundation grant

The abbreviation used are: CAR, constitutive androstane receptor; ERR, estrogen
receptor-related receptor; GR, glucocorticoid receptor; HNF4α, hepatocyte nuclear factor 4
alpha; PDK4, pyruvate dehydrogenase kinase 4; SMILE, small heterodimer partner
interacting leucine zipper protein.

                                                                                             Downloaded from by guest, on August 6, 2010



                                   FIGURE LEGENDS

    Figure 1. Interactions and colocalizations of SMILE with ERRγ. (A) In vitro GST
pull-down assays. 35S-radiolabeled ERRα, ERRβ or ERRγ 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. (B) In vivo interaction between exogenous ERRγ and SMILE. HepG2 cells
were cotransfected with pSG5-HA-ERRγ and pEBG-SMILE (GST-SMILE) or pEBG alone
(GST). Protein interactions were examined via in vivo GST pull-down. The top and middle
panels (GST puri) show GST beads-precipitated HA-ERRγ and GST fusion proteins,
respectively. The bottom panel shows the protein expression levels of HA-ERRγ in cell
lysates. (C) In vivo interaction of endogenous ERRγ and SMILE. Co-Immunoprecipitation
assays were performed using cell extract from HepG2 cells, mouse liver, kidney, and heart
tissues with anti-SMILE antibody. Endogenous SMILE was immunoprecipitated with ERRγ
(upper panels). The proteins in the cell lysates (middle and lower panels) were analyzed with

                                                                                                 Downloaded from by guest, on August 6, 2010
Western blot (WB) analysis using indicated antibodies. (E) Co-localizations of SMILE with
ERRγ. HeLa cells were transfected with 0.1 μg of expression vectors encoding GFP-SMILE
and HA-ERRγ. HA-fusion proteins were detected with dye Alexa 594-conjugated anti-HA
monoclonal antibody. The cell images were captured under 400 x magnifications. The data
shown are representative of at least three independent experiments.

    Figure 2. Effect of overexpression and knockdown of SMILE on ERRγ
transactivation. Reporter assays (A-D) were performed as described in the Materials and
Methods section. (A and B) The effect of SMILE on ERRγ-mediated transcriptional activity.
293T and HepG2 cells were cotransfected with 0.3 μg of pcDNA3-Flag-ERRγ, and 0.1 μg of
sft4-Luc reporter vectors, together with indicated amounts of plasmids expressing wild-type
(wt) SMILE, SMILE-L (SMILE-83Leu), and SMILE-S (SMILE-1Phe). (C and D) siSMILE
increases ERRγ transactivation. 293T and HepG2 cells were transfected with pSUPER
[control (con)], or pSUPER siSMILE-I (siSM#1), or pSUPER siSMILE-II (siSM#2), or
pSUPER siSHP (siSHP). After 24h, the cells were cotransfected with expression vector for
Flag-ERRγ and sft4-Luc reporter vectors. The luciferase activity was measured 48h after the
second transfection. The mean and standard deviation (n = 3) of a representative experiment
are shown. **P <0.01, using Student’s t-test. (E) The effects of overexpressed SMILE on the
protein levels of Flag-ERRγ. 293T cells were cotransfected with various plasmids as indicated.
The proteins of Flag-ERRγ, SMILE, and tubulin were detected by respective antibodies
though Western blot analysis. (F) Effect of siRNAs for SMILE or SHP on the expression of
SMILE and SHP. 293T and HepG2 cells were transfected with pSUPER siSMILE-I (siSM#1),
siSMILE-II (siSM#2), siSHP or pSUPER (con), and after 72 h the total RNA was isolated.
The mRNA levels of SHP and SMILE were measured via RT-PCR analysis, with β-actin
shown as a control. The data shown are representative of at least three independent

