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Molecular Endocrinology, doi:10.1210/me.2003-0339
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Molecular Endocrinology 18 (5): 1082-1095
Copyright © 2004 by The Endocrine Society

Differential Role of the Loop Region between Helices H6 and H7 within the Orphan Nuclear Receptors Small Heterodimer Partner and DAX-1

Yun-Yong Park, Han-Jong Kim, Joon-Young Kim, Mi-Young Kim, Kwang-Hoon Song, Ki Cheol Park, Kang-Yeol Yu, Minho Shong, Kyoung-Hee Kim and Hueng-Sik Choi

Hormone Research Center (Y.-Y.P., H.-J.K., J.-Y.K., M.-Y.K., K.-H.S., H.-S.C.), School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757; KOMED Institute for Life Science (K.-Y.Y.), Graduate School of Biotechnology, Korea University, Seoul 136-701; Laboratory of Endocrine Cell Biology (K.C.P., M.S.), Department of Internal Medicine, Chungnam National University School of Medicine, Daejon 301-721; and R&D Park (K.-H.K.), LG Life Sciences, Ltd., Daejeon 305-380, Republic of Korea

Address all correspondence and requests for reprints to: Hueung-Sik Choi, Ph.D., Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea. E-mail: hsc{at}chonnam.chonnam.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The orphan nuclear receptors small heterodimer partner (SHP) and dosage-sensitive sex-reversal adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1 (DAX-1) contain extra amino acids between helices H6 and H7 of LBD, and here we investigated a possible role of these additional amino acids. Transient transfection assay demonstrated that, in contrast to wild type, in mutant SHP {Delta}128–139 deletion of 12 extra amino acids in H6-H7 failed to repress the transactivity of orphan nuclear receptors such as estrogen receptor-related receptor-{gamma}, hepatocyte nuclear factor 4{alpha}, and constitutive androstane receptor. Interestingly, yeast two-hybrid and glutathione-S-transferase pull-down assays demonstrated that wild-type and SHP {Delta}128–139 have similar abilities to interact with estrogen receptor-related receptor-{gamma}, hepatocyte nuclear factor 4{alpha}, and constitutive androstane receptor. Unexpectedly, in wild-type DAX-1 and mutant DAX-1 {Delta}338–362, deletion of 25 extra amino acids in H6-H7 had no significant difference in the interaction and repression of steroidogenic factor 1 transactivation. Mutant SHP that contains DAX-1 extra amino acids or polyalanine stretch in H6-H7 showed indistinguishable pattern of repression from wild-type SHP. Interestingly, the swapped SHP mutant with DAX-1 extra amino acids interacted with EID-1 (E1A-like inhibitor of differentiation 1), which is characterized as an SHP-interacting corepressor. However, interaction between SHP {Delta}128–139 and EID-1 was significantly diminished. Moreover, SHP-mediated repression of constitutive androstane receptor transactivation was significantly released by down-regulation of EID-1 expression with EID-1 small interfering RNA. The present study suggests that H6-H7 loop regions of SHP and DAX-1 play a different role in the repression of nuclear receptor transactivation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR RECEPTOR superfamily is a key regulator of cellular processes including cell growth, differentiation, development, and homeostasis. Conventionally, nuclear receptors are composed of four independent interaction and functional modules, A/B domain, the DNA-binding domain (DBD), the hinge region (D), and the ligand-binding domain (LBD) (1). Small heterodimer partner (SHP) is an atypical orphan nuclear receptor that lacks a DBD and consists only of putative LBD (2). It has been reported that SHP represses the transcriptional activity of a number of nuclear receptors such as constitutive androstane receptor (CAR), estrogen receptor (ER), thyroid hormone receptor (TR), retinoid X receptor (RXR), hepatocyte nuclear factor 4{alpha} (HNF4{alpha}), androgen receptor (AR), liver receptor homolog 1 (LRH-1), ER-related receptor-{gamma} (ERR{gamma}), glucocorticoid receptor (GR), and liver X receptor (LXR) (2, 3, 4, 5, 6, 7, 8, 9). Interestingly, transactivation of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) is augmented by SHP (10). Although repression mechanism of SHP remains largely unclear, several reports demonstrate that SHP competes with coactivator on activation function 2 (AF-2) surface of nuclear receptors and recruits unidentified corepressors (4, 11, 12).

Physiologically, SHP is a direct target of farnesoid X receptor (FXR), and SHP gene expression is drastically up-regulated in the liver by bile acids. The increased level of SHP results in the complete repression of the CYP7A1 promoter and eventually the SHP promoter itself. In addition, the mechanism of SHP repression on both CYP7A1 and SHP promoters is due to the direct interaction of SHP with the orphan nuclear receptor LRH-1, suggesting that SHP plays a pivotal role in bile acid metabolism and in feedback repression of CYP7A1 (13, 14). In the view of glucose metabolism, genetic variations in the SHP gene contribute to increased body weight and reveal a pathway leading to common metabolic disorder in Japanese subjects (15).

The closet relative to SHP within the nuclear receptor family is DAX-1 [dosage-sensitive sex-reversal AHC (adrenal hypoplasia congenital) critical region on the X chromosome gene 1], which lacks conventional DBD and has high homology with LBD region of SHP (16). DAX-1 also interacts with and inhibits steroidogenic factor 1 (SF-1), ER, androgen receptor (AR) and LRH-1 (17, 18, 19, 20), and DAX-1 has a unique three-repeat region in the N terminus, which region is absent in SHP. Mutations in the human DAX-1 gene cause adrenal hypoplasia and abolish the potent repressive function of DAX-1 (16, 21). Although DAX-1 is closely related to SHP, DAX-1 inhibits nuclear receptors via a distinct repression mechanism from that of SHP. DAX-1 potentially recruits corepressor such as nuclear receptor corepressor and Alien, which directly interact with carboxyl-terminal domain of DAX-1 (22, 23), whereas SHP competes with coactivators such as p300 and steroid receptor coactivator (SRC)-1.

