Skip Navigation


Nucleic Acids Research Advance Access originally published online on May 8, 2009
Nucleic Acids Research 2009 37(12):4100-4115; doi:10.1093/nar/gkp333
This Article
Right arrow Abstract Freely available
Right arrow Print PDF (9509K) Freely available
Right arrow Screen PDF (1065K) Freely available
Right arrow Supplementary Data
Right arrowOA All Versions of this Article:
37/12/4100    most recent
gkp333v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Xie, Y.-B.
Right arrow Articles by Choi, H.-S.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xie, Y.-B.
Right arrow Articles by Choi, H.-S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Nucleic Acids Research, 2009, Vol. 37, No. 12 4100-4115
© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Molecular characterization of SMILE as a novel corepressor of nuclear receptors

Yuan-Bin Xie, Balachandar Nedumaran and Hueng-Sik Choi*

Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju, 500-757, Republic of Korea

*To whom correspondence should be addressed. Tel: +82 62 530 0503; Fax: +82 62 530 0506. Email: hsc{at}chonnam.ac.kr

Received March 25, 2009. Revised April 20, 2009. Accepted April 20, 2009.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a coregulator in ER signaling. In this study, we have examined the effects of SMILE on other NRs (nuclear receptors). SMILE inhibits GR, CAR and HNF4{alpha}-mediated transactivation. Knockdown of SMILE gene expression increases the transactivation of the NRs. SMILE interacts with GR, CAR and HNF4{alpha} in vitro and in vivo. SMILE and these NRs colocalize in the nucleus. SMILE binds to the ligand-binding domain or AF2 domain of the NRs. Competitions between SMILE and the coactivators GRIP1 or PGC-1{alpha} have been demonstrated in vitro and in vivo. Furthermore, an intrinsic repressive activity of SMILE is observed in Gal4-fusion system, and the intrinsic repressive domain is mapped to the C-terminus of SMILE, spanning residues 203–354. Moreover, SMILE interacts with specific HDACs (histone deacetylases) and SMILE-mediated repression is released by HDAC inhibitor trichostatin A, in a NR-specific manner. Finally, ChIP (chromatin immunoprecipitation) assays reveal that SMILE associates with the NRs on the target gene promoters. Adenoviral overexpression of SMILE represses GR-, CAR- and HNF4{alpha}-mediated target gene expression. Overall, these results suggest that SMILE functions as a novel corepressor of NRs via competition with coactivators and the recruitment of HDACs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Small heterodimer partner interacting leucine zipper protein (SMILE) belongs to basic region leucine zipper (bZIP) family (1,2). SMILE gene produces two isoforms, SMILE-L (long isoform of SMILE, also known as CREBZF) and SMILE-S (short isoform of SMILE, previously known as Zhangfei), from alternative usage of initiation codons (2). Although SMILE has the ability to homodimerize like other bZIP proteins, it cannot bind to DNA as a homodimer (1–3). SMILE has been identified as an interacting partner of herpes simplex virus-related host-cell factor (HCF) and inhibits the replication of the herpes simplex virus (1,3). SMILE has also been reported as a coactivator of ATF4 and as a corepressor of CREB3, another cellular HCF-binding transcription factor (4,5). Recently, we have reported that SMILE acts as a coregulator of estrogen receptor (ER) signaling (2), but its role in other nuclear receptors (NRs) signaling remains unknown.

NRs are transcription factors that modulate the expression of genes involved in embryonic development, maintenance of differentiated cellular phenotypes, metabolism and cell death [see references (6–8) for reviews]. Members of the NR superfamily include the conventional endocrine receptors, the adopted orphan receptors, for which ligands have been identified in recent years, and the orphan receptors, ligands of which have not yet been identified (8). Glucocorticoid receptor (GR) is a member of steroid receptor family and mediates the effect of glucocorticoids in a variety of cellular processes, including homeostasis, cell growth, development, stress response and inflammation (8). GR regulates the transcription of target genes either by binding to specific glucocorticoid response elements (GREs) within the target genes or by interacting with other DNA-bound transcription factors. The inactive GR resides in the cytoplasm bound to heat-shock protein. It dissociates from heat-shock protein upon ligand binding and enters the nucleus where it functions as a transcription factor (9). GR plays an important role in various metabolic pathways by regulating the expression of genes such as phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor-binding protein 1 (IGFBP1) (10,11).

Constitutive androstane receptor (CAR) is an adopted orphan NR which functions as heterodimers with the retinoid X receptor (RXR) (8). CAR evidences constitutive activity, and is expressed primarily in the liver, where it regulates many Phase I and Phase II biotransforming enzymes, including Cyp2b6, Sult2a1, SultN and Ugt1a1 (12,13). This xenobiotic receptor can also regulate the expression of membrane transporter proteins such as organic anion transporting peptide 2 (Oatp2) and multidrug resistance-associated proteins. CAR can be modulated by structurally diverse chemicals such as 1,4-bis-2[-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital (12–14). Hepatocyte nuclear factor 4 (HNF4) is an orphan nuclear receptor which is highly expressed in the liver, kidney, and pancreatic β-cells. HNF4 contains two subtypes in mammals, namely HNF4{alpha} and HNF4{gamma}, and binds to the DR-1 element of target gene promoters as homodimers (6,7). HNF4{alpha} plays critical roles not only in the specification of the hepatic phenotype during liver development but also in the transcriptional regulation of genes involved in glucose, cholesterol, fatty acids and xenobiotic metabolism (7,15), including PEPCK, cholesterol 7 alpha-hydroxylase (CYP7A1) and liver carnitine palmitoyl transferase CPT (L-CPT) (16–18). Mutations in the HNF4{alpha} gene have been associated with maturity-onset diabetes of the young (MODY) (7).

NR-mediated transcriptional effects are regulated by NR coregulators, including coactivators and corepressor (19). Coregulators modulate the transcription of NR target genes through taking part in chromatin remodeling or interacting with basal transcriptional machinery to influence the main steps in transcriptional initiation (20). In the presence of NR ligands, the SWI/SNF chromatin remodeling complex, the histone acetyltransferase (HAT) activity containing complexes CBP/p160/P/CAF, and the TRAP/DRIP/ARC complex are sequentially recruited to gene promoters to activate gene transcription (21–24). Coactivators of the p160 family, including SRC1/NCoA1 and TIF-2/GRIP1, interact with the ligand-binding domain (LBD)/activation function 2 (AF2) domain of receptors through an LXXLL motif or NR boxes (25). In the absence of NR ligands, on the other hand, many NRs prevent gene transcription via recruitment of corepressors such as N-CoR and SMRT, which have been proposed to antagonize the actions of coactivators and to maintain a more repressed state in the chromatin structure. Histone deacetylases (HDACs)-dependent and HDACs-independent mechanisms are involved in the transrepression induced by N-CoR and SMRT (26).

In this study, we have identified that SMILE represses the transcriptional activities of GR, CAR and HNF4{alpha} through direct interaction. We have demonstrated that SMILE represses the transactivities of the NRs via competition with coactivators and the recruitment of HDACs for its active repression. Overall, our findings suggest that SMILE acts as a novel corepressor of NRs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Plasmid and DNA construction
The plasmids of pCMV-β-gal, pcDNA3mCAR, pcDNA3 mCAR{Delta}AF2 and pcDNA3-HA-mPPAR{gamma}, -PGC-1{alpha}, pSG5HA-GRIP1, (NR1)X5-Luc, Gal4-tk-Luc and PPRE-Luc were described elsewhere (27–29). p(HNF4)8-tk-Luc and MMTV-Luc were kindly provided by Drs Akiyoshi Fukamizu and Yoon-Kwang Lee, respectively. pcDNA3-SMILE, pcDNA3-Flag-SMLE, pcDNA3-SMILE-83Leu, pcDNA3-SMILE-1Phe, pGEX4T-1, pGEX4T-1-SMILE, pEBG, pEGFP-SMILE, pSuper, pSuper-siSHP, pSuper-siSMILE-I and pSuper-siSMILE-II were described previously (2).

pcDNA3-Flag-mCAR, pcDNA3-HA-mCAR and pcDNA3-HA-HNF4{alpha} were constructed by inserting the full PCR fragments of the ORFs into the EcoRI/XhoI sites of pcDNA3-Flag, or pcDNA3-HA vector. pcDNA3-HA-mGR was generated by subcloning the full ORF of mouse GR into the XhoI/XbaI sites of pcDNA3-HA vector. Mouse GR deletion constructs, including pcDAN3-mGR-N (1–531 aa) and pcDNA3-mGR-LBD (532–783 aa), were subcloned via the insertion of the PCR fragments of mouse GR into pcDNA3 between the BamHI and XhoI sites. The pcDNA3-HA-HNF4{alpha}CD (1–370 aa) and pcDNA3-HA-HNF4{alpha}{Delta}LBD (1–174 aa) plasmids were constructed via subcloning the EcoRI-XhoI cDNA fragments of rat HNF4{alpha} into pcDNA3-HA vector. The SMILE leucine zipper region mutant SMILE-L (239–267)V was generated via PCR-mediated site-directed mutagenesis, and the PCR products were cloned into the EcoRI/XhoI sites of pcDNA3-Flag and the BamHI/KpnI sites of pEBG vector. pEBG-SMILE and pEBG-SMILE deletion constructs were constructed by inserting full length SMILE or appropriate SMILE deletion fragments into pEBG vector between BamHI and KpnI sites. All plasmids were confirmed via sequencing analysis.