   Figure 3. Interaction domains of ERRγ and SMILE. (A) Schematic representation of


the structures of ERRγ mutants. AF1, activation function-1 domain; DBD, DNA binding
domain; LBD, ligand binding domain; AF2, activation function-2 domain. The numbers in the
figure indicate the amino acid (aa) residues. (B) SMILE interacts with the AF2 domain of
ERRγ. 35S-radiolabeled ERRγ or ERRγ △AF2 proteins were incubated with GST, or
GST-SMILE fusion proteins. Protein interactions were detected via autoradiography. (C)
Schematic representation of the structures of SMILE mutants. bZIP indicates the basic region
leucine zipper domain. The numbers in the figure indicate the aa residues. (D) In vivo
interaction assays between ERRγ and SMILE mutants. HepG2 cells were cotransfected with
expression vectors for HA-ERRγ and pEBG alone (GST) or pEBG-SMILE (GST-SMILE)
fusions as indicated. Protein interactions were examined via in vivo GST pull-down. The top
and middle panels (GST puri) show GST beads-precipitated HA-ERRγ and GST fusions,
respectively. The bottom panel shows the protein expression levels of HA-ERRγ in cell
lysates. Wt, wild-type. The data shown are representative of at least three independent
experiments with similar results.

    Figure 4. Competition between SMILE and coactivators. Reporter assays in (A-C,

                                                                                                Downloaded from by guest, on August 6, 2010
upper panel) 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 μg of sft4-Luc reporter plasmids, together with indicated expression
vectors for Flag-ERRγ (0.2 μg), Flag-SMILE, HA-PGC-1α, HA-PGC-1β, and HA-GRIP1.
(A-C, lower panel) In vitro competition of SMILE with PGC-1α, HA-PGC-1β, or
HA-GRIP1. 35S-radiolabeled ERRγ were incubated with indicated GST, or GST-SMILE
fusion proteins, together with increasing amounts of unlabeled in vitro translated HA-PGC-1α,
HA-PGC-1β, or HA-GRIP1 (0, 3, 6, or 12 μl) proteins. The protein interactions were detected
via autoradiography. (D) Schematic representation of SMILE and LXXLL motif mutants of
SMILE. Upper arrows indicate the locations of four LXXLL motifs in SMILE and lower
arrows indicate the mutation of LXXLL motifs to AXXAL. m1, SMILE-m1 (first LXXLL
mutated to AXXAL); m2, SMILE-m2 (second LXXLL mutated to AXXAL); m3, SMILE-m3
(third LXXLL mutated to AXXAL); m4, SMILE-m4 (fourth LXXLL mutated to AXXAL);
m5, SMILE-m5 (all of the four LXXLL mutated to AXXAL). bZIP, basic region leucine
zipper domain. (E) Western blot analysis using specific antibodies for SMILE and tubulin,
with whole-cell extracts from HepG2 cells transfected with expression vectors encoding
wild-type (wt) Flag-SMILE, or Flag-SMILE-m1, -m2, -m3, -m4, -m5. (F) The effects of
SMILE LXXLL mutants on ERRγ-mediated transcriptional activity. HepG2 cells were
co-transfected with reporter vector sft4-Luc, together with indicated expression vector for
ERRγ, wild-type (wt) Flag-SMILE or Flag-SMILE LXXLL mutants. Luciferase activity was
measured after 48h of transfection. The mean and standard deviation (n = 3) of a
representative experiment are shown. (G) In vivo interactions of SMILE LXXLL mutants
with ERRγ. HepG2
                            were cotransfected with expression vectors for Flag-ERRγ and
wild-type pEBG-SMILE (GST-SMILE), or indicated GST-SMILE mutants, or pEBG alone
(GST). Protein interactions were examined via in vivo GST pull-down. The top and middle
panels (GST puri) show GST beads-precipitated Flag-ERRγ and GST fusions, respectively.
The bottom panel shows the protein expression levels of Flag-ERRγ in cell lysates. The data
shown are representative of at least three independent experiments with similar results.