In contrast to interaction of DAX-1 with corepressors, antagonism of p300 coactivation function via EID-1 is involved in the repression of nuclear receptors by SHP (12). EID-1 is a nuclear protein that interacts with Rb, Myo D, and p300 and EID-1 blocks muscle differentiation in skeletal muscle (24, 25). Interestingly, EID-1 interacts with p300 to repress transcriptional activity of MyoD, whereas EID-2 (EID-1-like inhibitor of differentiation-2) is associated with class I histone deacetylases, demonstrating that EID-1 and EID-2 possess distinct repression mechanism (26).

In this study, we demonstrated that the H6-H7 loop regions within SHP and DAX-1 have different roles in the interaction and repression of nuclear receptors. Moreover, modeling study on SHP structure suggested that the extra amino acids within helices H6 and H7 form a bulged loop extended from the surface of the protein and removal of the H6-H7 loop may have insignificant effect on the overall structure of SHP. However, the bulged loop may play a crucial role in the interaction of SHP with EID-1 by providing the additional recognition surface for EID-1. We propose that repression function of SHP and DAX-1 via extra amino acids between helices H6 and H7 is distinct and that extra amino acids of SHP is essential to give a space interacting with EID-1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SHP and DAX-1 Contain Extra Amino Acids between Helices H6 and H7
It has been reported that SHP and DAX-1 lack conventional DBD and have the typical structure of LBD (2, 16). In addition, DAX-1 possesses repeating region in N terminus (Fig. 1AGo). Human SHP includes 12 extra amino acids (aa 128–139) and human DAX-1 contains longer 25 extra amino acids (aa 338–362) between helices H6 and H7 (Fig. 1AGo). Sequence alignment with other orphan nuclear receptors showed that SHP and DAX-1 contain additional amino acids between helices H6 and H7 (Fig. 1BGo), whereas this insertion was absent in hHNF4{alpha}, RXR, FXR, nerve growth factor-inducible-B (NGFI-B), SF-1 and LRH-1 (Fig. 1BGo). Sequence alignment of SHP and DAX-1 demonstrated that additional amino acids between helices H6 and H7 are unique to the atypical orphan nuclear receptors SHP and DAX-1.



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  		Fig. 1. Structure of Helices H6 and H7 in SHP and DAX-1

A, Schematic view of SHP and DAX-1 LBDs and repeating region of DAX-1. Gray boxes indicate helices H6 and H7 region, and gray arrows indicate repeating region of DAX-1. Black triangles indicate additional insertion between helices H6 and H7 of SHP and DAX-1. The numbers represent respective amino acid positions. B, sequence alignment of the helices H6 and H7 motif in the LBD of SHP and DAX-1 with related orphan nuclear receptors. Italic letters indicate additional insertion between helices H6 and H7. C, Structural model of SHP. The x-ray structures of HNF4{alpha} (PDB ID 1M7W) and ERR{gamma} (PDB ID 1KV6) are shown in brown and in pink, respectively, and some of its secondary structures are labeled. SHP was modeled using the LBD structures of rat HNF4{alpha} and human ERR{gamma} as templates. The modeled SHP is shown in brown, and its secondary structures are labeled. The 12-residue insertion (in blue) is modeled as a bulged loop on the protein surface.

 
Because the crystal structure of SHP has not been reported yet, the computer homology modeling study was performed to gain a better insight into the structure of the additional amino acids of SHP. Based on the secondary structure alignments, the LBD structures of rat HNF4{alpha} and human ERR{gamma} are selected as homologous templates (27, 28). The structural homology is present throughout the molecule except the H2 region and the helix H6 that is present in ERR{gamma}, but not in HNF4{alpha} (Fig. 1CGo). The SHP model is generally in good agreement with HNF4{alpha} structure throughout the whole molecule. The helix H6 is modeled as a short 3/10 helix just before the insertion. The secondary structure predictions on additional amino acids in SHP suggested that the additional amino acids form neither {alpha} helix nor ß strand. The additional amino acids are modeled as a flexible loop that extends from helices H6 and H7.

SHP {Delta}128–139 Failed to Inhibit the Transcriptional Activities of Orphan Nuclear Receptors
Because SHP and DAX-1 contain additional amino acids between helices H6 and H7, we investigated the possible role of these amino acids in the repression of transcriptional activity of nuclear receptors by SHP and DAX-1. The 12 extra amino acids (aa 128–139) between helices H6 and H7 were deleted to generate SHP {Delta}128–139 (Fig. 2AGo) and repressive function of wild-type SHP and SHP {Delta}128–139 was examined in transient transfection assay. As shown in Fig. 2Go, B–D, wild-type SHP significantly inhibits the transcriptional activities of ERR{gamma}, mouse CAR (mCAR), and HNF4{alpha} as described previously (2, 4, 7). However, SHP {Delta}128–139 failed to inhibit ERR{gamma}, mCAR, and HNF4{alpha} transactivation (Fig. 2Go, B–D). Furthermore, the repressive function of SHP {Delta}128–139 dramatically also decreased in Gal4-mCAR and Gal-LRH-1 (data not shown), suggesting that extra amino acids between helices H6 and H7 are critical for SHP-mediated nuclear receptor repression.