Gal4-DBD fusion constructs were generated using the pCMX-Gal4N expression vector (30). To generate Gal4-DBD-SMILE, EcoRI/XhoI digested full-length SMILE fragments from pcDNA3-Flag-SMILE were cloned into EcoRI/SalI-digested pCMX-Gal4N vector. To construct the Gal4-DBD-SMILE deletion constructs, SMILE cDNA deletion fragments were obtained from pcDNA3-Flag-SMILE via PCR, and cloned into pCMX-Gal4N vector between the EcoRI and SalI sites. pSuper-siHDAC1, pSuper-siHDAC3 and pSuper-siHDAC4 constructs were constructed by inserting a 64-bp double-stranded oligonucleotide containing 5'-aagcagatgcagagattcaac-3' of the human HDAC1 cDNA sequence, or 5'-aagatgctgaaccatgcacct-3' of human HDAC3 cDNA sequence, or 5'-aatgtacgacgccaaagat-3' of human HDAC4 cDNA sequence into the pSUPER vector between BglII and Xho I sites. All plasmids were confirmed via sequencing analysis.

Cell culture, transient transfection assay and luciferase assay
HEK293T (293T), HepG2 and HeLa cells were obtained from the American-type culture collection (ATCC) and cultured according to the manufacturer's instructions. Transient transfection was performed using Superfect transfection reagent (Qiagen) as described previously (2). Total amounts of DNA in each transfection were maintained at the same levels using empty pcDNA3 vectors. Luciferase assays were performed as described previously (31). Fold activity was calculated considering the activity of reporter gene alone as 1.

In vitro glutathione S-transferase (GST) pull-down assay and competition assay
In vitro GST pull-down and competition assays were performed as described previously (31,32).

Co-immunoprecipitation (Co-IP) and western blot analysis
Co-IP and western blot analysis were performed as described previously (2,33). Antibodies used for Co-IPs were anti-GR (Santa Cruz, sc-8992), anti-CAR (Santa Cruz, sc-13065) and anti-HNF4{alpha} antibody (Santa Cruz, sc-8987). Control Co-IPs were carried out using rabbit IgG (Santa Cruz, sc-2027). In western blot assays, the following antibodies were used at dilution of 1:1000: anti-Flag M2 (Stratagene, #200472-21), anti-HA (12CA5) (Roche Molecular Biochemicals), anti-SMILE (Abcom, #ab28700), anti-GST (Santa Cruz, sc-33614) and anti-tubulin (Cell Signaling Technology, #2146) antibodies. In western blot analysis of immunoprecipitated proteins, conventional HRP conjugated anti-rabbit IgG was replaced with rabbit IgG TrueBlot (eBioscience, #18-8816) to eliminate signal interference by the immunoglobulin heavy and light chains.

In vivo GST pull-down assay
In vivo GST pull-down experiments were performed as described previously (2). In brief, 293T cells were transfected in 60 mm dishes with the indicated plasmids. Forty-eight hours after transfection, the whole-cell extracts were prepared and equal amounts of total protein were used for in vivo GST pull-down assays followed by western blot analysis.

Confocal microscopy
The confocal microscopy assays were carried out as described previously (2). In brief, the HeLa cells grown on gelatin-coated coverslips were transfected with indicated plasmids using Effectene transfection reagent (Qiagen) according to the manufacturer's instructions. Twelve hours after transfection, the cells were treated with or without 100 nM dexamethasone for 12 h followed by cell fixation and immunostaining. To detect HA-fusion proteins and nucleus, the cells were incubated with dye Alexa 594-conjugated anti-HA monoclonal antibody (1:500 dilution; Invitrogen) for 1 h at room temperature (25°C), washed three times in PBS, and incubated with 0.1 mg/ml of DAPI (Invitrogen) solution for 10 min. After three times washing with PBS, the cells were subjected to observation by confocal microscopy.

Preparation of recombinant adenovirus
The adenovirus-encoding human SMILE was described previously (2). The adenovirus-encoding rat HNF4{alpha} was constructed via the previously described method (2). In brief, the cDNA-encoding rat HNF4{alpha} was cloned into the KpnI/XbaI sites of the pAdTrack-CMV vector. The recombination of the AdTrack-CMV-rHNF4{alpha} (where rHNF4{alpha} is rat HNF4{alpha}) with adenoviral gene carrier vector was performed by transformation to pretransformed adEasy-BJ21-competent cells.

RNA interference
Knockdown of SMILE and histone deacetylases (HDACs) was performed using the pSuper vector system (2). 293T cells were transfected with siRNA constructs using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. siRNA-treated cells were subjected to reverse transcription-PCR (RT–PCR) or the second transfection as indicated in the figure legends.

RT–PCR analysis
Total RNA was isolated using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The mRNAs of SMILE, SHP, insulin-like growth factor-binding protein 1 (IGFBP1), CYP2B6 and cholesterol 7{alpha}-hydroxylase (CYP 7A1) were analyzed by RT–PCR as previously described (2), and the mRNA levels of β-actin served as an internal control for RT–PCR. The RT–PCR primers are provided in Supplementary Table 1.

Chromatin immunoprecipitation (ChIP) Assay
ChIP assay was performed as previously described (32). In brief, HepG2 cells seeded into 35 mm culture dishes were treated as indicated in the figure legends and then the cells were fixed with 1% formaldehyde, washed with ice-cold PBS, harvested and sonicated. The soluble chromatin was then subjected to immunoprecipitation using anti-GR, anti-CAR, anti-HNF4{alpha}, anti-SMILE (Santa Cruz, sc-49329), or acetyl-histone H3 (Lys9) antibody (Cell Signaling Technology, #9671) followed by protein A agarose/salmon sperm DNA (Upstate). DNA was recovered via phenol/chloroform extraction and amplified by PCR for 30–35 cycles using specific primer sets for the indicated specific regions of IGFBP1, CYP2B6 and CYP7A1 genes. The PCR primers for ChIP assay are provided in Supplementary Table 2.

Statistical analysis
Student's t-test was performed using GraphPad Prism version 3.0 for Windows and results were considered to be statistically significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
SMILE inhibits the transactivation of nuclear receptor GR, CAR and HNF4{alpha}
Previously, we have reported that SMILE regulates orphan NR small heterodimer partner (SHP)-repressed ER transactivation through direct interaction with SHP (2). To investigate whether SMILE interacts with other NRs, yeast two-hybrid interaction assays were performed. Of great interest, SMILE was determined to interact with many NRs, including GR, TR{alpha}, CAR, SF-1, ERR{alpha}, ERRβ, ERR{gamma}, HNF4{alpha} and Nur77 (Supplementary Table 3). For detailed study, GR, CAR and HNF4{alpha} were selected as a representative of classical endocrine receptors, adopted orphan receptors, and orphan receptors (8), respectively.

We have previously demonstrated that wild-type SMILE generates two isoforms, SMILE long form (SMILE-L) and SMILE short form (SMILE-S), which can be produced by the mutants SMILE-83Leu and SMILE-1Phe, respectively (2). To determine whether these isoforms can regulate the transactivation of GR, CAR and HNF4{alpha}, transient transfection was performed in 293T cells. Overexpression of wild-type SMILE dose-dependently repressed dexamethasone-stimulated GR transactivation (Figure 1A), as well as CAR and HNF4{alpha} transactivation (Figure 1B and C). Furthermore, overexpression of SMILE-L or SMILE-S through the aforementioned SMILE mutants evidenced similar inhibitory effects on the NRs (Figure 1A–C). However, SMILE did not show any significant effect on PPAR{gamma}-mediated transactivation (Figure 1D). Western blot analysis demonstrated that the overexpression of wild-type SMILE, SMILE-L or SMILE-S alone did not significantly change the protein expression of HA-GR and HA-CAR (Figure 1E and F). Taken together, these results suggest that both SMILE isoforms down-regulate GR, CAR and HNF4{alpha} transactivation. Since SMILE-L and SMILE-S show similar effects on those NRs, and SMILE-L is the major isoform in tested cell lines and tissues (2), we have focused on wild-type SMILE-generated SMILE-L (SMILE) for further investigations.


Figure 1
View larger version (53K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. SMILE represses GR-, CAR- and HNF4{alpha} transactivation. Reporter assays (A–D) were performed as described in the Materials and methods section. HEK293T (293T) cells were cotransfected with 0.1 µg of pcDNA3-HA-GR (A), pcDNA3-HA-CAR (B), pcDNA3-HA-HNF4{alpha} (C) or, pcDNA3-HA-PPAR{gamma} (D) and 0.1 µg of MMTV-Luc (A), (NR1)5-Luc (B), (HNF4)8-Luc (C) or, PPRE-Luc (D) luciferase reporter vectors, 0.1 µg of pCMV-β-gal as internal control, together with indicated amounts of plasmids expressing wild-type SMILE, SMILE-L (SMILE-83Leu) and SMILE-S (SMILE-1Phe). Twenty-four hours after transfection, the cells were treated with or without 100 nM of dexamethasone (Dex) (A) or rosiglitazone (Rosi) (D) as indicated for 24 h prior to the measurement luciferase activity. The effects of overexpressed SMILE on the protein levels of HA-GR (E) and HA-CAR (F). 293T cells were cotransfected with various plasmids as indicated. Fifty microgram of cellular extracts from the transient transfection assay were subjected to western blot analysis. The proteins of HA-GR, HA-CAR, SMILE and tubulin were detected as described in the Materials and methods section. wt, wild-type.