    Figure 5. Involvement of SIRT1 in SMILE repression of ERRγ. Reporter assays in
(A-D, H and I) 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 μg of indicated reporter plasmids, (HNF4)8-Luc (A) or sft4-Luc (B-D)
and 0.1 μg of pcDNA3-HA-HNF4α (A) or pcDNA3-Flag-ERRγ (B-D), together with or
without pcDNA3-Flag-SMILE ( 0.2 μg in A-C, and 0.1 μg in D), and indicated doses of
pcDNA3-Myc-SIRT1 or -SIRT1H363Y. 36h after transfection, the cells in A-C were left
untreated, or treated with HDAC inhibitor TSA, or SIRT1 inhibitors Sortinol (20 μM), EX527
(10 μM), or Nicotinamide (NAM, 20 mM) for 12 h prior to the measurement of luciferase
activity. (E) In vitro interaction of SMILE and SIRT1. 35S-radiolabeled SIRT1 protein was
incubated with GST, or GST-SMILE, or GST-ERRγ fusion proteins. Protein interactions were
detected via autoradiography. (F) In vivo interaction of exogenous SIRT1 and SMILE. HepG2
cells were cotransfected with wild-type pcDNA3-myc-SIRT1, or pcDNA3-myc-SIRT1H363Y
and pEBG-SMILE (GST-SMILE) or pEBG alone (GST). Protein interactions were examined
via in vivo GST pull-down. The top and middle panels (GST puri) show GST

                                                                                                   Downloaded from by guest, on August 6, 2010
beads-precipitated Myc-SIRT1 and GST fusions, respectively. The bottom panel shows the
protein expression levels of Myc-SIRT1 in cell lysates. (G) In vivo interaction of endogenous
SIRT1 and SMILE. Co-Immunoprecipitation assays were performed with cell extract from
HepG2 cells using anti-SMILE antibody. Endogenous SMILE was immunoprecipitated with
SIRT1 (upper panel). The proteins in the cell lysates (middle and lower panels) were analyzed
by Western blot (WB) analysis using indicated antibodies. (H and I) HepG2 cells were
transfected with pSUPER [control (con)], or pSUPER siSMILE-I (siSM#1), or pSUPER
siSMILE-II (siSM#2), or pSUPER siSIRT1 (siSIRT1). After 24h, the cells were cotransfected
with expression vector for Flag-ERRγ and sft4-Luc reporter vectors. 36h after the second
transfection, the cells were treated with or without indicated SIRT1 activators resveratrol (100
nM), or piceatannol (20 μΜ) for 12 h prior to the measurement of luciferase activity. (J)
Effect of siSMILE and siSIRT1 on the expression of SMILE and SIRT1. HepG2 cells were
transfected with pSUPER siSMILE-I (siSM#1), siSMILE-II (siSM#2), siSHP or pSUPER
(con), and after 72 h the total RNA was isolated. The mRNA levels of SHP and SMILE were
measured via RT–PCR analysis, with β-actin shown as a control. The data shown are
representative of at least three independent experiments.

    Figure 6. ERRγ inverse agonist GSK5182 enhances SMILE to down-regulate ERRγ
target PDK4. (A, B) GSK5182 represses ERRγ transactivation in a SMILE and
SIRT1-dependent manner. HepG2 cells were transfected with pSUPER siSMILE-II (siSM#2),
siSIRT1 or pSUPER (con) as indicated. 24h after transfection, the cells were cotransfected
with 0.1 μg of indicated reporter plasmids, sft4-Luc (A) or PDK4-Luc (B) and 0.2 μg of
pcDNA3-Flag-ERRγ, together with or without 0.1 μg of pcDNA3-Flag-SMILE (A-C). 36 h
after transfection, the cells were treated with chemicals [1μΜ of GSK5182, 10μM EX527 or
20 mM Nicotinamide (NAM)] as indicated for 12 h prior to the measurement of luciferase
activity. The mean and standard deviation (n=3) of a representative experiment are shown. (C)
GSK5182 treatment intensifies the interaction between ERRγ and SMILE. HepG2 cells were
cotransfected with pcDNA3-Flag-ERRγ and pEBG-SMILE (GST-SMILE) or pEBG alone