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  		Fig. 2. Deletion of Extra Loop between Helices H6 and H7 in SHP Failed to Inhibit Orphan Nuclear Receptors

A, Schematic view of SHP {Delta}128–139. Extra 12 amino acids were indicated as black triangle. SHP {Delta}128–139 represents absence of additional insertion between helices H6 and H7 of SHP. HEK 293 (B and E) and HepG2 (C and D) cells were cotransfected with 100 ng of indicated luciferase reporter, ERR{gamma}, mCAR, HNF4{alpha}, and SRC-1 with indicated doses of SHP and SHP {Delta}128–139. Approximately 40–48 h after transfection, the cells were harvested, and the luciferase activity was measured and normalized against ß-galactosidase activity. One representative experiment is shown. All values represent the mean of duplicate samples, and similar results were obtained in at least three independent experiments. F, pcDNA3 (a), pcDNA3/HA-SHP (b) and pcDNA3/HA-SHP {Delta}128–139 (c) were transfected and expressed in HEK 293 cells. Forty-eight hours later, the cell lysates were prepared and 100 µg of protein from each lysate were analyzed by anti-HA monoclonal antibody. Numbers on the left indicate molecular mass in kilodaltons.

 
Because it has been reported that SHP competes with coactivator such as SRC-1 (4), we examined whether SHP {Delta}128–139 has any effect on the SRC-1-dependent mCAR transactivation. The expression plasmids encoding wild-type SHP or SHP {Delta}128–139 were cotransfected with mCAR and SRC-1 expression vectors in HEK 293 cells. The result showed that SHP {Delta}128–139 had a reduced ability to repress SRC-1-dependent mCAR transactivation, compared with wild-type SHP (Fig. 2EGo).

To examine whether impairment of repression by deletion of extra amino acids between helices H6 and H7 is due to reduced protein expression, Western blot analysis was performed with whole cell extracts from HEK 293 cells transfected with pcDNA3/HA SHP and SHP {Delta}128–139. As shown in Fig. 2FGo, expression level of SHP {Delta}128–139 proteins was similar to that of wild-type SHP, indicating that the difference in protein expression level might not be responsible for impairment of repression by SHP{Delta}128–139. Taken together, these results demonstrated that additional amino acids between helices H6 and H7 play a critical role in the repression of nuclear receptors by SHP.

SHP {Delta}128–139 Possesses the Ability to Interact with Orphan Nuclear Receptors
To determine whether the loss of repressive function of SHP {Delta}128–139 is due to the impairment of interaction, we investigated interaction and colocalization of SHP {Delta}128–139 with orphan nuclear receptors. SHP has been shown to interact with AF-2 domain of nuclear receptors and competes with coactivator to repress nuclear receptor transactivation (4, 6). To determine whether SHP {Delta}128–139 still possesses the ability to interact with nuclear receptors via the AF-2 domain, yeast two-hybrid and glutathione-S-transferase (GST) pull-down assays were performed. As shown in Fig. 3Go, A and B, yeast two-hybrid assay demonstrated that SHP {Delta}128–139 interacts with ERR{gamma} and mCAR and that AF-2 domains of mCAR and ERR{gamma} are essential for the interaction with both SHP and SHP {Delta}128–139. Furthermore, no significant difference in the interaction between SHP and SHP {Delta}128–139 with mCAR, ERR{gamma}, and LRH-1 was observed in GST pull-down assay (Fig. 3CGo).



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  		Fig. 3. Interaction between SHP {Delta}128–139 and Orphan Nuclear Receptors

A and B, The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate LacZ reporter gene as described previously (7 ). AF-2 deletion of mCAR, in which C-terminal amino acids 352–359 are deleted (mCAR {Delta}AF-2). The data are representative of at least two similar experiments. The error bars indicate SD. C, GST-fused SHP and SHP {Delta}128–139 were isolated from bacterial culture and immobilized on glutathione-Sepharose beads. In vitro-transcribed and translated [35S]methionine-labeled ERR{gamma}, mCAR, and LRH-1 were incubated with purified GST-fused receptors or GST alone as indicated in the figure. The interaction complexes were resolved by a 10% denaturing SDS-PAGE and analyzed by autoradiography. D, Colocalization of SHP, ERR{gamma} and SHP {Delta}128–139, ERR{gamma}. Cos-7 cells were transiently transfected with pEGFP SHP, SHP {Delta}128–139 and pCDNA3 HA ERR{gamma} by Lipofectamine. GFP-fused SHP and SHP {Delta}128–139 were detected by autofluorescence, and pCDNA3 HA ERR{gamma} was detected by staining with primary anti-HA antibody and rhodamine-conjugated secondary antibody. The yellow stain in the merged image depicts colocalization of SHP, ERR{gamma}, and SHP {Delta}128–139, ERR{gamma}. Shown are representative cells from one of three independent experiments.

 
To determine whether the loss of SHP function is due to the different cellular localization of SHP {Delta}128–139, immunofluorescence confocal microscopy studies were performed in COS-7 cells cotransfected with pEGFP-SHP or pEGFP-SHP {Delta}128–139 and pCDNA3/HA-ERR{gamma}. SHP and ERR{gamma} were predominantly localized in the nucleus and similar pattern of cellular localization was also observed between SHP {Delta}128–139 and ERR{gamma}, as depicted in the merged image (Fig. 3DGo). Collectively, these results demonstrated that impairment of repression by SHP {Delta}128–139 is not due to the defect in the interaction with nuclear receptors via AF-2 domain nor alteration of cellular localization.