 
Knockdown of SMILE gene expression up-regulates GR, CAR and HNF4{alpha} transactivation
To determine whether endogenous SMILE negatively regulates GR, CAR and HNF4{alpha} transactivation, we investigated these NRs-mediated transcriptional activities after individually knocking down the expression of SMILE and SHP in HepG2 cells. As shown in Figure 2A, siSMILE-II (siSM-II) efficiently knocked down the mRNA levels of SMILE, whereas siSMILE-I (siSM-I) did not show any significant effect, and siSHP efficiently silenced the gene expression of SHP, which is a well-known corepressor of GR, CAR and HNF4{alpha} (2). In the reporter assay, knockdown of SMILE gene expression through siSMILE-II increased GR-, CAR- and HNF4{alpha}-mediated transactivation by 65–100%, which is similar to the effect of positive control siSHP (Figure 2B–D). Collectively, these results indicate that endogenous SMILE is a functional corepressor of receptor GR, CAR and HNF4{alpha}.


Figure 2
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. siSMILE increases GR-, CAR- and HNF4{alpha} transactivation. (A) Effect of siRNAs for SMILE or SHP on the expression of SMILE and SHP. HepG2 cells were transfected with pSUPER siSMILE-I (siSM#1), siSMILE-II (siSM#2), siSHP or pSUPER [control (con)], and after 72 h the total RNA was isolated. The mRNA levels of SMILE and SHP were measured via RT–PCR analysis, with β-actin shown as a control. The data shown are representative of at least three independent experiments. (B–D) SMILE siRNA induces GR-, CAR- and HNF4{alpha}-mediated transactivation in HepG2 cells. HepG2 cells were transfected with pSUPER, pSUPER siSMILE-I (siSM#1), or pSUPER siSMILE-II (siSM#2). After 24 h, the cells were cotransfected with expression vector for GR (B), CAR (C) or HNF4{alpha} (D), and MMTV-Luc (B), (NR1)5-Luc (C) or (HNF4)8-Luc (D) luciferase reporter vectors, together with pCMV-β-gal as internal control. Twenty-four hours after transfection, the cells in (B) were treated for 24 h with or without 100 nM Dex prior to the measurement of luciferase activity. The mean and standard deviation (n = 3) of a representative experiment are shown. *P < 0.05; **P < 0.01, using Student's t-test.

 
SMILE interacts with GR, CAR and HNF4{alpha} in vitro and in vivo
To determine whether SMILE inhibits GR, CAR and HNF4{alpha} transactivation through protein–protein interaction, in vitro and in vivo GST pull-down experiments were performed. For the in vitro GST pull-down assays, bacteria-expressed GST only, or GST-SMILE proteins were incubated with in vitro translated 35S-labeled GR, CAR, or HNF4{alpha}. We found that 35S-labeled GR was able to bind to GST-tagged SMILE in the presence of dexamethasone, but not in the absence of the ligand (Figure 3G, upper panel), indicating that SMILE can interact with GR in a ligand-dependent manner. Moreover, 35S-labeled CAR, and HNF4{alpha} were also observed to bind to GST-SMILE (Figure 3G, lower panel). These results suggest that SMILE can interact with GR, CAR and HNF4{alpha} in vitro. For the in vivo GST pull-down assays, mammalian expression vectors encoding either pEBG (GST) alone or pEBG-SMILE (GST-SMILE) together with indicated pcDNA3-HA-GR, pcDNA-Flag-mCAR, or pcDNA3-HA-HNF4{alpha} were cotransfected into 293T cells. As shown in Figure 3A, HA-GR was detected in the coprecipitate only in the presence of its ligand when coexpressed with GST-SMILE but not with GST alone. The expression levels of GST, GST-SMILE and HA-GR were confirmed by western blot analysis (Figure 3A, middle and bottom panels, respectively). These results demonstrate that ectopically expressed SMILE interacts with exogenous GR in a ligand-dependent manner in 293T cells. Similarly, interactions of exogenous SMILE with CAR (Figure 3B) and HNF4{alpha} (Figure 3C) were verified using in vivo GST pull-down assays. To further examine whether endogenous SMILE and these NRs can interact in vivo, co-immunoprecipitation assays were performed. Endogenous SMILE proteins were found to be co-precipitated with GR in a ligand-dependent manner (Figure 3D), while with CAR in a ligand-independent manner (Figure 3E). In addition, endogenous SMILE was co-precipitated with endogenous HNF4{alpha} (Figure 3F), confirming the interaction between that endogenous SMILE and the NRs. Collectively, these results indicate that SMILE can interact with GR, CAR and HNF4{alpha} both in vitro and in vivo.


Figure 3
View larger version (66K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Interactions and colocalizations of SMILE with NRs. (A–C) In vivo interactions of exogenous GR (A), CAR(B) and HNF4{alpha} (C) with exogenous SMILE. 293T cells were cotransfected with expression vectors for HA-GR (A), Flag-mCAR (B), or HA-HNF4{alpha} (C) with pEBG-SMILE (GST-SMILE) or pEBG alone (GST). The in vivo GST pull-down assays were performed in the presence or absence of the GR ligand Dex (100 nM) as indicated (A). The complex formation (top panel in A–C, GST puri.) and the amount of HA-GR, Flag-mCAR or HA-HNF4{alpha} used for the in vivo binding assay (bottom panel in A–C, Lysate) were determined via western blot using an anti-HA or anti-Flag antibody. The same blot was stripped and reprobed with an anti-GST antibody (middle panel in A–C) to confirm the expression levels of the GST fusion protein (GST-SMILE) and the GST control (GST). In vivo interactions of endogenous GR (D), CAR (E) and HNF4{alpha} (F) with endogenous SMILE. Co-immunoprecipitation assays were performed using cell extract from HepG2 cells using indicated antibodies in the presence or absence of 100 nM Dex or 250 nM of TCPOBOP. Endogenous SMILE was immunoprecipitated with GR, CAR and HNF4{alpha} (upper panels). The proteins in the cell lysates (middle and lower panels) were analyzed with western blot analysis using indicated antibodies. (G) In vitro GST pull-down assays. Upper panel, 35S-radiolabeled GR protein was incubated with GST, or GST-SMILE fusion proteins in the presence of 100 nM Dex or vehicle (DMSO). Lower panel: 35S-radiolabeled HNF4{alpha}, or CAR proteins were incubated with GST, or GST-SMILE fusion proteins. The input lane represents 10% of the total volume of in vitro-translated proteins used for binding assay. Protein interactions were detected via autoradiography. (H–J) Co-localizations of SMILE with NRs. Hela cells grown on coverslips on 12-well plates were transfected with 0.1 µg of expression vectors encoding GFP-SMILE and HA-GR (H), HA-CAR (I) or HA-HNF4{alpha} (J). Twelve hours after transfection, the cells (H) were treated with 100 nM Dex for 12 h. For the immunofluorescence of fixed cells, the HA-fusion proteins were detected with dye Alexa 594-conjugated anti-HA monoclonal antibody. The cell images were captured under 400 x magnifications. The data shown are representative of at least three independent experiments.

 
To examine whether SMILE and its binding partners (GR, CAR, or HNF4{alpha}) are colocalized to the same subcellular compartments, confocal microscopic studies were performed. HeLa cells were cotransfected with the expression plasmids pEGFP-SMILE along with pcDNA3-HA-GR, or pcDNA3-HA-mCAR, or pcDNA3-HA-HNF4{alpha}, stained with dye Alexa 594-conjugated anti-HA antibody and DAPI, and analyzed via confocal microscopy. As shown in Figure 3H, GFP-SMILE was predominantly localized within the nucleus, and was also weakly detected in the cytoplasm, which was consistent with the results of our previous study (2). In the presence of ligand, GR was detected predominantly in the nucleus (Figure 3H). CAR (Figure 3I) and HNF4{alpha} (Figure 3J) were also detected mainly in the nucleus. The merged images indicated that SMILE and GR, CAR, or HNF4{alpha} were colocalized to the nucleus (Figure 3H–J). Collectively, these data reveal that SMILE interacts and colocalizes with receptor GR, CAR and HNF4{alpha} in vivo.

Dimerization of SMILE is not required for its repressive function
It was reported that SMILE can homodimerize through the leucine zipper region like other bZIP proteins (1,34) and this homodimerization is important for the function of leucine zipper protein (35). To determine whether the homodimerization is essential for the repressive function of SMILE, site-directed mutational analysis and in vivo GST pull-down assays were performed. We generated a mutant SMILE [SMILE-L (239–267) V], in which five consecutive leucine residues in the leucine zipper region were mutated to valine (Figure 4A). Next, the possibility of homodimer formation was investigated via in vivo GST pull-down assays. As expected, Flag-SMILE was shown to be coprecipitated with GST-SMILE. However, Flag-SMILE-L (239–267)V was not coprecipitated with GST-SMILE-L (239–267)V (Figure 4B, upper panel), although the mutant proteins were expressed to comparable levels as the wild-type SMILE (Figure 4B, middle and lower panel). These results indicate that SMILE is capable of forming homodimers, and the mutation of the five consecutive leucine residues in the leucine zipper region destroyed the homodimerization. Next, the functional effects of the mutation were assessed using reporter assays. SMILE-L (239–267)V repressed GR- and CAR-mediated transcriptional activity as profoundly as the wild-type SMILE (Figure 4C and D). Although the repression of SMILE-L (239–267)V on HNF4{alpha} was not so strong as wild-type SMILE, SMILE-L (239–267)V still significantly inhibited HNF4{alpha} (Figure 4E). Collectively, these results indicate that SMILE homodimerization is not essential for its repressive function.