(GST), 36 h after transfection, the cells were treated with or without 1μΜ of GSK5182. Cell
extracts were prepared and subjected to in vivo GST pull-down assays in the absence or
presence of GSK5182 as indicated. The top and middle panels (GST puri) show GST
beads-precipitated Flag-ERRγ and GST fusions, respectively. The bottom panel shows the
protein expression levels of Flag-ERRγ in the cell lysates. (D) Recruitment of SMILE by
GSK5182 on PDK4 promoters is correlated with PGC-1α dissociation and histone 3
deacetlyation in ChIP assays. HepG2 cells were treated with or without indicated chemicals
(0.1% DMSO, 1 μΜ of GSK5182, 0.3 μM of TSA and 10 μM of EX527). Chromatin
fragments prepared from the treated HepG2 cells were immumoprecipitated with the
indicated specific antibodies. Unrelated immunoglobin G (IgG) was used as a negative
control. DNA fragments covering an ERRE on human PDK4 promoter is indicated in the
upper panel. The occupancy of ERRγ, SMILE, PGC1α, SIRT1, acetylated histone H3
(Ace-H3) on ERRγ-binding region (lower left panel) was analyzed by amplifying the
corresponding regions using quantitative real-time PCR. A control region on the PDK4
promoter was used to check the specific binding of those proteins (lower right panel). Data
are representative of at least two independent IP and three independent PCR amplifications.

                                                                                                  Downloaded from by guest, on August 6, 2010
Values are presented as mean ± SD. (E) Relative mRNA expression levels of ERRγ,
SMILE, PDK4, and PDK2 analyzed by quantitative real-time PCR (standardized using
beta-actin). Normalized basal levels of each transcript were assigned an arbitrary value of 1.0
for comparison. HepG2 cells were infected with indicated adenovirus vector (Ad-Null,
Ad-ERRγ, Ad-SMILE and Ad-SIRT1H355A) at a concentration of 100 pfu/cell. After 36 h of
infection, the cells were stimulated with or without indicated chemicals (0.1% DMSO, 1μΜ
of GSK5182, and 10 μM of EX527) for 12 h before total RNA were isolated. Data shown are
representative of three independent experiments. (G) Structure of ERRγ inverse agonist

    Figure 7. Autoregulatory loop controlling SMILE gene expression by ERRγ. (A)
Upper panel shows the relative ERRγ and SMILE mRNA levels analyzed by quantitative
real-time PCR (standardized using beta-actin). Normalized basal levels of each transcript
were assigned an arbitrary value of 1.0 for comparison. Lower panel shows the protein
expression levels of ERRγ and SMILE. Tubulin was used as a loading control. HepG2 cells
were infected with adenovirus vector (Ad-Null and Ad-ERRγ) at indicated multiplicity of
infection (0, 50 or 100). (B) ERRγ activates human SMILE promoter activity. HepG2 cell
were cotransfected with 0.1μg of SMILE-Luc reporter vector and indicated amount of
expression vectors encoding ERRα, ERRβ, or ERRγ. (C) SMILE inhibits ERRγ-mediated
and PGC-1α-enhanced SMILE promoter activity. ΗepG2 cells were cotransfected with 0.2μg
of SMILE-Luc reporter together with or without 0.2μg of ERRγ expression vector, and
indicated amount of expression plasmids for SMILE and PGC-1α. (D) Schematic
representation of wild-type and mutant hSMILE promoter constructs. The putative ERRγ
binding sites are shown and the mutated ERRE is indicated with “X”. (E) ERRE2 is essential
for the activation of SMILE promoter by ERRγ. ΗepG2 cells were cotransfected with 0.2μg
of wild-type or mutant SMILE promoter constructs along with or without 0.2μg of
ERRγ expression vector. Luciferase activity was measured 48h after transfection. (F)
ERRγ binds to SMILE promoter in ChIP assays. HepG2 cells were transfected with