DAX-1 {Delta}338–362 Represses SF-1 Transactivation
Because DAX-1 also contains additional amino acids between helices H6 and H7, we investigated whether extra amino acids between helices H6 and H7 of DAX-1 are required for repressive function. To evaluate whether the additional amino acids are involved in repressive function of DAX-1, 25 extra amino acids (aa 338–362) between helices H6 and H7 were deleted to generate DAX-1 {Delta}338–362 (Fig. 4AGo). Based on the previous report that DAX-1 specifically interacts with SF-1 and represses transactivity of SF-1 (17, 20, 22), we determined whether wild-type DAX-1 and DAX-1 {Delta}338–362 differentially interact with and inhibit SF-1 transactivation. DAX-1 {Delta}338–362, as well as wild-type DAX-1, interacted with SF-1 in yeast two-hybrid assay (Fig. 4BGo). Similar pattern of interaction between DAX-1 and SF-1 or DAX-1 {Delta}338–362 and SF-1 was also observed in the GST pull-down assay (Fig. 4CGo). Furthermore, transient transfection assay was performed to examine whether DAX-1 and DAX-1 {Delta}338–362 differentially inhibit SF-1 transactivation. Unexpectedly, DAX-1 {Delta}338–362 strongly inhibited SF-1 and pattern of repression was similar to that of wild-type DAX-1 (Fig. 4DGo). Taken together, these results indicate that additional amino acids between helices H6 and H7 region of DAX-1 are not essential for interaction with SF-1 and repressive function of DAX-1.



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  		Fig. 4. Deletion of Extra Loop between Helices H6 and H7 in DAX-1 Has No Effect on SF-1 Transactivation

A, Schematic of construct for DAX-1 {Delta}338–362. Additional 25 amino between helices H6 and H7 were indicated as black triangle. Gray boxes indicate helices H6 and H7 region. Gray arrows indicate repeating region of DAX-1. DAX-1 {Delta}338–362 represents absence of additional insertion in loop region of DAX-1. B, EGY48 yeast cells were transformed with LexA DAX-1 or LexA DAX-1 {Delta}338–362 and B42 or B42-fused SF-1. Transformants were selected on plates with appropriate selection markers, and the ß-galactosidase activity was measured. C, GST-fused SF-1 was isolated from bacterial culture and immobilized on glutathione-Sepharose beads. In vitro-transcribed and translated [35S] methionine-labeled DAX-1 and DAX-1 {Delta}338–362 were incubated with purified GST-fused receptors or GST alone as indicated in the figure. The interaction complexes were resolved by a 10% denaturing SDS-PAGE and analyzed by autoradiography. D, HepG2 cells were cotransfected with 100 ng of indicated luciferase reporter and SF-1 with indicated doses of DAX-1 and DAX-1 {Delta}338–362. Approximately 40–48 h after transfection, the cells were harvested, and the luciferase activity was measured and normalized against ß-galactosidase activity. Similar results were obtained in at least three independent experiments.

 
The Effect of Mutual Substitution of Additional Amino Acids between SHP and DAX-1
Because additional amino acids play a different role in the repressive function of SHP and DAX-1, we investigated whether specificity or length of sequence in extra amino acids has any effect on the repressive function of SHP and DAX-1. To examine the effects of substitution of additional amino acids between SHP and DAX-1, we constructed swapping receptors in which the additional insertion region of SHP was replaced with that of the DAX-1 and vice versa (Fig. 5AGo). SHP Swap interacted with mCAR, but not with SF-1, and DAX-1 Swap also interacted with SF-1, but not with mCAR in the GST pull-down assay, (Fig. 5Go, B and C). Transient transfection assay demonstrated that SHP Swap inhibited the transcriptional activity of mCAR and the pattern of inhibition was indistinguishable from that of wild-type SHP (Fig. 5DGo). In addition, DAX-1 Swap also inhibited SF-1 similarly to wild-type DAX-1 (Fig. 5EGo), whereas DAX-1 Swap and SHP Swap had no significant effects on the inhibition of mCAR and SF-1 transactivation, respectively (Fig. 5Go, D and E). These results demonstrate that specificity and length of sequence of additional amino acids between helices H6 and H7 may not be required for the repressive function and interaction of SHP and DAX-1 with nuclear receptors.



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  		Fig. 5. Substitution of Additional Insertion between SHP and DAX-1

A, Schematic of swapping constructs of SHP and DAX-1. Additional insertion of SHP (aa 128–139) and DAX-1 (aa 338–362) is swapped for DAX-1 and SHP. Black triangle indicates that additional insertion of SHP and DAX-1. Gray boxes indicate helices H6 and H7. B and C, GST-fused mCAR and SF-1 were isolated from bacterial culture and immobilized on glutathione-Sepharose beads. In vitro-transcribed and translated [35S] methionine-labeled SHP Swap and DAX-1 Swap were incubated with purified GST-fused receptors or GST alone as indicated in Fig. 3Go, B and C. The interaction complexes were resolved by a 10% denaturing SDS-PAGE, and analyzed by autoradiography. D and E, HepG2 cells were cotransfected with 100 ng of indicated luciferase reporter, mCAR and SF-1 with indicated doses of SHP, DAX-1, SHP Swap, and DAX-1 Swap. All values represent the mean of duplicate samples, and similar results were obtained in at least three independent experiments. One representative experiment is shown.

 
Sequence Specificity of Amino Acid Is Not Related to SHP Repression
To confirm whether the composition or length of additional insertion in H6-H7 has any effect on the repressive function of SHP, we substituted four (SHP Ala 4), 8 (SHP Ala 8), and 12 (SHP Ala 12) polyalanine for the 12 additional amino acids (Fig. 6AGo). To determine the effect of these SHP mutants on nuclear receptor transactivation, SHP Ala 4, 8, and 12 were cotransfected with mCAR. Interestingly, all SHP polyalanine stretched mutants were able to repress mCAR transactivation as strongly as wild-type SHP (Fig. 6BGo). We also observed the similar effect of these SHP mutants on the HNF4{alpha} transactivation (data not shown), suggesting that four-alanine stretch in H6-H7 is fully enough for SHP to repress transactivation of nuclear receptors. Taken together, these results indicate that sequence specificity of additional amino acid in H6-H7 may not be required for the repressive function of SHP.