Figure 4
View larger version (29K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Homodimerization of SMILE is not essential for the repressive function. (A) Structure of human SMILE. The basic region and leucine zipper domains are shown. The leucine zipper mutant SMILE-L (239–267)V indicates the leucine residues between positions 239 and 267 were mutated to valine, as indicated by the arrows. The numbers in the figure indicate the amino-acid residues. (B) In vivo homodimerization possibility analysis of wt SMILE and SMILE-L (239–267)V. 293T cells were cotransfected with expression vectors for Flag-SMILE, Flag-SMILE-L (239–267)V with pEBG-SMILE (GST-SMILE wt) or pEBG-SMILE-L (239–267)V or pEBG alone (GST) as indicated. The complex formation (top panel, GST puri.) and the amount of Flag-SMILE, Flag-SMILE-L (239–267)V used for the in vivo-binding assay (bottom panel, Lysate) were determined by western blot using anti-Flag antibody. The same blot was stripped and reprobed with an anti-GST antibody (middle panel) to verify the expression levels of the GST fusion proteins (GST-SMILE) and the GST control (GST). (C–E) The effect of SMILE-L (239–267)V on GR, CAR and HNF4{alpha}-mediated transactivation. HepG2 cells were cotransfected with expression vector for GR (C), CAR (D), or HNF4{alpha} (E) and MMTV-Luc (C), (NR1)5-Luc (D), or (HNF4)8-Luc (E) luciferase reporter vectors, pCMV-β-gal as internal control, together with expression vectors for wt SMILE or SMILE-L (239–267)V as indicated. Twenty-four hours after transfection, the cells in (C) were treated with 100 nM Dex for 24 h. Forty-eight hours after tranfection, luciferase activity was measured. wt, wild type. The mean and standard deviation (n = 3) of a representative experiment are shown.

 
Interaction domain mapping of SMILE with GR, CAR and HNF4{alpha}
To identify the interaction domain of SMILE with the NRs, a series of SMILE deletion fragments (Figure 5A) were cloned into in vivo GST vector and in vivo GST pull-down assays were performed. We found that the mutant GST-SMILE-N2 (1–202 aa) and GST-SMILE were associated strongly with GR, CAR and HNF4{alpha}, whereas the mutants GST-SMILE-N1 (1–112 aa), GST-SMILE-{Delta}202 (203–354 aa) and GST-SMILE-{Delta}268 (269–354 aa) were not significantly associated with the NRs (upper panel in Figure 5B–D). Moreover, all the GST SMILE fusion proteins and GR, CAR, HNF4{alpha} proteins were expressed properly (middle and lower panel in Figure 5B–D), indicating that the differences in the interactions between the SMILE mutants and the NRs are not the result of differences in protein expression. Taken together, these results indicate that the region spanning residues 113–202 of SMILE is responsible for the interactions with the NRs.


Figure 5
View larger version (43K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Interaction domain of SMILE. (A) Schematic representation of the structures of SMILE mutants. bZIP indicates the basic region leucine zipper domain. The numbers in the figure indicate the amino acid residues. (B–D) In vivo interaction assays between wt SMILE or SMILE mutants and GR (B), CAR (C) or HNF4{alpha} (D). 293T cells were cotransfected with expression vectors for HA-GR (B), Flag-CAR (C) or HA-HNF4{alpha} (D) with pEBG alone (GST) or pEBG-SMILE (GST-SMILE) fusions as indicated. The in vivo GST pull-down assays in (B) was performed in the presence or absence of GR ligand Dex (100 nM). The complex formation (top panel in B–D, GST puri.) and the amount of HA-GR, Flag-mCAR or HA-HNF4{alpha} used for the in vivo-binding assay (bottom panel in B–D, Lysate) were determined by western blot using an anti-HA or anti-Flag antibody. The same blot was stripped and reprobed with an anti-GST antibody (middle panel in B–D) to confirm the expression levels of the GST fusion proteins (GST-SMILE fusions) and the GST control (GST). wt, wild type. The data shown are representative of at least three independent experiments with similar results.

 
To identify the region of GR, CAR and HNF4{alpha} involved in the interactions with SMILE, in vitro GST pull-down experiments were performed using various GR (Figure 6A), mCAR (Figure 6C) and HNF4{alpha}-deletion constructs (Figure 6E). GST-SMILE was observed to bind to 35S-labeled full length GR in the presence of ligand (Figure 6B, upper panel), as well as 35S-labeled GR-LBD (532–783 aa) (Figure 6C, lower panel), but did not bind to GR-N (1–531 aa) (Figure 6B, middle panel), indicating the LBD of GR is important for the ligand-dependent interaction between GR and SMILE. In addition, deletion of AF2 domain abolished the interaction of CAR with SMILE (Figure 6D) indicating that the AF2 domain of CAR is essential for the interaction. In the case of HNF4{alpha}, GST-SMILE was capable of interacting with full length HNF4{alpha} and HNF4{alpha}CD (1–370 aa) (Figure 6F, upper and middle panel), which harbor the complete LBD domain, but was not able to interact with HNF4{alpha}{Delta}LBD (1–174 aa) (Figure 6F, lower panel), thereby indicating that the LBD domain of HNF4{alpha} is required for the interaction between HNF4{alpha} and SMILE. Collectively, these results suggest that the LBD/AF2 domain of GR, CAR and HNF4{alpha} are essential for the interactions with SMILE.


Figure 6
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. SMILE interacts with LBD/AF2 domain of the NRs. (A, C and E) Schematic representation of the structures of the GR (A), CAR (C) and HNF4{alpha} (E) mutants. AF1, activation function-1 domain; DBD, DNA-binding domain; LBD, ligand-binding domain; AF2, activation function-2 domain. (B) 35S-radiolabeled GR proteins were incubated with GST, or GST-SMILE fusion proteins in the presence of ligand Dex (100 nM) or vehicle (DMSO). (D and F) 35S-radiolabeled CAR (D) or HNF4{alpha} proteins (F) were incubated with GST, or GST-SMILE fusion proteins. The input lane represents 10% of the total volume of in vitro-translated proteins used for the binding assay. Protein interactions were detected via autoradiography. The data shown represent at least three independent experiments with similar results.

 
SMILE competes with coactivators
It has been well established that a host of coactivators, including PGC-1{alpha}, CBP/p300 and GRIP1, can interact with the LBD/AF2 region of NRs to form LBD-coactivator complexes and positively regulate NR-mediated transcription (25,36,37). The aforementioned results that SMILE interacts with the LBD/AF2 region of GR, HNF4{alpha} and CAR prompted us to determine whether SMILE could compete with coactivators. In the presence of ligand, overexpression of GRIP1 increased GR-stimulated transcriptional activity (Figure 7A), which is consistent with previous report (37), and overexpression of SMILE reduced the coactivation in a dose-dependent manner (Figure 7A). In a reciprocal experiment, the transfection of increasing quantities of GRIP1 expression vector induced a gradual release of SMILE repression on GR (Figure 7A). Interestingly, SMILE overexpression also reduced PGC-1{alpha}-enhanced HNF4{alpha}-, and CAR-stimulated transactivation in a dose-dependent fashion, and overexpression of PGC-1{alpha} recovered the inhibitory effect of SMILE on HN4{alpha} and CAR (Figure 7B and C). These results indicate that SMILE can compete with the coactivators GRIP1 and PGC-1{alpha} functionally in vivo. To further confirm the competition between SMILE and either GRIP1 or PGC-1{alpha}, in vitro GST pull-down assays were performed. In the presence of the ligand, increasing amounts of the cold competitor, HA-GRIP1, reduced the binding of 35S-methionine-labeled GR protein to GST-SMILE (Figure 7D, upper panel). Moreover, increasing amounts of the cold competitor, HA-PGC-1{alpha}, reduced the association of GST-SMILE with 35S-HNF4{alpha} and 35S-CAR (Figure 7D, lower panel). Taken together, these results indicate that coactivator competition is one mechanism underlying for the repression of GR, HN4{alpha} and CAR by SMIILE.


Figure 7
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7. SMILE competes with coactivators GRIP1 and PGC-1{alpha}. Reporter assays in (A–C) were performed as described in the Materials and methods section. The mean and standard deviation (n = 3) of a representative experiment are shown. HepG2 cells were cotransfected with 0.1 µg of indicated reporter plasmids, MMTV-luc (A), (NR1)5-luc (B), or (HNF4)8-Luc (C), and 0.1 µg of pcDNA3-HA-GR (A), pcDNA3-HA-mCAR (B) or pcDNA3-HA-HNF4{alpha} (C), together with the indicated quantities of pcDNA3-Flag-SMILE, pSG5-HA-GRIP1 (A) or pcDNA3-HA-PGC-1{alpha} (B and C). Twenty-four hours after transfection, the cells were treated with or without GR ligand Dex (100 nM) for 24 h prior to the measurement of luciferase activity. (D) In vitro competition between SMILE and GRIP1 or PGC-1{alpha}. 35S-radiolabeled GR (in the presence of 100 nM DEX), or CAR, or HNF4{alpha} proteins were incubated with GST, or GST-SMILE fusion proteins, together with an increasing amounts of unlabeled in vitro translated GRIP1 (0, 3, 6 or 12 µl, upper panel) or PGC-1{alpha} (0, 3, 6 or 12 µl, middle and lower panel) proteins. After pull-down, the beads were washed and the samples separated on a 12% SDS–PAGE gel and the protein interactions were detected via autoradiography. The data shown represent at least three independent experiments.