expression vector for HA or HA-ERRγ. Chromatin fragments were prepared from the
transfeced cells and immumoprecipitated with anti-HA antibody or unrelated immunoglobin
G (IgG) as indicated. DNA fragments covering ERRE (-996 to -735bp) on SMILE promoter
and a control region (-220 to -19bp) were PCR-amplified. Data shown are representative of
three experiments. (G) A schematic representation of the autoregulatory loop controlling the
expression of SMILE by ERRγ and the mechanisms of SMILE repression on
ERRγ: recruitment of SIRT1 and dissociation of coactivator PGC-1α.

                                                                                               Downloaded from by guest, on August 6, 2010



      Figure 1.

       A                                          B
                                                      GST              +       -


                                                      GST-SMILE        -       +
                 t (1


                                                                       +       +

                                    35   S-ERRα
                                                      GST puri                       GST-SMILE
                                    35   S-ERRβ
                                    35   S-ERRγ       Lysate                         HA-ERRγ

                    HepG2                                      liver   kidney        heart


           IP                                         IP




                               WB: ERRγ                                                         WB: ERRγ
                               WB: SMILE                                                        WB: SMILE
       Lysate                                     Lysate
                               WB: ERRγ                                                         WB: ERRγ

                GFP-SMILE                 HA-ERRγ              DAPI                      Merge


  Figure 2.
   A                                                                   B
                                                 293T sft4-Luc                                                       HepG2    sft4-Luc
                                180                                                                       33
                Fold Activity

                                                                                          Fold Activity
                                120                                                                       22
                                 60                                                                       11
             0                                                                       0
          ERRγ -                           + + + + + + +                          ERRγ -                            + + + + + + +
   wt SMILE(μg)                              0.1 0.2                       wt SMILE(μg)                               0.1 0.2
    SMILE-L(μg)                                      0.1 0.2               SMILE-L(μg)                                        0.1 0.2
    SMILE-S(μg)                                              0.1 0.2       SMILE-S(μg)                                                0.1 0.2

   C                                                                   D
                                                                                                56             HepG2              **
                          350         293T           **                                                                         **
                                                                                Fold Activity
                          280                                                                   42
          Fold Activity

                          210                                                                   28
              0                                                                  ERRγ                          - - -      - + + +         +
            ERRγ                      - - - +         + +                          Con                         + - -      - + - -         -
             Con                      + - - +         - -                      siSM#1                          - + -      - - + -         -
          siSM#1                      - + - -         + -                      siSM#2                          - - +      - - - +         -
          siSM#2                      - - + -         - +                        siSHP                         - - -      + - - -         +
   E                                                                   F
       Flag-ERRγ                          + + + +          + + +
       wt SMILE(μg)                       0 0.1 0.2 0      0 0 0             293T                                         HepG2
       SMILE-L (μg)                       0 0 0 0.1       0.2 0 0
                                                                        siS #1

                                                                                                                       siS #2
                                                                                                                       siS #1

       SMILE-S (μg)                       0 0 0 0          0 0.1 0.2







       SMILE-L                                                                                            SMILE                          SMILE
       SMILE-S                                                                                            β-actin                        SHP
       tubulin                                                                                                                           β-actin


  Figure 3.
   A                                               B
                          BD                           Input


                                                       (10%) GST GST-SMILE


   ERRγ             1                       458                            35   S-ERRγ
   ERRγ △AF2        1                       448                            35   S-ERRγ △AF2

  C                                                D

                                                                   △ 02
                                 bZIP              GST-SMILE -

                                                   GST       +     -   -   -      -   -
   SMILE     1                               354                                  +   +
                                                   HA-ERRγ   +     +   +   +
   N1(1-112) 1            112                                                             HA-ERRγ
   N2(1-202) 1                   202               GST-puri                               GST-SMILE
   △202 (203-354)          203               354                                          fusions
   △268 (269-354)                                                                         GST
                                 269         354   Lysate                                 HA-ERRγ