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  		Fig. 6. Effects of Polyalanine Stretch on H6-H7 Loop Region in SHP

A, Schematic view of SHP polyalanine stretch. Gray arrows indicate the inserted polyalanine in H6-H7 region and the numbers 4, 8, and 12 indicate the inserted alanine residue. Black triangle indicates additional insertion of SHP. Gray boxes indicate helices H6 and H7. B, HepG2 cells were cotransfected with 100 ng of (NR1)X5 luciferase reporter and mCAR expression vector, together with indicated doses of plasmids encoding wild-type SHP and mutant SHPs.

 
Interaction between SHP {Delta}128–139 and EID-1
Based on the results showing that additional amino acids play a pivotal role in the repressive function of SHP, but not in that of DAX-1 and this insertion has no effect on the interaction of SHP with nuclear receptors and localization of SHP, we suspected that another factor may be involved in impairment of repression by SHP {Delta}128–139. Previously, we carried out yeast two-hybrid screening of A20 cell and mouse testis cDNA library using SHP as a bait, and we isolated coregulators EID-1 and DP103 (Song, K. H., and H. S. Choi, unpublished data) as SHP-interacting proteins. It was reported that SHP inhibits nuclear receptors by recruiting EID-1 and blocking the function of p300 (12). To determine whether the EID-1 or DP103 is involved in the impairment of repression by SHP, a yeast two-hybrid assay was performed. Interestingly, interaction between EID-1 and SHP {Delta}128–139 was markedly diminished, whereas DP103 showed a strong interaction with both wild-type SHP and SHP {Delta}128–139 (Fig. 7AGo). Moreover, GST pull-down assay results also showed that EID-1 failed to interact with SHP {Delta}128–139 (Fig. 7BGo). Wild-type DAX-1 and DAX-1 {Delta}338–362 did not interact with EID-1 in GST pull-down and yeast two-hybrid assays (data not shown). To confirm whether EID-1 retains capacity to interact with DAX-1 swap and SHP swap, we performed GST pull-down assay. Interestingly, EID-1 still interacted with SHP swap, but not with DAX-1 swap (Fig. 7BGo), indicating that additional amino acids in H6-H7 are not sufficient for the interaction of SHP with EID-1.



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  		Fig. 7. Effect of EID-1 on the SHP-Mediated Repression

A, EGY48 yeast cells were transformed with LexA SHP or LexA SHP {Delta}128–139 and B42-fused EID-1 or DP103. Transformants were selected on plates with appropriate selection markers, and the ß-galactosidase activity was measured. The results shown are the mean of ß-galactosidase value from six independent transformant colonies. The error bars indicate SD. B, GST-fused EID-1 was isolated and immobilized above described. In vitro-transcribed and translated [35S]methionine-labeled SHP, SHP {Delta}128–139, SHP Swap, and DAX-1 Swap were incubated with purified GST-fused EID-1 or GST alone as indicated in Fig. 3CGo. The interaction complexes were resolved by a 10% denaturing SDS-PAGE, and analyzed by autoradiography. C, Endogenous EID-1 gene expression was inhibited by transfection of a 19-nucleotide RNA duplex (siRNA-EID-1 I and II) in HepG2 cells. The effects of siRNAs on the EID-1 expression were assayed by RT-PCR for EID-1 (460 bp) and ß-actin gene (231 bp) as a control. D, HepG2 cells were transfected with siRNA EID-1 I or II. Forty-eight hours after transfection, the cells were transfected with (NR1)X5 and mCAR together with indicated dose of SHP expression vectors.

 
To determine whether EID-1 is essential for the repressive function of SHP, we designed EID-1 small interfering (si) RNA to inhibit EID-1 gene expression. EID-1 siRNA was transfected in HepG2 cells and the effect of siRNA on the EID-1 gene expression was analyzed by RT-PCR. EID-1 II siRNA (si EID-1 II) treatment significantly inhibits EID-1 gene expression, whereas EID-1 I siRNA (si EID-1 I) was not able to (Fig. 7CGo). Next, we tested the effect of si EID-1 II on the repressive function of SHP. As shown in Fig. 7DGo, SHP-mediated repression of CAR transactivation was significantly released by treatment of si EID-1 II. Consistent with the result from Fig. 7CGo, si EID-1 I had no effect on the repressive function of SHP. Taken together, these results demonstrate that additional amino acids in H6-H7 within SHP are necessary but not sufficient for the interaction with EID-1 and that EID-1 plays an important role in the SHP-mediated repression of nuclear receptor transactivation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
It has been reported that several orphan nuclear receptors including SHP, DAX-1, FXR-like orphan receptor, and human CAR contain extra amino acids in helix H7 region (29, 30) and the significance of additional amino acids in this region has not been fully characterized yet. However, our sequence alignment results revealed that extra amino acids reside not in helix H7 but between helices H6 and H7. In present study, we investigated the function of additional amino acids in SHP and DAX-1, and we concluded that additional amino acids play a pivotal role in the repression of SHP by maintaining structural space. Therefore, the loss of interaction of SHP with EID-1 by deleting additional amino acids may cause the impairment of repressive function of SHP.

Although SHP and DAX-1 show high sequence homology in LBD (2, 16), repression mechanism of SHP and DAX-1 is largely different. SHP efficiently inhibits various nuclear receptors using two steps repression mechanism, whereas DAX-1 recruits corepressors such as nuclear receptor corepressor and Alien (22, 23). In the first step, SHP displaces coactivators by competing for binding to the AF-2 surface of nuclear receptors. In the second step, the transrepression domain within SHP is required for the full repressive function of SHP via an unknown mechanism (4, 6). In the structural aspect, SHP and DAX-1 possess loop region between helices H6 and H7 in LBD and the H6-H7 loop of SHP and DAX-1 is also conserved in lower vertebrates such as Nile tilapia, although sequence and length of extra amino acids of loop region are different from fish to mammalian SHP and DAX-1 (31). Interestingly, vitamin D receptor also contains extra amino acids between helices H1and H3, but the insertion domain is not involved in the main functions of vitamin D receptor such as ligand binding, dimerization with RXR and ligand-dependent transactivation (32).