 
SMILE has intrinsic repressive activity
Many corepressors, including SHP (38,39), and RIP140 (40), were reported to inherently possess transcriptional repressive activity. To determine whether SMILE also has an intrinsic repressive function, the transcriptional activities of a set of Gal4-SMILE deletion constructs were investigated (Figure 8A). The reporter plasmid Gal4-tk-Luc, and indicated expression vectors encoding Gal4-DBD alone, Gal4-SMILE, or Gal4-SMILE deletions were cotransfected into 293T cells. As indicated in Figure 8B, Gal4-SMILE, Gal4-SMILE-{Delta}112 (113–354 aa), -{Delta}202 (203–354 aa) showed only ~10% of Gal4-DBD-stimulated reporter activity, and {Delta}268 (269–354 aa) showed only ~15% of the activity by Gal4-DBD. However, Gal4-SMILE-N1 (1–112 aa) and Gal4-SMILE-N2 (1–202 aa) displayed 2–3-fold activity of that by Gal4-DBD, and Gal4-SMILE-{Delta}NC (113–202 aa) displayed a comparable effect to Gal4-DBD. Moreover, all of the Gal4-fusions were expressed properly (Figure 8C), indicating the distinct reporter activities stimulated by the Gal4-SMILE fusions were not the consequence of different protein levels. Taken together, these results indicate that the SMILE N-terminus (1–112 aa) has intrinsic activation activity, whereas the C-terminus (203–354 aa) has intrinsic repression. As a whole, SMILE showed repression activity, indicating the intrinsic repression derived from the C-terminus predominates.


Figure 8
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 8. Intrinsic repressive function of SMILE. (A) Schematic representation of wt SMILE and its deletion mutants fused in-frame to the yeast Gal4 DBD-(1–147 amino acid). bZIP, basic region leucine zipper domain. (B) 293T cells were cotransfected with the reporter plasmid Gal4-tk-Luc and the indicated expression vectors of Gal4-SMILE or Gal4-SMILE deletion mutants together with pCMV-β-gal vector. Forty-eight hours after transfection, the luciferase activity was measured as described in the Materials and methods section. The normalized luciferase activity values are shown as the percentage of the Gal4-tk-Luc reporter activity stimulated by Gal4-DBD. The mean and standard deviation (n = 3) of a representative experiment are shown. (C) Western blot analysis of Gal4-SMILE and Gal4-SMILE deletion mutants. 293T cells were transfected with the indicated Gal-SMILE chimeras. Whole cell extracts (50 µg) were analyzed via western blot using anti-Gal4 rabbit polyclonal antibody.

 
SMILE recruits HDACs in a NR-specific manner
It has been reported previously that the recruitment of HDAC contributes to the intrinsic repressive function of corepressors, including RIP140 (41), and SHP (42). To determine whether SMILE could also recruit HDACs, the effect of the HDAC-specific inhibitor trichostatin A (TSA) on SMILE-mediated repression was examined. The results showed that TSA treatment partially but significantly reversed the repression of GR and HNF4{alpha} by SMILE (Figure 9A and B), whereas TSA treatment did not significantly affect the repression of CAR (Figure 9C). These results demonstrate that the recruitment of HDACs is required for the inhibition of SMILE on GR and HNF4{alpha}, but not required for the inhibition on CAR, indicating that the recruitment of HDACs by SMILE might be NR-specific.


Figure 9
View larger version (50K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 9. SMILE recruits HDACs. (A–C) TSA releases SMILE-mediated repression on GR and HNF4{alpha}. HepG2 cells were cotransfected with 0.1 µg of reporter plasmids, MMTV-luc (A), (HNF4)8-luc (B) or (NR1)5-Luc (C), and 0.1 µg of pcDNA3-HA-GR (A), pcDNA3-HA-HNF4{alpha} (B) or pcDNA3-HA-mCAR (C), together with or without 0.2 µg of pcDNA3-Flag-SMILE as indicated. Twenty-four hours after transfection, the cells were treated for 12 h with or without 100 nM of Dex. Then the cells were treated with indicated concentration of TSA for 12 h in the absence or presence of Dex (100 nM). In vivo interactions of HDAC1 (D), HDAC3 (E) and HDAC4 (F) with SMILE. 293T cells were cotransfected with expression vectors for Flag-HDAC1, or HA-HDAC3 or Flag-HDAC4 with pEBG-SMILE (GST-SMILE) or pEBG alone (GST). The complex formation (top panel in D–F, GST puri.) and the amount of Flag- or HA-tagged HDAC fusion proteins used for the in vivo-binding assay (bottom panel in D–F, Lysate) were determined using anti-Flag or anti-HA antibody, respecitively. The same blot was stripped and reprobed with an anti-GST antibody (middle panel in D–F) to confirm the expression levels of the GST fusion protein (GST-SMILE) and the GST control (GST). (G and H) The effect of HDAC siRNAs on the inhibition of GR- and HNF4{alpha}-mediated transactivation by SMILE. HepG2 cells were transfected with pSuper siHDAC1, siHDAC3, or siHDAC4 as indicated and after 24 h, the cells were cotransfected with 0.1 µg of indicated reporter plasmids, MMTV-luc (G), or (HNF4)8-Luc (H), and 0.1 µg of pcDNA3-HA-GR (G) or pcDNA3-HA-HNF4{alpha} (H), together with 0.1 µg of pcDNA3-Flag-SMILE and after 24 h, the cells were treated with or without 100 nM of Dex for 24 h prior to the measurement of luciferase activity. The mean and standard deviation (n = 3) of a representative experiment are shown. *P < 0.05, using Student's t-test. (I) Effect of siRNAs on the expression of HDACs. HepG2 cells were transfected with pSUPER siHDAC1, siHDAC3, siHDAC4 or pSUPER [control (con)], and after 72 h the total RNA was isolated. The mRNA levels of HDACs were measured via RT–PCR analysis, with β-actin shown as a control. The results are representative of three experiments.

 
To further determine the HDACs involved in the repression of GR and HNF4{alpha} by SMILE, the potential interactions between HDACs (HDAC1, HDAC2, HDAC3, HDAC4, HDAC5 and HDAC6) and SMILE were investigated via in vivo GST pull-down assays. HDAC1 (Figure 9D), and HDAC3 (Figure 9E), as well as HDAC4 (Figure 9F) were detected in the coprecipitate only when coexpressed with the GST-SMILE but not with GST alone. The expression levels of GST, GST-SMILE, Flag-HDAC1, HA-HDAC3 and Flag-HDAC4 were confirmed via western blot analysis (middle and bottom panel in Figure 9D–F). However, Flag-HDAC2, Flag-HDAC5 and Flag-HDAC6 were not detected in the coprecipitate (data not shown). These results demonstrate that SMILE specifically interacts with HDAC1, HDAC3 and HDAC4 in vivo, thereby indicating that the recruitment of HDAC1, HDAC3 and HDAC4 may play a role in the SMILE-mediated repression of GR and HNF4{alpha}.

To further investigate whether HDAC1, HDAC3 and HDAC4 are also involved in the repressive effect of SMILE on GR and HNF4{alpha}, reporter assays combined with siRNA-mediated knockdown of the HDACs gene expression were performed. As shown in Figure 9G, siHDAC3 or siHDAC4 alone induced a slight reduction in the repression of GR by SMILE, but siHDAC1 exerted no detectable effects. Moreover, the combination of siHDAC3 and siHDAC4 additively and significantly attenuated the repression. In the case of HNF4{alpha}, only the combination of siHDAC1, siHDAC3 and siHDAC4 significantly attenuated the repression of HNF4{alpha} by SMILE (Figure 9H). In addition, all the siRNAs for HDAC1, HDAC3, or HDAC4 were demonstrated to knockdown the specific HDAC gene expression effectively (Figure 9I). Taken together, these results indicate that HDAC3 and HDAC4 contribute to the inhibition of GR by SMILE, and HDAC1, HDAC3 and HDAC4 contribute to the repression of HNF4{alpha} by SMILE.