 Figure 4.
 A                                                                            B
          Fold Activity    40               HepG2 sft4-Luc                                         33                  HepG2      sft4-Luc

                                                                                   Fold Activity

          0                                                                            0
      ERRγ -                      + + + + + + + + +                                ERRγ -                    + + + + + + + + +
  SMILE (μg) 0                    0 0 0.05 0.1 0.2 0.2 0.2 0.2 0.2             SMILE (μg) 0                  0 0 0.05 0.1 0.2 0.2 0.2 0.2 0.2
 PGC-1α (μg) 0                    0 0.2 0.2 0.2 0.2 0 0.1 0.2 0.4             PGC-1β (μg) 0                  0 0.2 0.2 0.2 0.2 0 0.1 0.2 0.4

                                        GST-SMILE+                                         Input       GST-SMILE+
          Input                                                                                  GST Cold HA-PGC-1β
                GST                    Cold HA-PGC-1α                                      (10%)
                                                           35   S-ERRγ                                                                    35   S-ERRγ

 C                                                                            D
                           44              HepG2     sft4-Luc

           Fold Activity

                                                                              SMILE 1                                                             354
                                                                                                   LTKLL LADLL LQRLL                       LADLL
                                                                                                   ATKAL AADAL AQRAL                       AADAL
                                                                                                    m1    m2    m3                          m4
      ERRγ -                      + + + + + + + + +                                                m5=m1234
  SMILE (μg) 0                    0 0 0.05 0.1 0.2 0.2 0.2 0.2 0.2
  GRIP1 (μg) 0                    0 0.2 0.2 0.2 0.2 0 0.1 0.2 0.4

                           Input          GST-SMILE+                          E
                                                                                   wt               m1   m2       m3   m4   m5
                           (10%) GST     Cold HA-GRIP1
                                                                35   S-ERRγ                                                            Tubulin

 F                                                                       G
                                      HepG2     sft4-Luc
     Fold Activity





                                                                          GST-SMILE -

                                                                          GST       +                    -    -    -   -    -     -
                                                                          Flag-ERRγ +                    +    +    +   +    +     +
                                                                          GST-puri                                                      GST-SMILE
        0                                                                 Lysate                                                        Flag-ERRγ
      ERRγ -                      +    + + + + + +
     SMILE                             wt m1 m2 m3 m4 m5

Figure 5.
A                                                                      B                                                                    C
                                                                                                                                                                      50           HepG2    sft4-Luc
                                      HepG2 (HNF4)8-Luc
                      30                                                                               40       HepG2       sft4-Luc                                  40

                                                                                                                                                      Fold Activity
      Fold Activity

                                                                                       Fold Activity   30                                                             30
                      10                                                                                                                                              10
                                                                                                                                                   ERRγ                     -   + + + + + + +          +
      0                                                                     0                                                                                               -   - + - - - + -          -
  HNF4α - + + + + +                                                      ERRγ - + + + + +                                                         EX527                     -   - - + - - - +          -
  SMILE - - + + + +                                                     SMILE - - + + + +                                                           NAM                     -   - - - + - - -          +
TSA (μM) 0 0 0 0.030.1 0.3                                            TSA (μM) 0 0 0 0.03 0.1 0.3                                                 SMILE                     -   - - - - + + +          +

D                                                                                                      E                                              F
                                           HepG2           sft4-Luc
                                                                                                                                                                      GST       + - -
                      Fold Activity