It has been reported that the repressive function of SHP and DAX-1 is perturbed by impairment of localization in cells when mutations are introduced to specific domain or several motifs (8, 33). For example, when AF-2 domain or LXXLL motif is mutated in SHP and DAX-1, SHP and DAX-1 are dominantly distributed in cytoplasm and impair repression, demonstrating that LXXLL motif and AF-2 domain are crucial for repression and localization of SHP and DAX-1 in mammalian cells (8). In this study, we showed that impairment of repression by SHP {Delta}128–139 is neither due to the loss of interaction with orphan nuclear receptors nor to alteration of cellular localization (Fig. 3Go). Confocal microscopic analysis demonstrated that at least localization of SHP is not perturbed by deletion of H6-H7 loop, indicating that the loop region is not required for the localization of SHP. In addition, we showed that SHP {Delta}128–139 maintains normal interaction with orphan nuclear receptors and represses SRC-1-mediated mCAR transactivation. However, the repressive effect of SHP {Delta}128–139 was markedly reduced compared with wild-type SHP, suggesting that in addition to competition with coactivators, recruitment of EID-1 to SHP may be necessary for the full repressive function of SHP.

Previous studies reported that several naturally occurring mutants of DAX-1 (16, 17, 20, 21, 22) had significant effects on the recruitment of NCo-R, resulting in the impairment of repressive function of DAX-1. Here, we showed that SHP {Delta}128–139 lost the repressive function, whereas DAX-1 {Delta}338–362 still possessed repressive function (Figs. 2Go and 4Go). In addition, our results are consistent with a previous report that DAX-1 {Delta}345–352 corresponding to small part of the 25 additional amino acids has no effect on the repressive function of DAX-1 (23). In an effort to identify SHP-interacting proteins, we isolated corepressors EID-1 and DP103 as SHP-interacting proteins. DP103 is expressed predominantly in the testis and governs the transcriptional activity of SF-1 (34). However, the functional significance between DP103 and SHP is currently under investigation. It was previously reported that inhibition of nuclear receptors by SHP involves EID-1 antagonism of CBP/p300-dependant coactivator functions (12). In addition, EID-1 inhibits transcription factors that use p300 as a coactivator (25). We showed that EID-1 and DP103 had different interaction pattern to SHP {Delta}128–139 (Fig. 7Go), suggesting that the H6-H7 loop in SHP is critical for the interaction with EID-1 but not with DP103. Thus, we propose that SHP and DAX-1 possess functionally distinct repression mechanism via additional insertion between helices H6 and H7.

As shown in structural model of SHP, the additional amino acids form a bulged loop extended from the protein and its removal may not perturb the overall structure of SHP, supported by our result showing that SHP {Delta}128–139 maintains normal interaction with nuclear receptors. In addition, we showed that EID-1 interacts with SHP Swap, but not with DAX-1 Swap, indicating that the H6-H7 loop region of SHP may not be the only recognition site for EID-1 and EID-1 may recognize other structural elements of SHP in addition to the H6-H7 loop region. This idea is supported by the report that several amino acids in H3 to H5 and AF-2 domain of SHP are involved in interaction with EID-1 (12). In addition, we demonstrated that SHP Swap possesses normal repressive function, although the H6-H7 loop of DAX-1 in SHP Swap may form more protruded loop than that of SHP. Moreover, substitution experiment, replacement of 12 additional amino acids with polyalanine stretch from 4–12 amino acids in H6-H7 loop of SHP, demonstrated that a four-amino acid insertion is enough for the repressive function of SHP and that sequence specificity of amino acids in H6-H7 is not involved in SHP function. Therefore, we propose that the length and composition of amino acids within SHP may not be important for the repressive function and interaction with EID-1. However, the presence of a bulged H6-H7 loop and its space filling in SHP might be important for the repression of nuclear receptor transactivation by SHP via interaction with EID-1.

It has been previously reported that SHP directly interacts with nuclear receptors, whereas EID-1 does not interact with nuclear receptors (12). Thus, we predict that SHP may form a triple complex with EID and nuclear receptors and SHP may act as a bridge molecule between EID-1 and nuclear receptors. Here, we showed that treatment of EID-1 siRNA significantly released SHP repression, suggesting that EID-1 plays an important role in the SHP mediated repression of nuclear receptor transactivation. Finally, we propose a model showing that SHP represses nuclear receptors by recruiting EID-1 and H6-H7 in SHP is involved in EID-1 interaction. However, when the H6-H7 loop of SHP is deleted, SHP loses its ability to interact with EID-1 and to repress the nuclear receptors, although the interaction of SHP with nuclear receptors is not perturbed by deletion of H6-H7 (Fig. 8Go).



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  		Fig. 8. Schematic Model for the Repression Mechanism of SHP

Wild-type SHP, which contains additional amino acids as a bulged loop between helices H6 and H7, directly interacts with nuclear receptors and SHP represses nuclear receptor transactivation only in the presence of EID-1. However, when additional insertion (aa 128–139) in loop region is deleted, SHP interacts with nuclear receptors, but SHP fails to interact with EID-1. Deletion of additional insertion makes EID-1 free from SHP and inactive form of SHP lost the repression function.

 
In summary, the present study demonstrated that the H6-H7 loop is essential for the repressive function of SHP and for the maintenance of structural spacing for the interaction with EID-1. We also propose that the H6-H7 loop within SHP and DAX-1 plays a different role in the regulation of nuclear receptor transactivation. Our observation may provide new insights into the mechanism of SHP and DAX-1 action on the regulation of nuclear receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and DNA Construction
pCDNA3/HA-ERR{gamma}, pCI LRH-1, pCEP4 SF-1, pRC-CMV DAX-1, pCMX HNF4{alpha}, pCR3.1 SRC-1, murine SHP promoter Luc, ß-RARE Luc, (sft4)X3 Luc, pGEX4T-1 SF-1, B42 ERR{gamma}, B42 ERR{gamma}-{Delta}AF2 and (NR1)X5 Luc were as described previously (2, 4, 7, 17, 35, 36, 37). pHNF4-tk-luc and pEGFP SHP were kind gifts from Dr. Akiyoshi Fukamizu (University of Tsukuba, Tsukuba, Japan) and Dr. Jae Woon Lee (Baylor College of Medicine, Houston, TX), respectively. pcDNA3 mCAR was constructed by PCR from CDM8-mCAR and resulting product was subcloned into pGEXT4T-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK), B42 (BD Biosciences, CLONTECH, Palo Alto, CA) and pCMX-Gal4-DBD at EcoRI and XhoI. mCAR {Delta}AF-2 (352–359) construct was generated by PCR deleting from pCDAN3 mCAR and cloned into pcDNA3, the pCDNA3 mCAR {Delta}AF-2 was then digested with EcoRI and XhoI and resulting fragment was cloned in frame into B42 yeast vector. SHP {Delta}128–139, DAX-1 {Delta}338–362, SHP Swap, DAX-1 Swap, and polyalanine-stretch SHP mutants, SHP Ala 4, SHP Ala 8, and SHP Ala 12 were created by PCR-based site-directed mutagenesis from pCDAN3 SHP and pRC-CMV DAX-1, the resulting mutant PCR products were cloned into pCDNA3 at EcoRI and XhoI. SHP {Delta}128–139, DAX-1 {Delta}338–362, SHP Swap, and DAX-1 Swap mutants in pCDNA3 were subcloned into pGEX4T-1 and LexA at EcoRI and XhoI sites encoding the open reading frame. pEGFP SHP {Delta}128–139 was made by inserting PCR products encoding open reading frame of SHP {Delta}128–139 containing a 5' EcoRI and a 3' XhoI sites into pEGFP (BD Biosciences, CLONTECH). B42 EID-1 and B42 DP103 were obtained by yeast two-hybrid screening in mouse A20 cDNA library and mouse testis cDNA library respectively using SHP as bait. GST-fused construct of EID-1 was made by inserting PCR products encoding the open reading frame of EID-1 with EcoRI and XhoI sites into pGEX4T-1. All of the clones were verified by sequencing.

In Vitro Translation
mCAR, ERR{gamma}, LRH-1, SHP, SHP {Delta}129–139, DAX-1, DAX-1 {Delta}338–362, DAX-1 Swap, and SHP Swap were transcribed and translated in vitro using a coupled rabbit reticulocyte system (Promega Corp., Madison, WI) in the presence of [35S]methionine according to manufacturer’s instructions. The translated proteins were analyzed on 10% sodium dodecyl sulfate-polyacrylamide gels and visualized by autoradiography.

GST Pull-Down Assay
GST SHP, SHP {Delta}128–139, SF-1, mCAR, and EID-1 fusion proteins or GST protein only were expressed in Escherichia coli BL21 (DE3) pLys bacterial culture and induced by 0.2 mM isopropyl-1-thio-D-galactopyranoside and cells were extracted. GST fusion proteins were prebound with a 30-µl aliquot of glutathione-Sepharose beads and beads were incubated with transcribed and translated [35S]methionine-labeled proteins for 3–4 h at 4 C. Beads were washed three times with the washing buffer, and analyzed by sodium dodecyl sulfate-polyacrylamide gels and visualized by a phosphorimaging analyzer (Fuji, Tokyo, Japan). Ten percent of in vitro-translated proteins were used as input.

Cell Culture and Transient Transfection Assay
HEK 293, COS-7 (monkey kidney), and HepG2 cells (human hepatoma) were maintained with DMEM in the presence of 10% fetal bovine serum and antibiotics in humidified air containing 5% CO2 at 37 C. After incubation, cells were transfected, using Superfect reagent (QIAGEN GmbH, Hilden, Germany), according to the manufacturer’s instructions. Total DNA used in each transfection was adjusted to 1 µg by adding appropriate amount of pcDNA3 vector. Approximately 40–48 h post transfection, cells were harvested and the luciferase activity was measured and normalized by ß-galactosidase activity. Experiments were performed three times in duplicate.

Yeast Two-Hybrid Interaction Assay
Yeast two-hybrid interaction assays were performed as described previously (38). Briefly, LexA only or LexA-fused full-length human SHP, SHP {Delta}128–139, DAX-1 {Delta}338–362, SHP Swap, and DAX-1 Swap and B42-AD or ERR{gamma}, ERR{gamma} {Delta}AF-2, mCAR, mCAR-{Delta}AF-2, DP103, and EID-1 were transformed into Saccharomyces cerevisae EGY48 strain containing the ß-galactosidase reporter plasmid 8H18–34, and the transformants were selected on plates with appropriate selection markers. The ß-galactosidase assay on plates was carried out as described elsewhere (38).

Western Blot Analysis
pcDNA3/HA-SHP and -SHP {Delta}128–139 (5 µg) were transfected into HEK 293 cells, in 6-cm dishes using Superfect reagent. The cell lysates were prepared 48 h after transfection 100 µg of protein from each cell lysates were loaded and separated on a 10% denaturing polyacrylamide gel. The proteins were blotted on to Hybond-C extra nylon membranes and visualized with monoclonal HA antibody and ECL detection kit (Amersham Biosciences, Piscataway, NJ).

Confocal Microscopy
COS-7 cells were grown on coverslips and transfected with pEGFP-SHP, pEGFP SHP {Delta}128–139, and pCDNA3/HA-ERR{gamma} by the Lipofectamine method (Life Technologies, Inc., Gaithersburg, MD). At 24 h after transfection, cells were washed three times with cold PBS and fixed in 3.7% formaldehyde for 40 min. Fixed cells were mounted on glass slides with PBS and observed with a laser-scanning confocal microscope (Olympus Corp., Lake Success, NY). For detection of HA-pCDNA3 ERR{gamma}, cells mounted on glass slides were permeablized with 2 ml PBS containing 0.1% Triton X-100 and 0.1 M glycine at room temperature, incubated for 15 min, washed three times with 1x PBS, and blocked with 3% (wt/vol) BSA in PBS for 10 min at RT. Cells were incubated with primary anti-HA antibody for 1 h at 37 C, washed three times with 1x PBS, and incubated for 1 h with rhodamine-conjugated antirabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 37 C.

siRNA Experiments
The siRNAs for EID-1 were chemically synthesized (Dharmacon Research, Lafayette, CO), deprotected, annealed, and transfected according to the manufacturer’s instructions. The siRNA sequences are after: si EID-1 I, ACGGAGCCTTGCTAACGGGdTdT; si EID-1 II GAGCTTTTTTCACTGATGGdTdT. For the experiments in Fig. 7Go, C and D, HepG2 cells were transfected with siRNA using Oligofectamine reagent (QIAGEN). Forty-eight hours after transfection, cells were extracted for RT-PCR for EID-1 (30 cycles) and for ß-actin (25 cycles) as a control.

Structure Modeling
The BLAST search of SHP protein sequence without the additional amino acids against the Protein Data Bank resulted in four structures with more than 35% sequence homology. Based on the secondary structure alignments, the LBD structures of rat HNF4{alpha} (PDB ID 1M7W) and human ERR{gamma} (1KV6) are selected as homologous templates (27, 28). HNF4{alpha} and ERR{gamma} showed 41% and 37% sequence similarities over C-terminal 188 and 184 residues, respectively. The root mean square deviation between the two structures was 1.9 over 192 superimposed C{alpha} positions. The multiple sequence alignments were performed with CLUSTAL W (39). Models were generated with the MODELER module of QUANTA software (Accelrys, San Diego, CA) using established protocols. The root mean square deviations between the model and HNF4{alpha} and between the model and ERR{gamma} were 0.6 and 1.7 over 188 and 184 superimposed C{alpha} positions, respectively.


   ACKNOWLEDGMENTS
 
We thank Dr. Akiyoshi Fukamizu and Dr. Jae Woon Lee for generously providing their valuable DNA constructs. We also thank Dr. Kyung-Tae Kim for technical assistance on siRNA experiment.


   FOOTNOTES
 
This work was supported by Hormone Research Center Grant 2001G0201, the Brain Korea 21 program in 2003 (to H.S.C.) and KOSEF Grant 2003-0508.

Abbreviations: aa, Amino acid; AF-2, activation function 2; CAR, constitutive androstane receptor; CYP7A, cytochrome P450 7A1; DAX-1, dosage-sensitive sex-reversal AHC critical region on the X chromosome gene 1; DBD, DNA-binding domain; EID-1, E1A-like inhibitor of differentiation 1; ER, estrogen receptor; ERR{gamma}, ER-related receptor {gamma}; FXR, farnesoid X receptor; GST, glutathione-S-transferase; H, helix; HNF 4{alpha}, hepatocyte nuclear factor 4{alpha}; LBD, ligand-binding domain; LRH-1, liver receptor homolog 1; mCAR, mouse CAR; RXR, retinoid X receptor; SF-1, steroidogenic factor 1; SHP, small heterodimer partner; si, small interfering; SRC, steroid receptor coactivator.

Received for publication September 5, 2003. Accepted for publication February 5, 2004.


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NURSA Molecule Pages Link:

Nuclear Receptors:   DAX1  |  SHP  |  CAR  |  HNF4α  |  ERRγ  |  SF-1  |  LRH-1
Coregulators:   SRC-1



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 Nucleic Acids ResHome page
Y.-Y. Park, S.-W. Ahn, H.-J. Kim, J.-M. Kim, I.-K. Lee, H. Kang, and H.-S. Choi
An autoregulatory loop controlling orphan nuclear receptor DAX-1 gene expression by orphan nuclear receptor ERR{gamma}
Nucleic Acids Res., November 28, 2005; 33(21): 6756 - 6768.
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 Mol. Endocrinol.Home page
M. G. Yeo, Y.-G. Yoo, H.-S. Choi, Y. K. Pak, and M.-O. Lee
Negative Cross-Talk between Nur77 and Small Heterodimer Partner and Its Role in Apoptotic Cell Death of Hepatoma Cells
Mol. Endocrinol., April 1, 2005; 19(4): 950 - 963.
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 Mol. Endocrinol.Home page
J.-Y. Kim, H.-J. Kim, K. T. Kim, Y.-Y. Park, H.-A Seong, K. C. Park, I.-K. Lee, H. Ha, M. Shong, S. C. Park, et al.
Orphan Nuclear Receptor Small Heterodimer Partner Represses Hepatocyte Nuclear Factor 3/Foxa Transactivation via Inhibition of Its DNA Binding
Mol. Endocrinol., December 1, 2004; 18(12): 2880 - 2894.
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