Adenovirus-mediated overexpression of SMILE down-regulates the expression of GR, CAR and HNF4{alpha} target genes
Next, we performed ChIP assays to determine whether SMILE can associate with the NRs on the promoter of the IGFBP1, CYP2B6 and CYP7A1 genes, which are known targets of GR, CAR and HNF4{alpha}, respectively (11,12,17). As shown in Figure 10A, low levels of GR and SMILE were associated on IGFBP1 promoter in the absence of dexamethasone (upper panel, lanes 5 and 9). We observed an increased occupancy of GR after 1 h of dexamethasone treatment (upper panel of Figure 10A, compare lanes 5 and 6), whereas the occupancy of SMILE significantly increased after 2 h of dexamethasone treatment (upper panel of Figure 10A, compare lanes 9 and 11). On the promoter of CYP2B6, the occupancy of CAR did not significantly changed upon the treatment of CAR agonist TCPOBOP (upper panel of Figure 10B, compare lanes 6–7 to lane 5), whereas the association of SMILE was increased after 12 h TCPOBOP treatment (upper panel of Figure 10B, compare lanes 9 and 11). On the promoter of CYP7A1, the occupancy of HNF4{alpha} was significantly increased after adenovirus (Ad)-mediated overexpression of HNF4{alpha} (upper panel of Figure 10C, compare lanes 6–7 to lane 5), whereas the occupancy of SMILE increased after 24 h of Ad-HNF4{alpha} infection (upper panel of Figure 10C, compare lanes 9 and 11). However, no recruitment was observed in the nonregulatory regions of target gene promoters (Figure 10, lower panels of A–C, see lanes 5–12). These results indicate that SMILE dynamically forms complex with GR, CAR, or HNF4{alpha} on their target gene promoters.


Figure 10
View larger version (62K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 10. SMILE down-regulates the transcription of IGFBP1, CYP2B6 and CYP7A1 gene. (A–C) The recruitment of SMILE on IGFBP-1, CYP2B6 and CYP7A1 promoters is associated with histone deacetlyation. (A and B) HepG2 cells were stimulated with or without 100 nM of Dex (A) or 250 nM of TCPOBOP (B) in the presence or absence of 300 µM TSA as indicated. (C) HepG2 cells were infected with or without adenovirus-HNF4{alpha} (Ad-HNF4{alpha}) in the presence or absence of 300 µM TSA as indicated. Cell lysates from the treated HepG2 cells were then collected for Chip assays. Chromotian fragments were prepared and immumoprecipitated with the indicated specific antibodies. DNA fragments covering a GRE on IGFBP1 promoter (A, upper panel), or CAR-binding site on CYP2B6 promoter (B, upper panel), BARE-I and BARE-II element on CYP7A1 promoter (C, upper panel) were PCR-amplified as described in the Materials and methods section. The lower panels in A–C indicate the amplification of the control regions. (D–F) Shown are RT–PCR carried out using PCR primers for SMILE, IGFBP1, CYP2B6, CYP7A1, HNF4{alpha} and β-actin, using total RNA prepared from HepG2 cells infected with indicated adenovirus vector (Ad-null, Ad-SMILE and Ad-HNF4{alpha}) (100 pfu/cell). After 24 h of infection, the cells were stimulated with vehicle (DMSO) or 100 nM of Dex (D) for 2 h or 250 nM of TCPOBOP (E) for 24 h before total RNA was isolated. Data shown are representative of three experiments.

 
Since SMILE interacted with HDACs (Figure 9), we assume that the recruitment of SMILE to the target gene promoters may lead to histone deacetylation. To test this hypothesis, ChIP assays were performed using antibodies against acetylated lysine 9 of histone H3. One hour dexamethasone treatment increased the acetylation of histone H3 on the GR-binding region of IGFBP1 promoter, whereas theacetylation decreased to basal level after 2 h treatment of dexamethasone, which coincides with the timing of increased SMILE association. Interestingly, the decline of the acetylated histone H3 was recovered by HDAC inhibitor (TSA) treatment (upper panel of Figure 10A, see lanes 9–16). Moreover, 2 h TCPOBOP treatment resulted in increased acetylation of histone H3 on the CAR-binding region of CYP2B6 promoter and the acetylation diminished after 12 h TCPOBOP treatment. Although this deacetylation of histone H3 occured in line with the recruitment of SMILE, it did not change upon TSA treatment (upper panel of Figure 10B, see lanes 9–16). In addition, acetylated histone H3 on the HNF4{alpha}-binding region of CYP7A1 promoter increased 12 h after Ad-HNF4{alpha} infection and reduced to basal level 24 h after Ad-HNF4{alpha} infection, which also coincides with the recruitment of SMILE. Similar to the case of IGFBP1 promoter, the decrease in acetylated histone H3 on CYP7A1 promoter was prevented by the treatment of TSA (upper panel of Figure 10C, see lanes 9–16). Collectively, these results demonstrate that the recruitment of SMILE on these target gene promoters is associated with chromatin histone deacetylation.

As the aforementioned data show that SMILE is able to inhibit the transactivation of GR, CAR and HNF4{alpha}, and these three NRs form complex with SMILE on IGFBP1, CYP2B6 and CYP7A1 promoters, respectively, we speculated that SMILE may repress IGFBP1, CYP2B6 and CYP7A1 gene expression. As expected, the overexpression of SMILE in HepG2 cells using adenovirus vector markedly reduced dexamethasone-induced as well as the basal mRNA levels of IGFBP1 (Figure 10D, compare lane 4 to lane 3 and lane 2 to lane 1). Moreover, SMILE overexpression blocked CAR agonist TCPOBOP-mediated increase in CYP2B6 mRNA levels (Figure 10E, compare lane 4 to lane 3). In addition, SMILE overexpression also inhibited the basal and Ad-HNF4{alpha}-mediated increase in CYP7A1 mRNA levels (Figure 10F, compare lane 2 to 1 and lane 4 to 3). Taken together, these results reveal that SMILE is capable of down-regulating GR, CAR and HNF4{alpha} target gene expression.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 
Previous results have demonstrated that the bZIP protein SMILE plays an important role in repressing the replication of the herpes simplex virus (1,3) and serves as a coregulator in ER signaling (2). The results presented in this study extend the role of SMILE in NR signaling. SMILE inhibited GR-, HNF4{alpha}- and CAR-mediated transcriptional activity through direct binding to the LBD/AF2 domain of the NRs. Moreover, the knockdown of SMILE gene expression increased the GR, HNF4{alpha} and CAR transactivation. Furthermore, the overexpression of SMILE via adenovirus vector inhibited the transcription of the NRs’ target genes, including IGFBP-1, CYP2B6 and CYP7A1. In addition, SMILE also inhibited the transactivation by receptor LXR, FXR, Nur77 and ERR{gamma} through direct interactions (data not shown). These findings indicate that SMILE may be an important modulator of NR signaling.

We have investigated the roles of potential functional domains of SMILE for its repressive function, including the leucine zipper motif (1), the HCF-binding motif (HBM) (1,3,5) and the LXXLL motifs (NR boxes) (25,43). The leucine zipper region is known to be essential for the dimerization and functions of b-zip proteins (44). For instance, the leucine zipper of cyclic AMP response element-binding (CREB) protein is required for the dimerization and transcriptional activation (35). By way of contrast, our findings support the notion that the bZIP region of SMILE is required for the homodimerization, but is not essential for the repressive effect of SMILE on GR and CAR (Figure 4). It has been reported that Jun dimerization protein 2 (JDP-2) functions as a progesterone receptor (PR) coactivator through direct interaction via the DBD of PR and the bZIP region of JDP-2 (45). However, the domain-mapping results have demonstrated that the bZIP region of SMILE is not involved in the interactions with GR, CAR and HNF4{alpha} (Figure 5). Although HBM-mediated association of SMILE with HCF is required for SMILE to repress CREB3 (5), our reporter assay results have shown that wild-type SMILE and HBM-defective SMILE mutant (Y306A), which was demonstrated not able to interact with HCF (1), have similar inhibitory effect on GR, CAR and HNF4{alpha} (Supplementary Figure 1), indicating the repression of the NRs by SMILE is independent of HBM.

LXXLL motif is commonly found in NR coregultors and has been reported to be important for coregulators function through interaction with the LBD/AF2 domain of NRs (25,43). The results of domain-mapping analysis manifests that SMILE binds to the LBD/AF2 domain of GR, CAR and HNF4{alpha} through the region spanning residues 113–202, which contain a LXXLL motif. Surprisingly, we found that the repressive effects of SMILE on GR, CAR and HNF4{alpha} were not significantly changed by single mutation or combinatorial mutation of four LXXLL motifs (Supplementary Figure 2), indicating that LXXLL motifs are not essential for the interactions and repressive effects of SMILE in the cases of GR, CAR and HNF4{alpha}. Interestingly, this LXXLL-independent interaction was also observed between proline-rich nuclear receptor coregulatory protein (PNRC) and LBD of ER{alpha} (46). In addition of using LXXLL motifs to interact with NRs, corepressor RIP140 also uses its C-terminus, which contains no LXXLL motifs, to interact with LBD of NRs (40). However, it remains to be determined whether the LXXLL motifs are also dispensible for the repressive effect of SMILE on other NRs, such as Nur77, LXR and FXR.

We have recently reported that SMILE functions as a coregulator in ER signaling in association with SHP. The regulation of ER by SMILE depends on the existence of SHP in breast cancer MCF-7 cells (2). In contrast, the results of our siRNA knockdown experiments indicate that SHP is not involved in the SMILE-mediated repression of GR, CAR and HNF4{alpha} (data not shown). In our previous study, SMILE regulates the inhibition of ER by SHP in a cell-type specific manner (2). However, the repression of GR, CAR and HNF4{alpha} by SMILE is not cell-type specific, since similar repressive effects were observed in 293T, HepG2 and HeLa cells (data not shown).

Our results suggest that multiple mechanisms are involved in SMILE-mediated repression. One such mechanism could be competition with coactivators such as GRIP and PGC-1{alpha}, which is a common mechanism among certain NR corepressors, including SHP (31), DAX-1 (29), RIP140 (43) and the ligand-dependent corepressor (LCoR) (47). Interestingly, besides coactivator competition, SMILE has an intrinsic repressive function, like the corepressors SHP (42) and RIP140 (41). Moreover, we found that SMILE specifically interacts with HDAC1, HDAC3 and HDAC4. The inhibition of HDAC activity using the HDAC inhibitor TSA, or the knockdown of the HDACs gene expression through siRNA partially released the repression of GR and HNF4{alpha} by SMILE. In contrast, TSA showed little effect on the repression of CAR by SMILE, indicating HDAC-dependent and -independent mechanism of repression. Consistently, our ChIP assay results also evidenced that TSA was able to prevent SMILE-associated deacetylation of histone H3 on GR and HNF4{alpha} target gene promoters, but not on CAR target gene promoter. Of note, the TSA-sensitive and -insensitive actions of SMILE are similar to several other corepessors, including RIP140 (41) and LCoR (47). In addition, HDAC1, HDAC3 and HDAC4 are required for the repression of HNF4{alpha} by SMILE, whereas HDAC1 is not essential for the repression of GR, indicating that SMILE associations with HDACs exhibits promoter specificity. Similar phenomenon has been reported with the corepressors NCoR and SMRT (26).

It is worth noting that the inhibition of DNA binding is one of the common repression mechanisms utilized by certain corepressors. For instance, this mechanism underlies the inhibition of TR and GR by tumor suppressor p53 (48,49), and the inhibition of hepatic nuclear factor-3 (HNF3) family by the corepressor SHP (30). However, our results indicate that the inhibition of DNA binding is not involved in the repression of GR, CAR, and HNF4{alpha} by SMILE, as the recruitment of SMILE exerted no detectable effect on the binding of the NRs to the promoters of IGFBP1, CYP2B6 and CYP7A1 (Figure 10A–C). Whether this mechanism is involved in the inhibitory effect of SMILE on other NRs, including Nur77, LXR and FXR, still needs to be clarified.

GR, CAR and HNF4{alpha} are crucial for liver function, including the regulation and processing of glucose, lipids, amino acids and drug metabolism, as well as bile acid homeostasis (14,15,50). Therefore, the repression of their transcriptional activity by SMILE indicates that SMILE may function as a negative coregulator in the aforementioned physiological processes. It has been reported that as integrators of various biological processes, several transcriptional coregulators are regulated by distinct nutritional and hormonal signals (51). For example, activation of cAMP signaling by fasting induces the coactivator PGC-1{alpha} expression in hepatocytes, whereas the activation of insulin-signaling pathway by refeeding exhibits quite opposite effect (51). Increased bile acid levels switch on the feedback pathway of bile acid synthesis through induction of the corepressor SHP (52). Therefore, it would be necessary to study the regulation of SMILE gene expression by diverse physiological settings and intracellular signaling pathways, which is currently under investigation. Moreover, to better understand the function of SMILE in those aforementioned physiological processes, the SMILE knockout and transgenic animal model will be useful. In addition, the identification of more SMILE-interacting proteins and the elucidation of SMILE crystal structure will be helpful to illuminate the detailed mechanism of SMILE-mediated repression.

In summary, we have identified that SMILE represses GR-, CAR- and HNF4{alpha}-mediated transactivation through direct interaction. At least two mechanisms are involved in SMILE-mediated repression of the NRs, competition with coactivators, and active repression through the recruitment of HDACs. Taken together, these observations indicate that SMILE is novel corepressor and may play an important role in NR signaling.


   SUPPLEMENTARY DATA
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
FUNDING
 REFERENCES
 
Supplementary Data are available at NAR Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
REFERENCES
 
National Research Laboratory grant (ROA-2005-000-10047-0) and the Korea Research Foundation grant (KRF-2006-005-J03003). Funding for open access charge: Brain Korea 21 programme.

Conflict of interest statement. None declared.


   ACKNOWLEDGEMENTS
 
We thank Drs Richard W. Hanson, Keesook Lee, Changsoo Kim, Hee-Sae Park and Eungseok Kim for helpful discussions and comments on the project. We would like to acknowledge Dipanjan Chanda and Dr Seok-Yong Choi for critical reading of the manuscript.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUPPLEMENTARY DATA
 FUNDING
 REFERENCES
 

  1. Lu R, Misra V. Zhangfei: a second cellular protein interacts with herpes simplex virus accessory factor HCF in a manner similar to Luman and VP16. Nucleic Acids Res. (2000) 28:2446–2454.[Abstract/Free Full Text]

  2. Xie YB, Lee OH, Nedumaran B, Seong HA, Lee KM, Ha H, Lee IK, Yun Y, Choi HS. SMILE, a new orphan nuclear receptor SHP interacting protein, regulates SHP-repressed estrogen receptor transactivation. Biochem. J. (2008) 416:463–473.[CrossRef][Web of Science][Medline]

  3. Akhova O, Bainbridge M, Misra V. The neuronal host cell factor-binding protein Zhangfei inhibits herpes simplex virus replication. J. Virol. (2005) 79:14708–14718.[Abstract/Free Full Text]

  4. Hogan MR, Cockram GP, Lu R. Cooperative interaction of Zhangfei and ATF4 in transactivation of the cyclic AMP response element. FEBS Lett. (2006) 580:58–62.[CrossRef][Web of Science][Medline]

  5. Misra V, Rapin N, Akhova O, Bainbridge M, Korchinski P. Zhangfei is a potent and specific inhibitor of the host cell factor-binding transcription factor Luman. J. Biol. Chem. (2005) 280:15257–15266.[Abstract/Free Full Text]

  6. Glass CK. Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. Endocr. Rev. (1994) 15:391–407.[Abstract/Free Full Text]

  7. Giguère V. Orphan nuclear receptors: from gene to function. Endocr. Rev. (1999) 20:689–725.[Abstract/Free Full Text]

  8. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science (2001) 294:1866–1870. review.[Abstract/Free Full Text]

  9. Bresnick EH, Dalman FC, Sanchez ER, Pratt WB. Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor. J. Biol. Chem. (1989) 264:4992–4997.[Abstract/Free Full Text]

  10. Sengupta S, Wasylyk B. Ligand-dependent interaction of the glucocorticoid receptor with p53 enhances their degradation by Hdm2. Genes Dev. (2001) 15:2367–2380.[Abstract/Free Full Text]

  11. Suh DS, Rechler MM. Hepatocyte nuclear factor 1 and the glucocorticoid receptor synergistically activate transcription of the rat insulin-like growth factor binding protein-1 gene. Mol. Endocrinol. (1997) 11:1822–1831.[Abstract/Free Full Text]

  12. Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P, Negishi M. The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. J. Biol. Chem. (1999) 274:6043–6046.[Abstract/Free Full Text]

  13. Maglich JM, Watson J, McMillen PJ, Goodwin B, Willson TM, Moore JT. The nuclear receptor CAR is a regulator of thyroid hormone metabolism during caloric restriction. J. Biol. Chem. (2004) 279:19832–19838.[Abstract/Free Full Text]

  14. Timsit YE, Negishi M. CAR and PXR: the xenobiotic-sensing receptors. Steroids (2007) 72:231–246.[CrossRef][Web of Science][Medline]

  15. Watt AJ, Garrison WD, Duncan SA. HNF4: a central regulator of hepatocyte differentiation and function. Hepatology (2003) 37:1249–1255.[CrossRef][Web of Science][Medline]

  16. Wang JC, Strömstedt PE, Sugiyama T, Granner DK. The phosphoenolpyruvate carboxykinase gene glucocorticoid response unit: identification of the functional domains of accessory factors HNF3 beta (hepatic nuclear factor-3 beta) and HNF4 and the necessity of proper alignment of their cognate binding sites. Mol. Endocrinol. (1999) 13:604–618.[Abstract/Free Full Text]

  17. Stroup D, Chiang JY. HNF4 and COUP-TFII interact to modulate transcription of the cholesterol 7alpha-hydroxylase gene (CYP7A1). J. Lipid Res. (2000) 41:1–11.[Abstract/Free Full Text]

  18. Louet JF, Hayhurst G, Gonzalez FJ, Girard J, Decaux JF. The coactivator PGC-1 is involved in the regulation of the liver carnitine palmitoyltransferase I gene expression by cAMP in combination with HNF4 alpha and cAMP-response element-binding protein (CREB). J. Biol. Chem. (2002) 277:37991–38000.[Abstract/Free Full Text]

  19. Lonard DM, O’malley BW. Nuclear receptor coregulators: judges, juries, and executioners of cellular regulation. Mol. Cell (2007) 27:691–700.[CrossRef][Web of Science][Medline]

  20. Perissi V, Rosenfeld MG. Controlling nuclear receptors: the circular logic of cofactor cycles. Nat. Rev. Mol. Cell Biol. (2005) 6:542–554.[CrossRef][Web of Science][Medline]

  21. Belandia B, Orford RL, Hurst HC, Parker MG. Targeting of SWI/SNF chromatin remodelling complexes to estrogen-responsive genes. EMBO J. (2002) 21:4094–4103.[CrossRef][Web of Science][Medline]

  22. McKenna NJ, O’Malley BW. Combinatorial control of gene expression by nuclear receptors and coregulators. Cell (2002) 108:465–474.[CrossRef][Web of Science][Medline]

  23. Rachez C, Freedman LP. Mediator complexes and transcription. Curr. Opin. Cell Biol. (2001) 13:274–280.[CrossRef][Web of Science][Medline]

  24. Rosenfeld MG, Lunyak VV, Glass CK. Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response. Genes Dev. (2006) 20:1405–1428.[Abstract/Free Full Text]

  25. Heery DM, Kalkhoven E, Hoare S, Parker MG. A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature (1997) 387:733–736.[CrossRef][Medline]

  26. Rosenfeld MG, Glass CK. Coregulator codes of transcriptional regulation by nuclear receptors. J. Biol. Chem. (2001) 276:36865–36868.[Free Full Text]

  27. Park YY, Kim HJ, Kim JY, Kim MY, Song KH, Park K, Yu KY, Shong M, Kim KH, Choi HS. Differential role of the loop region between helices H6 and H7 within the orphan nuclear receptors small heterodimer partner and DAX-1. Mol. Endocrinol. (2004) 18:1082–1095.[Abstract/Free Full Text]

  28. Sanyal S, Kim JY, Kim HJ, Takeda J, Lee YK, Moore DD, Choi HS. Differential regulation of the orphan nuclear receptor small heterodimer partner (SHP) gene promoter by orphan nuclear receptor ERR isoforms. J. Biol. Chem. (2002) 277:1739–1748.[Abstract/Free Full Text]

  29. Kim GS, Lee GY, Nedumaran B, Park YY, Kim KT, Park SC, Lee YC, Kim JB, Choi HS. The orphan nuclear receptor DAX-1 acts as a novel transcriptional corepressor of PPARgamma. Biochem. Biophys. Res. Commun. (2008) 370:264–268.[CrossRef][Web of Science][Medline]

  30. Kim JY, Kim HJ, Kim KT, Park YY, Seong HA, Park KC, Lee IK, Ha H, Shong MH, Park SC, et al. Orphan nuclear receptor small heterodimer partner represses hepatocyte nuclear factor 3/Foxa transactivation via inhibition of its DNA binding. Mol. Endocrinol. (2004) 18:2880–2894.[Abstract/Free Full Text]

  31. Kim JY, Chu K, Kim HJ, Seong H, Park KC, Sanyal S, Takeda J, Ha HJ, Shong MH, Tsai MJ, et al. Orphan nuclear receptor small heterodimer partner, a novel corepressor for a basic helix-loop-helix transcription factor BETA2/NeuroD. Mol. Endocrinol. (2004) 18:776–790.[Abstract/Free Full Text]

  32. Kim HJ, Kim JY, Park YY, Choi HS. Synergistic activation of the human orphan nuclear receptor SHP gene promoter by basic helix-loop-helix protein E2A and orphan nuclear receptor SF-1. Nucleic Acids Res. (2003) 31:6860–6872.[Abstract/Free Full Text]

  33. Zhang X, Ozawa Y, Lee H, Wen YD, Tan TH, Wadzinski BE, Seto E. Histone deacetylase 3 (HDAC3) activity is regulated by interaction with protein serine/threonine phosphatase 4. Genes Dev. (2005) 19:827–839.[Abstract/Free Full Text]

  34. Cockram GP, Hogan MR, Burnett HF, Lu R. Identification and characterization of the DNA-binding properties of a Zhangfei homologue in Japanese pufferfish, Takifugu rubripes. Biochem. Biophys. Res. Commun. (2006) 339:1238–1245.[CrossRef][Web of Science][Medline]

  35. Dwarki VJ, Montminy M, Verma IM. Both the basic region and the ‘leucine zipper’ domain of the cyclic AMP response element binding (CREB) protein are essential for transcriptional activation. EMBO J. (1990) 9:225–232.[Web of Science][Medline]

  36. Vega RB, Huss JM, Kelly DP. The coactivator PGC-1 cooperates with peroxisome proliferator-activated receptor {alpha} in transcriptional control of nuclear genes encoding mitochondrial fatty acid oxidation enzymes. Mol. Cell Biol. (2000) 20:1868–1876.[Abstract/Free Full Text]

  37. Hong H, Kohli K, Garabedian MJ, Stallcup MR. GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol. Cell Biol. (1997) 17:2735–2744.[Abstract]

  38. Seol W, Choi HS, Moore DD. An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors. Science (1996) 272:1336–1339.[Abstract]

  39. Lee YK, Dell H, Dowhan DH, Hadzopoulou-Cladaras M, Moore DD. The orphan nuclear receptor SHP inhibits hepatocyte nuclear factor 4 and retinoid X receptor transactivation: Two mechanisms for repression. Mol. Cell Biol. (2000) 20:187–195.[Abstract/Free Full Text]

  40. Lee CH, Wei LN. Characterization of receptor-interacting protein 140 in retinoid receptor activities. J. Biol. Chem. (1999) 274:31320–31326.[Abstract/Free Full Text]

  41. Castet A, Boulahtouf A, Versini G, Bonnet S, Augereau P, Vignon F, Khochbin S, Jalaguier S, Cavaillès V. Multiple domains of the receptor-interacting protein 140 contribute to transcription inhibition. Nucleic Acids Res. (2004) 32:1957–1966.[Abstract/Free Full Text]

  42. Gobinet J, Carascossa S, Cavaillès V, Vignon F, Nicolas JC, Jalaguier S. SHP represses transcriptional activity via recruitment of histone deacetylases. Biochemistry (2005) 44:6312–6320.[CrossRef][Web of Science][Medline]

  43. Heery DM, Hoare S, Hussain S, Parker MG, Sheppard H. Core LXXLL motif sequences in CREB-binding protein, SRC1, and RIP140 define affinity and selectivity for steroid and retinoid receptors. J. Bio.l Chem. (2001) 276:6695–6702.[Abstract/Free Full Text]

  44. Deppmann CD, Alvania RS, Taparowsky EJ. Cross-species annotation of basic leucine zipper factor interactions: Insight into the evolution of closed interaction networks. Mol. Biol. Evol. (2006) 23:1480–1492.[Abstract/Free Full Text]

  45. Wardell SE, Boonyaratanakornkit V, Adelman JS, Aronheim A, Edwards DP. Jun dimerization protein 2 functions as a progesterone receptor N-terminal domain coactivator. Mol. Cell Biol. (2002) 22:5451–5466.[Abstract/Free Full Text]

  46. Zhou D, Ye JJ, Li Y, Lui K, Chen S. The molecular basis of the interaction between the proline-rich SH3-binding motif of PNRC and estrogen receptor alpha. Nucleic Acids Res. (2006) 34:5974–5986.[Abstract/Free Full Text]

  47. Fernandes I, Bastien Y, Wai T, Nygard K, Lin R, Cormier O, Lee HS, Eng F, Bertos NR, Pelletier N, et al. Ligand-dependent nuclear receptor corepressor LCoR functions by histone deacetylase-dependent and-independent mechanisms. Mol. Cell (2003) 11:139–150.[CrossRef][Web of Science][Medline]

  48. Yap N, Yu CL, Cheng SY. Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53. Proc. Natl. Acad. Sci. USA (1996) 93:4273–4277.[Abstract/Free Full Text]

  49. Maiyar AC, Phu PT, Huang AJ, Firestone GL. Repression of glucocorticoid receptor transactivation and DNA binding of a glucocorticoid response element within the serum/glucocorticoid-inducible protein kinase (sgk) gene promoter by the p53 tumor suppressor protein. Mol. Endocrinol. (1997) 11:312–329.[Abstract/Free Full Text]

  50. Heitzer MD, Wolf IM, Sanchez ER, Witchel SF, DeFranco DB. Glucocorticoid receptor physiology. Rev. Endocr. Metab. Disord. (2007) 8:321–330.[CrossRef][Web of Science][Medline]

  51. Feige JN, Auwerx J. Transcriptional coregulators in the control of energy homeostasis. Trends Cell Biol. (2007) 17:292–301.[CrossRef][Web of Science][Medline]

  52. Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, Auwerx J, Mangelsdorf DJ. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol. Cell (2000) 6:507–515.[CrossRef][Web of Science][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
Y.-B. Xie, J.-H. Park, D.-K. Kim, J. H. Hwang, S. Oh, S. B. Park, M. Shong, I.-K. Lee, and H.-S. Choi
Transcriptional Corepressor SMILE Recruits SIRT1 to Inhibit Nuclear Receptor Estrogen Receptor-related Receptor {gamma} Transactivation
J. Biol. Chem., October 16, 2009; 284(42): 28762 - 28774.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Chanda, T. Li, K.-H. Song, Y.-H. Kim, J. Sim, C. H. Lee, J. Y. L. Chiang, and H.-S. Choi
Hepatocyte Growth Factor Family Negatively Regulates Hepatic Gluconeogenesis via Induction of Orphan Nuclear Receptor Small Heterodimer Partner in Primary Hepatocytes
J. Biol. Chem., October 16, 2009; 284(42): 28510 - 28521.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Print PDF (9509K) Freely available
Right arrow Screen PDF (1065K) Freely available
Right arrow Supplementary Data
Right arrowOA All Versions of this Article:
37/12/4100    most recent
gkp333v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Xie, Y.-B.
Right arrow Articles by Choi, H.-S.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xie, Y.-B.
Right arrow Articles by Choi, H.-S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?