                                      33                                                               Input                                                          GST-SMILE - + +
                                                                                                       (10%) GST GST-SMILE                                            Myc-SIRT1 + wt H363Y
                                      22                                                                                                                                                 Myc-SIRT1
                                                                                                                                 35   S-SIRT1
                                                                                                                                                                      GST puri
                                      11                                                               Input                                                                                    GST-SMILE
                                                                                                        10% GST GST-ERRγ
          0                                                                                                                      35   S-SIRT1
      ERRγ                                 - + + +           +    + +                                                                                                 Lysate                    Myc-SIRT1
     SMILE                                 - - - -           +    + +
  SIRT1 (μ g)                              0 0 0.2 0         0   0.2 0
SIRT1H363Y                                 0 0 0 0.2         0    0 0.2
        (μ g)

G                                                                     H                                                                I
                                              IL                                                                                                             50            HepG2     sft4-Luc
                                                                                                           HepG2    sft4-Luc
                                         G -SM
      IP                              Ig   α                                           45                                                                    40
                                                                                                                                            Fold Activity

                                             WB: SIRT1
                                                                       Fold Activity

                                              WB: SMILE
    Lysate                                                                             30
                                              WB: SIRT1                                                                                                      20

J                                                                                      15                                                                    10
          siS #1
          siS #2


                                                                                                                                             ERRγ                          - + + + + + +          + + +

                                                                           0                                                                  Con                          + + + + - - -          - - -
                                                       SMILE                                                -
                                                                        ERRγ                                    +   +   +    +             siSM#1                          - - - - + + +          - - -
                                                       SIRT1              Con                               +   +   -   +    -             siSM#2                          - - - - - - -          + + +
                                                                      siSIRT1                               -   -   +   -    +         Resveratrol                         - - + - - + -          - + -
                                                                       SMILE                                -   -   -   +    +         Piceatannol                         - - - + - - +          - - +

Figure 6.
A                                                                                         B                                                                         C
                                    60                HepG2            sft4-Luc                               16           HepG2              PDK4-Luc
                    Fold Activity

                                                                                              Fold Activity
                                    45                                                                        12
                                                                                                                                                                      GST-SMILE -         -   + +
                                    30                                                                         8                                                      GST       +         +   - -
                                                                                                                                                                      Flag-ERRγ +         +   + +
                                                                                                                                                                      GSK5182   -         +   - +
                                    15                                                                         4
                                                                                                                                                                        GST-puri                     GST-SMILE
      0                                                                                          0
    ERRγ                                      -   +   +    +   +   +    +   +   + + +          ERRγ                -   +   +   +     +        +   +   +   + + +
  SMILE                                       -   -   +    -   +   -    -   -   - - -        SMILE                 -   -   +   -     +        -   -   -   - - -                                      GST
GSK5182                                       -   -   -    +   +   -    +   -   + + +      GSK5182                 -   -   -   +     +        -   +   -   + + +         Lysate                       Flag-ERRγ
  siSM#2                                      -   -   -    -   -   +    +   -   - - -        siSM#2                -   -   -   -     -        +   +   -   - - -
 siSIRT1                                      -   -   -    -   -   -    -   +   + - -       siSIRT1                -   -   -   -     -        -   -   +   + - -
     Con                                      +   +   +    +   +   -    -   -   - + +           Con                +   +   +   +     +        -   -   -   - + +
   EX527                                      -   -   -    -   -   -    -   -   - + -         EX527                -   -   -   -     -        -   -   -   - + -
    NAM                                       -   -   -    -   -   -    -   -   - - +          NAM                 -   -   -   -     -        -   -   -   - - +
                                              1   2   3    4   5   6    7   8   9 10 11                            1   2   3   4     5        6   7   8   9 10 11

                                                          -1699 bp              -1493 bp                                       -1056 bp                     -886 bp
                                                                                                                                             control region                           PDK4
                                                          ERRE region (-1699 --- -1469 bp)                                                                  Control region (-1056 ---- -886bp)
                                    20                         IP ERRγ               1. DMSO                                                 10                 IP ERRγ              1. DMSO
                                                               IP SMILE              2. GSK5182                                                                 IP SMILE             2. GSK5182
                                                               IP PGC-1α             3. GSK5182 + TSA                                         8                 IP PGC-1α            3. GSK5182 + TSA
                                    15                         IP SIRT1                                                                                         IP SIRT1
                  % Input

                                                                                     4. GSK5182 + EX527                                                                              4. GSK5182 + EX527
                                                                                                                                   % Input

                                                               IP Ace-H3                                                                                        IP Ace-H3
                                                               IP IgG
                                    10                                                                                                                          IP IgG
                                        0                                                                                                     0
                                             1234 1234                  1234 1234 1234 1234                                                       1234 1234              1234 1234         1234 1234

E                                                                                                                                                                                F
                                                                                ERRγ                          1. Ad-Null + DMSO
                                                                                SMILE                         2. Ad-SMILE + DMSO
                                                                                PDK4                          3. Ad-ERRγ + DMSO
                                                                                PDK2                          4. Ad-ERRγ + Ad-SMILE + DMSO
                                                                                                              5. Ad-ERRγ + GSK5182                                                                  N
                             66                                                                                                                                                               O
                                                                                                              6. Ad-ERRγ + EX527
    Relative folds of mRNA

                                                                                                              7. Ad-ERRγ + Ad-SIRT1H355A
                                                                                                              8. Ad-ERRγ + Ad-SMILE + GSK5182
                                                                                                              9. Ad-ERRγ + Ad-SMILE + GSK5182 + EX527
                                                                                                              10. Ad-ERRγ + Ad-SMILE + Ad-SIRT1H355A
                                                                                                                  + GSK5182
                             22                                                                                                                                                                     OH

                             11                                                                                                                                                           GSK5182
                                            1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

Figure 7.
A.                                                  B.                                                               C.
                                                                            15   HepG2 SMILE-Luc                                             15
     Relative folds

                                                                                                                                                  HepG2   SMILE-Luc
       of mRNA

                      42        SMILE

                                                            Fold Activity

                                                                                                                             Fold Activity
                      28                                                    10                                                               10
         0                                                                   5                                                                5
 Ad-Null 0 100 50 0
Ad-ERRγ 0 0 50 100
  ERRγ                                                      0                                                                 0
                                                     ERRα (μ g) 0 0.1 0.2 0 0 0 0                                         ERRγ - + + + + + +
 SMILE                                               ERRβ (μg) 0 0 0 0.1 0.2 0 0                                      SMILE (μg) 0 0 0.1 0.2 0 0.1 0.2
 Tubulin                                             ERRγ (μg) 0 0 0 0 0 0.1 0.2                                     PGC-1α (μg) 0 0 0 0 0.2 0.2 0.2

D                                                                                                E
     - 1131bp    - 879bp     - 448bp
                                                                                                                           HepG2                   -1131 SMILE-Luc
           ERRE2       ERRE1                                                                                                                        -879 SMILE-Luc
                                       -1131 SMILE-Luc                                                               18
          AGGTCA      AGGTGG       Luc                                                                                                              -448 SMILE-Luc
                                        ERRE1                                                                                                      -SMILE-mtERRE2-Luc

                                                                                                     Fold Activity
                                                                             -879 SMILE-Luc
                                                           Luc                                                       12
                                                                             -448 SMILE-Luc
                                            x                                                                         6
                      ERRE2            ERRE1
    -1131bp                           AAATGG
                       ERRE2          ERRE1                                                              0
                                                                            -SMILE-mtERRE2-Luc         ERRγ - +                              - +    - +       - +   - +

F                                                                                   G
                      - 996bp           - 735 bp
                               AGGTCA                                                                                ERRγ
                                ERRE                     SMILE

                      HA          +     -       +    -       +               -
                 HA-ERRγ          -     +       -    +       -               +          PGC-1α                              SMILE SIRT1
     (-996--- -735bp)
                                                                                                   PGC-1α                                    GSK5182
       (-220--- -19bp)                                                                                                    ERRγ
                                  Input IP: α-HA IP: ΙgG                                                                                               PDK4


Shared By: