Identification of a Novel Testicular Orphan Receptor-4 (TR4)-associated Protein as Repressor for the Selective Suppression of TR4-mediated Transactivation*

Yue YangDagger §, Xin WangDagger , Tiefei DongDagger , Eungseok KimDagger , Wen-Jye LinDagger , and Chawnshang ChangDagger ||

From the Dagger  George Whipple Laboratory for Cancer Research Departments of Pathology, Urology, Radiation Oncology, and Cancer Center, University of Rochester Medical Center, Rochester, New York 14642, the § Department of Surgery, Beijing Institute for Cancer Research, Beijing Cancer Hospital, Peking University 100036 Beijing, China, and the  Department of Surgery, First Hospital, Peking University, 100034, Beijing, China

Received for publication, July 16, 2002, and in revised form, December 16, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although many co-activators have been identified for various nuclear receptors, relatively fewer co-repressors have been isolated and characterized. Here we report the identification of a novel testicular orphan nuclear receptor-4 (TR4)-associated protein (TRA16) that is mainly localized in the nucleus of cells as a repressor to suppress TR4-mediated transactivation. The suppression of TR4-mediated transactivation is selective because TRA16 shows only a slight influence on the transactivation of androgen receptor, glucocorticoid receptor, and progesterone receptor. Sequence analysis shows that TRA16 is a novel gene with 139 amino acids in an open reading frame with a molecular mass of 16 kDa, which did not match any published gene sequences. Mammalian two-hybrid system and co-immunoprecipitation assays both demonstrate that TRA16 can interact strongly with TR4. The electrophoretic mobility shift assay suggests that TRA16 may suppress TR4-mediated transactivation via decreased binding between the TR4 protein and the TR4 response element on the target gene(s). Furthermore, TRA16 can also block the interaction between TR4 and TR4 ligand-binding domain through interacting with TR4-DNA-binding and ligand-binding domains. These unique suppression mechanisms suggest that TRA16 may function as a novel repressor to selectively suppress the TR4-mediated transactivation.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Steroid hormones play important physiological roles in cellular differentiation, development, and homeostasis, which function through binding to the specific receptors that belong to the nuclear receptor superfamily (1-5). The superfamily members may act as transcriptional activators or repressors for the modulation of gene expression in a wide variety of biological processes via binding to the hormone response element that is located mainly on the 5' promoter of target genes (2, 6, 7).

The human testis orphan receptor (TR4),1 a member of the nuclear receptor superfamily, was initially isolated from human prostate, testis, and hypothalamus cDNA libraries (8). Sequence analysis shows that TR4 has 615 amino acids with a calculated molecular mass of 67 kDa and has a very high homology with TR2 orphan receptor (9, 10). Northern blot analysis and in situ hybridization indicate that TR4 was expressed in a variety of tissues, including the central neural system (habenula, hippocampal pyramidal cell, and granule cells of both hippocampus and cerebellum) and peripheral organs (most abundantly expressed in spermatocytes of testis, with lower amounts in adrenal cortex, spleen, thyroid, prostate, and pituitary gland) (8, 11-13). TR4 may function as a transcriptional factor to regulate many signal transduction pathways. For example, it has been shown that TR4 can function as repressor to suppress the retinoic acid receptor-, retinoid X receptor-, vitamin D receptor-, androgen receptor (AR)-, and estrogen receptor-mediated transactivation (14-17). A recent report (18) demonstrated that TR4 could suppress the expression of the steroid 21-hydroxylase gene, which belongs to the cytochrome P450 superfamily and is one of the key enzymes in biosynthesis of adrenal steroid hormones, leading to the production of cortisol and aldosterone. On the other hand, TR4 also can function as enhancer to induce the ciliary neurotrophic factor receptor-alpha and thyroid hormone receptor (TR) (12, 19). A recent report (20) has shown that the TR4 is an important regulator of myeloid progenitor cell proliferation and development. The hormone response element for the TR4 is AGGTCA with a variety of direct repeats (DRs) (16, 21). The detailed mechanisms of how TR4 can differentially modulate its target genes, however, remain unclear. Whether some of those mechanisms may involve the association of co-repressors also remains unclear.

Here we show the isolation and characterization of a novel protein, TR4-associated protein (TRA16), localized in the nucleus that may function as a repressor for suppression of TR4-mediated transactivation via decreased binding between TR4 protein and TR4 response element (TR4RE) and blockage of TR4 dimerization.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
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Screening of Human Testis cDNA Library by Yeast Two-hybrid System-- The Cyto Trap yeast two-hybrid system (Stratagene) and a pMyr plasmid library (Stratagene) that consists of a DNA sequence encoding the myristylation membrane localization signal, which is fused to human testis cDNA, were used in the yeast two-hybrid screening. The pSos vector containing hSos gene was fused with the full length of TR4 cDNA using the BamHI cloning site as bait. As previously described (22), the library was screened by transformation with the pSos-TR4 bait constructor, and then the transformed yeast cells were selected for growth on both synthetic dropout/glucose minus uronolactone (-UL) agar and synthetic dropout/galactose (-UL) agar plates and cultured at 25 and 37 °C. The positive clones grow at 25 °C both on synthetic dropout/glucose (-UL) and synthetic dropout/galactose (-UL) plates but at 37 °C grow on synthetic dropout/galactose (-UL) plates and not on synthetic dropout/glucose (-UL) plates. One positive clone was selected that encodes about 700 bp of an unknown cDNA sequence and was called fragment TRA16. PMyr-TRA16 fragment and pSos-TR4 were co-transformed into the yeast to verify the interaction between these two proteins. All of the positive and negative controls were set up following the instruction manual of the system.

Rapid Amplification of cDNA Ends (RACE-PCR) for Full Length of TRA16-- The missing 5'-coding region was isolated by RACE-PCR technology (8). The gene-specific antisense primer used for 5' RACE-PCR was 5'-TCACACCTTCTCCCCAAGCACCCGCAGGTGGTA-3'. The PCR reaction condition was 95 °C for 2 min; 30 cycles of 95 °C for 20 s, 58 °C for 30 s, and 72 °C for 30 s; and then 72 °C for 7 min. The PCR product was sequenced for confirmation.

Northern Blot Analysis-- A human multiple tissues RNA blot, purchased from Clontech (catalog number 7760-1) was hybridized against a TRA16 cDNA probe labeled with [alpha -32P]dCTP. The beta -actin was used as a control for normalization, and the results were analyzed by autoradiography.

Large Scale Expression and Purification of TR4-DL and TRA16-- Plasmids TR4-DL containing DBD and LBD or the full length of TRA16 cDNAs were constructed into the pET expression system, and then large scale expression and purification were carried out from transformed Escherichia coli BL21 (DE3) bacteria following the manufacturer's procedures (Qiagen, Chatsworth, CA). Both proteins of TR4-DL and TRA16 were then confirmed directly on 15% SDS-PAGE followed by Coomassie Blue staining.

Production of Polyclonal Antibody against Human TRA16-- The large amount of TRA16 using pET expression system was purified from transformed E. coli BL21 (DE3) bacteria and used directly for immunizing rabbits.

The polyclonal TRA16 antibody was obtained from Cocalico Biologicals, Inc. (Reamstown, PA). It was diluted 1:100 in phosphate-buffered saline for immunofluorescence assay and 1:1000 for Western blot assay.

Immunocytofluorescence Assay-- Human cancer cell lines, H1299, MCF-7, and LNCaP, and monkey kidney cell line COS-1 were seeded on two-well Lab Tek Chamber slides (Nalge) for 48 h. Immunostaining was performed by incubating with anti-TRA16 polyclonal antibody or with anti-TR4 monoclonal antibody (antibody 17), followed by incubating with either fluorescein-conjugated goat anti-rabbit or anti-mouse antibodies. Coverslips were fixed on the glass slides with a drop of DAPI to stain the nucleus. The slides were observed under 40-fold magnification using a fluorescence microscope or confocal fluorescence microscope. The green signal represents the localization of TRA16, the red signal represents the localization of TR4, and the blue signal represents the DAPI-stained nucleus.

Cell Culture, Transient Transfection, and Reporter Gene Assay-- H1299, DU145, and COS-1 cells were cultured with Dulbecco's minimal essential medium containing penicillin (25 units/ml), streptomycin (25 µg/ml), and 10% fetal calf serum. Transfection was performed by modified calcium phosphate precipitation as previously described (22) or using SuperFect according to the manufacturer's procedures (Qiagen). The dual luciferase (LUC) reporter assay system was conducted according to the manufacturer's instructions (Promega). The Renilla luciferase reporter plasmid-simian virus 40 (pRL-SV40) was used as an internal control. The chloramphenicol acetyltransferase (CAT) assay was performed as described previously (23). The beta -galactosidase expression gene was co-transfected to normalize the transfection efficiency. A PhosphorImager visualized the CAT activity. For all of the transfections, the total amount of plasmids in each transfection was adjusted to be equal by the addition of backbone vectors.

Mammalian Two-hybrid Assay-- COS-1 cells were transiently co-transfected with 3 µg of reporter plasmid pG5-LUC and 4 µg of both the GAL4DBD and VP16-hybrid expression plasmids as described previously (22). The pRL-SV40 plasmid (10 ng) was co-transfected for normalization of transfection efficiency. The LUC assay was performed 24 h after transfection.

Coupled in Vitro Transcription and Translation-- Plasmids containing the full-length TR4 or TRA16 cDNAs were in vitro transcribed and translated directly by a TNT-coupled reticulocyte lysate system (Promega) as previously described (23). The in vitro translated products were then analyzed directly by SDS-PAGE.

The in Vitro or in Vivo Co-immunoprecipitation of TR4, TR4-N, or TR4-DL and TRA16-- For in vitro co-immunoprecipitation, the TR4, TR4-N terminus, DL (containing DBD and LBD), TRA16, and pcDNA4c-His control proteins, transcribed and translated by the TNT-coupled reticulocyte lysate system, were purified as instructed by the manufacturer (Promega). For immunoprecipitation (IP) of each mixture, 5 µl of each in vitro translated 35S-labeled proteins were incubated for 1 h at 30 °C in various combinations as indicated and then incubated with an antibody bound to protein G-agarose beads (Santa Cruz Biotechnology) at 4 °C for another 2 h. After washing each mixture four times, each complex was loaded onto 15% SDS-PAGE gel and visualized by autoradiography. For in vivo co-IP, 10 µg of pcDNA4c-His or pcDNA4c-His-TRA16 was transiently transfected in H1299 cells for 48 h, and the lysates were harvested. Then anti-His probe antibody and protein G-agarose beads were added to 500 µg of protein lysate for 2 h for IP of pcDNA4c-His-TRA16 and endogenous TR4 complex. After washing each mixture four times, 15% SDS-PAGE gel was used to separate the complex. After transferring to the membrane, the expression was detected by anti-TR4 antibody or anti-His probe antibody. Each cell lysate input was loaded to show the expression of TR4 and pcDNA4c-His-TRA16.

Stable Transfection of COS-1 Cells-- COS-1 cells, which are TR4-negative and show little expression of TRA16, were transfected with pBig or pBig-TRA16 using SuperFect (Qiagen). The cells were then selected using 100 µg/ml hygromycin B (24), and a single colony was chosen, amplified, and confirmed by reporter gene assay.

Nuclear Extract Preparation-- The nuclear protein extraction was prepared as described previously (25). Briefly, the cells were collected and pelleted with 10 s of centrifugation and then resuspended in 5 volumes of cold Buffer A. The samples were put on ice for 10 min, vortexed for 10 s, and centrifuged for 10 s, and the supernatant fraction was separated (cytosol protein). The pellet was resuspended in 100 µl of cold Buffer C and incubated on ice for 20 min for high salt extraction, centrifugation for 2 min at 4 °C was used to remove the cellular debris, and then the supernatant fraction containing the nuclear protein was used for experimentation.

Electrophoretic Mobility Shift Assay (EMSA)-- The EMSA was performed as described previously (19, 21). Briefly, the reaction was performed by incubating the 32P-labeled DR1-TR4RE probe with 10 µg nuclear extracts of different cells or only with the purified proteins of TR4-DL and TRA16 from bacteria. For the antibody supershift analyses, 1 µl of anti-TR4 antibody (antibody 15 or C-16) was added to the reaction, DNA-protein complexes were resolved on a 5% native gel for electrophoresis, and the gel was dried and then analyzed by autoradiography.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Orphan Receptor TR4-associated Protein, TRA16-- To further understand the function and mechanism of TR4, a human TR4 was used as bait with the yeast two-hybrid system to fish out the interacting proteins from testis cDNA library. One clone, which can interact with TR4, was isolated and named human TRA16 (Fig. 1). Using a specific primer (5'-TCACACCTTCTCCCCAAGCACCCGCAGGTGGTA-3'), we applied the RACE-PCR technology with the isolated cDNA insert as template to amplify the full-length human TRA16 from the Marathon human testis cDNA library. Sequence analysis revealed that TRA16 is a novel protein whose sequence did not match any known published genes. The open reading frame between the first ATG and terminal TGA encodes 139 amino acids with the predicted molecular mass of 16 kDa (GenBankTM accession number AY101377). Genomic sequence analysis indicates that human TRA16 is located at human chromosome 19p12.


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Fig. 1.   cDNA sequences and deduced amino acid sequences of human TRA16. The sequence data have been deposited into GenBankTM (GenBankTM accession number AY101377). The nucleotide sequences and the deduced amino acid sequences of TRA16 are presented and numbered on the left.

Distribution of TRA16 and TR4-- Northern blot analysis indicated that TRA16 was expressed as a mRNA transcript of about 1 kb in several human tissues, such as heart, placenta, liver, skeletal muscle, kidney, and pancreas as shown in Fig. 2A. Interestingly, the distribution of TRA16 was found to be relatively higher in heart, skeletal muscle, and pancreas. In contrast, the expression of TRA16 was lowest in normal human brain and lung. To further check the TRA16 at protein level, we generated the anti-TRA16 antibody (Cocalico Biologicals, Inc.) to stain TRA16 protein. As shown in Fig. 2B, both the endogenous TRA16 and transiently transfected pcDNA4c-TRA16 expressed TRA16 are recognized by anti-TRA16 antibody. Furthermore, we determine the subcellular localization of the TRA16 in several cell lines using immunocytofluorescence assay. As shown in Fig. 2C, we found that TRA16 was located mainly in the nucleus detected by anti-TRA16 antibody.


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Fig. 2.   Distribution of endogenous TRA16 and TR4. A, Northern blot analysis of TRA16 mRNA levels in human normal tissues. Human multiple tissue Northern blot (Clontech, catalog number 7760-1) was used to determine the expression of TRA16 in different human tissues, including heart, whole brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. The mRNA species migrating ~1 kb of TRA16 was detected. The beta -actin expression was used an equal loading control. B, Western blot assay of endogenous TRA16 only and transfected TRA16 protein level. H1299 cells were transiently transfected either with 10 µg of pcDNA4c-His or pcDNA4c-His-TRA16 as indicated for 24 h. After harvesting, 50 µg of whole cell lysates were run in the 15% SDS-PAGE gel. The anti-TRA16 antibody or anti-His tag antibody was used to immunoblot the membrane to detect the expression of TRA16. beta -Actin expression level was used as a loading control. C, immunocytofluorescence detection of TRA16 in different cell lines. H1299, COS-1, MCF-7, and LNCaP cells were prepared for immunostaining with anti-TRA16 polyclonal antibody, followed by incubation with fluorescein-conjugated goat anti-rabbit (ICN). The green signal represents the localization of TRA16, and the blue signal represents the DAPI-stained nucleus. D, Western blot analysis of endogenous TR4 protein from various cell lines. The cells indicated were cultured in 10-cm dishes and harvested, and 50 µg of total protein was run in a 10% SDS-PAGE gel. After transferring to the membrane, the anti-TR4 antibody was used to immunoblot the membrane to detect the expression of TR4. The beta -actin expression level was used as a loading control.

For comparison, we also showed the expression of TR4 in various cell lines with Western blot assay. As shown in Fig. 2D, the expression of TR4 protein in H1299, PC-3, 293T (human kidney cell line), LNCaP, and CWR22 cell lines (lanes 1, and 5-8) is higher compared with the expression in DU145, HTB-14, and COS-1 cell lines, which expressed little TR4 (lanes 2-4).

The in Vivo and in Vitro Interaction between TR4 and TRA16-- To further confirm that the interaction between TR4 and TRA16 in yeast cells may also occur in mammalian cells, we applied the immunocytofluorescence assay to test whether both TR4 and TRA16 are localized in the same cells. As shown in Fig. 3A, the majority of TRA16 signal (panel 2, green) could be detected together with TR4 signal (panel 1, red) on the nuclear membrane (panel 3, yellow). This observation provided strong in vivo evidence that TRA16 co-localizes with TR4 on the nuclear membrane. Using the mammalian two-hybrid system to assay their direct interaction in COS-1 cells, we also found that the GAL4-TRA16 can interact strongly with VP16-TR4 (Fig. 3B, lane 4). We then used co-IP with in vitro translated TR4 and His tag fused TRA16 to assay their interaction. As shown in Fig. 3C (lane 3), the precipitated complex containing in vitro translated 35S-labeled TR4 and in vitro translated 35S-labeled pcDNA4c-His was immunoprecipitated with the polyclonal anti-His tag antibody. The TR4 protein could be clearly detected in the immunoprecipitated complex of both in vitro translated 35S-labeled TR4 and in vitro translated 35S-labeled TRA16 (Fig. 3C, lane 4). To further test that the endogenous TR4 could interact with the exogenous TRA16, the whole cell extracts from H1299 cells only transfected with TRA16 were immunoprecipitated with polyclonal anti-His tag antibody, and the results show that TR4 and TRA16 can definitely co-immunoprecipitate (Fig. 3D). Together, the results from Fig. 3 (A-D), using four different interaction assays, demonstrate that TR4 can interact with TRA16 in mammalian cells.


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Fig. 3.   Interaction between TR4 and TRA16. A, immunocytofluorescence detection of co-localization of TR4 and TRA16 in H1299 cells. The H1299 cells were prepared for immunostaining with anti-TRA16 polyclonal antibody or anti-TR4 monoclonal antibody (antibody 17) and then followed by incubating with either fluorescein-conjugated goat anti-rabbit or anti-mouse antibodies. The green signal represents the localization of TRA16, the red signal represents the localization of TR4, the yellow signal represents co-localization of the both TRA16 and TR4, and the blue signal represents the DAPI-stained nucleus. B, TRA16 can interact with TR4 in mammalian two-hybrid system. COS-1 cells in 60-mm dishes were transiently co-transfected with 3 µg of reporter plasmid pG5-LUC and 4 µg each of GAL4DBD, VP16, VP16-TR4, and GAL4-TRA16 in various combinations as indicated. The luciferase assay was performed 24 h after transfection. All of the values represent the means ± S.D. of three independent experiments. C, in vitro co-IP of TR4 and TRA16. 5 µl of each complex in vitro translated 35S-labeled TR4, TRA16, or pcDNA4c-His control proteins (mock) described under "Experimental Procedures" were loaded onto 15% SDS-PAGE gel as indicated and visualized by autoradiography. D, 10 µg of pcDNA4c-His or pcDNA4c- His-TRA16 was transiently transfected in H1299 cells as indicated for 48 h, the lysates were harvested, and then anti-His probe antibody and protein G-agarose beads were added to 500 µg of protein lysate for 2 h for IP of pcDNA4c-His-TRA16 and endogenous TR4 complex. 15% SDS-PAGE gel was used to separate the complex. After transferring to the membrane, the expression was detected by anti-TR4 antibody or anti-His sprobe antibody. Each cell lysate input was loaded to show the expression of TR4 and pcDNA4c-His-TRA16.

TRA16 Represses TR4-mediated Transactivation-- The potential effects of TRA16 on TR4-mediated transactivation were tested through LUC and CAT reporter assays. The full-length TRA16 and TR4 in eukaryotically expressed vectors (pSG5-TRA16, or pcDNA4c-TRA16 and pCMX-TR4) were co-transfected with a luciferase reporter HCR-1-LUC containing a TR4RE2 in COS-1 cells. As shown in Fig. 4A, the TR4RE-LUC activity induced by pCMX-TR4 could be significantly suppressed by TRA16 in a dose-dependent manner. Using H1299 cells, which express stronger endogenous TR4 protein levels (shown in Fig. 2D), LUC reporter assays confirm that exogenously transfected TRA16 can suppress TR4-mediated transactivation (Fig. 4B). To reduce any potential artifact effect related to any particular TR4RE reporter assay, we also replaced TR4 HCR-1-LUC reporter with other reporters containing different TR4REs, such as CpFL4-LUC and DR4-TK-CAT (16). As shown in Fig. 5A and B, addition of TRA16 can repress TR4-mediated transactivation in all reporter assays in a dose-dependent manner.


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Fig. 4.   TRA16 can suppress TR4 target gene expression in a dose-dependent manner. A, COS-1 cells were transiently co-transfected with 3 µg of reporter plasmid HCR-1LUC, 1 µg of TR4, and increasing amounts of TRA16 expression plasmid as indicated for 24 h. B, H1299 cells were transiently co-transfected with 3 µg of reporter plasmid HCR-1LUC and increasing amounts of TRA16 expression plasmid for 24 h. All of the values represent the means ± S.D. of three independent experiments.


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Fig. 5.   TRA16 suppresses TR4-mediated transactivation with different reporter assay and in TRA16 stably transfected COS-1 cells. A, TRA16 repression of TR4-mediated CpFL-LUC transcriptional activity comparison with HCR-1LUC. COS-1 cells were co-transfected with 3 µg of reporter plasmid CpFL-LUC with both ratios of pCMX-TR4 and pSG5-TRA16 (1:3 and 1:6). B, COS-1 cells were transiently co-transfected with 3 µg of reporter plasmid DR4-TK-CAT, 1 µg of TR4, and increasing amounts of TRA16 expression plasmid for 24 h. A PhosphorImager visualized the CAT activity. C, COS-1 (pBig-TRA16) and COS-1 (pBig) stably transfected cells were transiently co-transfected with 3 µg of HCR-1 LUC reporter plasmid, 10 ng of SV40-pRL internal control plasmid, and 1 µg of TR4 for 16 h. The cells were then treated with 6 µl of Me2SO (as negative control) or 6 µg/ml doxycycline for another 24 h and then harvested for luciferase assay. All of the values represent the means ± S.D. of three independent experiments.

To avoid the effect of transiently transfected TRA16, we then stably transfected COS-1 cells with pBig-TRA16 using a doxycycline-inducible system. As shown in Fig. 5C, treatment of the stably transfected pBig-TRA16 COS-1 cells with doxycycline results in significant suppression of TR4-mediated transactivation. In contrast, COS-1 cells stably transfected with the pBig vector shows little effect on TR4-mediated transactivation in the presence of doxycycline.

To test the specificity of TRA16 suppressive effect on other nuclear receptor transactivation, we used mouse mammary tumor virus (MMTV)-LUC reporter (22) to determine the influence of TRA16 on the transactivation of AR, progesterone receptor, and glucocorticoid receptor. Interestingly, in contrast to suppressing TR4-mediated transactivation, TRA16 has only a slight modulation effect on these three classic steroid receptors transactivation in COS-1 cells (Fig. 6). Together, these data demonstrate clearly that the suppression of TR4-mediated transactivation by TRA16 is selective.


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Fig. 6.   The suppression of TR4-mediated transactivation by TRA16 is selective. COS-1 cells were transiently co-transfected with 3 µg of reporter plasmid MMTV-LUC and 0.5 µg of different steroid receptors (AR, progesterone receptor, or glucocorticoid receptor) and doses (1.5 and 3 µg) of pSG5-TRA16. After 24 h of transfection, the cells were treated with 10 nM of synthetic steroids (dihydrotestosterone for AR, progesterone for progesterone receptor or dexamethasone for glucocorticoid receptor) and then harvested after 24 h for luciferase assay. All of the values represent the means ± S.D. of three independent experiments.

The Potential Mechanism for the TRA16 to Suppress TR4-mediated Transactivation-- We applied Western blot analysis to see whether the addition of TRA16 could influence the TR4 expression at the protein level. As shown in Fig. 7 (A and B), the addition of TRA16 at different doses shows little influence on either the transfected TR4 protein level in COS-1 cells or the endogenous TR4 protein level in H1299 cells. We then assayed the potential influence of TR4 nuclear translocation by the addition of TRA16. As shown in Fig. 7C, there is little influence in the endogenous TR4 protein in whole H1299 cell extract in the presence or the absence of TRA16 (lane 2 versus lane 1), which is consistent with results in Fig. 7B. Furthermore, the amount of TR4 protein remains relatively consistent in either nuclear fraction or cytosol fraction after the addition of TRA16 (Fig. 7C, lane 4 versus lane 3 and lane 6 versus lane 5). Together, the results from Fig. 7C suggest that TRA16 has little influence on the nuclear translocation of TR4.


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Fig. 7.   TRA16 effects on TR4 protein expression, localization, and abrogation through HDAC activity. A, COS-1 cells were transiently co-transfected with 1 µg of TR4 and two doses (6 and 12 µg) of TRA16 for 24 h, and then the medium was changed for another 16 h. After harvesting, 50 µg of whole cell lysate from each sample was run in the 10% SDS-PAGE gel and transferred to the membrane, and the anti-TR4 antibody was used to immunoblot the membrane to detect the expression of TR4. Anti-beta -actin antibody was used to detect beta -actin expression level as a loading control. B, H1299 cells were transiently transfected with increasing amounts of TRA16 (2-6 µg) for 38 h. After harvesting, 50 µg of whole cell lysate from each sample was run on the 10% SDS-PAGE gel and transferred to the membrane, and the anti-TR4 antibody was used to immunoblot the membrane to detect the expression of TR4. C, H1299 cells were transiently transfected without/with 12 µg of TRA16. After harvesting, the nuclear protein extraction was prepared as described under "Experimental Procedures," and cytosol protein and whole cell lysate were prepared. 20 µg of different samples as indicated were run in the 15% SDS-PAGE gel and transferred to the membrane, and the anti-TR4 and anti-His-tag antibodies were used to immunoblot the membrane to detect the expression of TR4 and TRA16. D, COS-1 cells were transiently co-transfected with 3 µg of reporter plasmid HCR-1LUC with both ratios of TR4 and TRA16 (1:3 and 1:6) for 24 h. The cells were treated with 50 or 100 nM of TSA or ethanol for another 24 h and then harvested for the luciferase assay. All of the values represent the means ± S.D. of three independent experiments.

To test the hypothesis that suppression of TR4-mediated transactivation by TRA16 might involve the modulation of histone deacetylase (HDAC) activity, we first examined the effect of trichostatin A (TSA), a specific inhibitor of HDACs (26), on the TRA16 suppression of TR4-mediated transactivation. As shown in Fig. 7D, the addition of TSA can dramatically enhance TR4-mediated transactivation in a dose-dependent manner (lanes 2 versus lanes 6 and 10). However, the addition of TSA cannot reverse significantly the TRA16 suppressed TR4-mediated transactivation in COS-1 cells (Fig. 7D, lane 6 versus lanes 7 and 8 or lane 10 versus lanes 11 and 12), suggesting that HDAC activity may not play major roles in the TRA16 suppressed TR4-mediated transactivation.

We then focused on the influence of TRA16 on the TR4 binding to the TR4RE of the target genes. Using 32P-labeled DR1-TR4RE (AGGTTAAAGGTCT) as probe, we applied the EMSA to see whether adding TRA16 can influence the TR4 binding to DR1-TR4RE. As shown in Fig. 8A, TR4 binds specifically to DR1-TR4RE, and this specific binding (open arrow, lanes 3 and lane 4) can be supershifted by adding anti-TR4 antibody (solid arrow, lane 4). In contrast, TR4 failed to bind to mutant DR1-TR4RE (AGGTTAAATGACT), even when adding anti-TR4 antibody (lanes 1 and 2). The addition of different doses of TRA16 can then reduce the binding between TR4 and DR1-TR4RE as shown in Fig. 8B (lane 1 versus lanes 2 and 3), even if supershifted by adding anti-TR4 antibody (Fig. 8B, lane 4 versus lanes 5 and 6). Then we demonstrated that the addition of TRA16 could reduce the binding between DR1-TR4RE and endogenous TR4 expressed in H1299 cells as shown in Fig. 8C (lane 2 versus lane 3 and lane 4 versus lane 5). To avoid any potential effects involved in the DNA binding assay, we also purified two proteins of TR4-DL (containing DBD and LBD of TR4) and TRA16 from E. coli strain DE3 and then checked with EMSA. As shown in Fig. 8D, TRA16 definitely can decrease TR4 binding to its target gene, even with the addition of anti-TR4 antibody (lane 3 versus lane 4 and lane 5 versus lane 6). Together, the results from Fig. 8 clearly demonstrate that TRA16 may suppress TR4-mediated transactivation via the interruption of the binding between TR4 and TR4RE on its target gene.


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Fig. 8.   Inhibition of TR4 DNA binding by TRA16. A, DU145 cells were transiently transfected with 1 µg of TR4 for 24 h. Then the medium was changed for another 16 h, and the cells were harvested; the nuclear protein extraction was prepared as described under "Experimental Procedures." EMSA was performed using the 32P-radiolabeled wild-type DR1-TR4RE (AGGTTAAAGGTCT) (wt) or mutated DR1-TR4RE (AGGTTAAATGACT) (mt) oligonucleotides as probe with 10-µg nuclear extracts isolated from DU145 cells as indicated. Addition of the TR4 (antibody 15) monoclonal antibody produced a TR4-DNA supershift band (solid arrow) in lane 4 compared with DR1-TR4RE-binding proteins (open arrow) in lane 3. B, COS-1 cells were transiently co-transfected with 1 µg of TR4 and doses of TRA16 (6 and 12 µg), and nuclear protein extraction was prepared as described under "Experimental Procedures." EMSA was performed using the 32P-radiolabeled DR1-TR4RE oligonucleotides with 10 µg of nuclear extracts. Addition of the TR4 (antibody 15) monoclonal antibody produced a TR4-DNA supershift band (solid arrow) in lanes 4-6 and separated TR4 from the rest of DR1-TR4RE-binding proteins (open arrow) in lanes 1-3. C, H1299 cells were transiently transfected with 12 µg of TRA16 only, and nuclear protein extract was prepared as described under "Experimental Procedures." EMSA was performed using the 32P-radiolabeled DR1-TR4RE oligonucleotides with 10 µg of nuclear extracts isolated from H1299 cells. Addition of the TR4 (antibody 15) monoclonal antibody produced a TR4-DNA supershift band (solid arrow) in lanes 4 and 5 and separated TR4 from the rest of DR1-TR4RE-binding proteins (open arrow) in lanes 2 and 3. 32P-Labeled DR1-TR4RE oligonucleotides probe only was used as a loading control in lane 1. D, EMSA was performed using the 32P-labeled DR1-TR4RE oligonucleotides with both TR4-DL containing DBD and LBD of TR4, and TRA16 purified from the E. coli strain DE3 bacteria (1:5). Addition of the TR4 (antibody C-16) polyclonal antibody produced a TR4-DNA supershift band (solid arrow) in lanes 5 and 6 and separated TR4 from the rest of DR1-TR4RE-binding proteins (open arrow) in lanes 3 and 4. 32P-Labeled DR1-TR4RE oligonucleotides probe with mock or mock and TR4 antibody was used as negative control in lanes 1 and 2.

We also checked whether dimerization of TR4 might play any role in the TRA16 suppression of TR4 transactivation. We first demonstrated that TR4 can form the dimers via the interaction between TR4 and TR4-LBD (amino acids 224-615) in the mammalian two-hybrid assay as shown in Fig. 9A (lane 4). Interestingly, the addition of TRA16 could then suppress the interaction between TR4 and TR4-LBD significantly (Fig. 9, lane 5). In contrast, the addition of AR showed little influence on the interaction between TR4 and TR4-LBD (Fig. 9A, lane 6). Our early report suggested that AR could also function as a repressor to suppress TR4 transactivation (16). These contrasting effects between TRA16 and AR strongly suggest that different TR4 repressors may go through different mechanisms to suppress TR4-mediated transactivation. To determine which region of TR4 dimerizes with TR4-LBD and mediates the protein-protein interaction with TRA16, the TR4 N terminus (amino acids 1-125) and TR4-DL (amino acids 125-615) including DBD and LBD of TR4 were constructed as shown in Fig. 9B. With the mammalian two-hybrid assay, we found that TR4-DL can interact with TR4-LBD as shown in Fig. 9B (lane 6). In vitro co-IP experiments were performed to demonstrate that TRA16 could be co-immunoprecipitated with TR4DL but not TR4-N (Fig. 9C), which was a competitor to interfere with the interaction between TR4-DL and TR4-LBD.


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Fig. 9.   TRA16 blocks the TR4 dimerization in mammalian two-hybrid assay. A, COS-1 cells were transiently co-transfected with 3 µg of reporter plasmid pG5-LUC and 3 µg each of GAL4DBD, VP16, VP16-TR4, GAL4-TR4-LBD, pcDNA4c, pcDNA4c-TRA16, and pSG5-AR in various combinations as indicated. The luciferase assay was performed 24 h after transfection. All of the values represent the means ± S.D. of three independent experiments. B, diagram of each construct and localization of the interaction domain within TR4. COS-1 cells were transiently co-transfected with 3 µg of reporter plasmid pG5-LUC and 3 µg each of GAL4DBD, VP16, VP16-TR4-N, VP16-TR4-DL, and GAL4-TR4-LBD in various combinations as indicated. The luciferase assay was performed 24 h after transfection. All of the values represent the means ± S.D. of three independent experiments. C, in vitro co-IP to detect which region of TR4 interacts with TRA16. 5 µl of each complex in vitro translated 35S-labeled TR4-N, TR4-DL, or TRA16 immunoprecipitated with anti-TR4 antibodies (antibody 15 is recognized by TR4-N; antibody c-16 is recognized by TR4-DL) described under "Experimental Procedures" was loaded onto a 15% SDS-PAGE gel as indicated and visualized by autoradiography.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nuclear hormone receptors comprise a huge family of transcriptional factors that represent important regulators in cellular proliferation, differentiation, development, and homeostasis (7, 27). The transcriptional activity of nuclear hormone receptors has been generally recognized to be modulated by co-activators or co-repressors (28, 29). To date, many co-activators have been identified for various nuclear receptors, but relatively fewer co-repressors have been isolated and characterized.

In general, nuclear receptor co-activators (such as p160/SRC, p300/CBP, and P/CAF) act through an inherent histone acetyltransferase activity to increase the level of histone acetylation and enhance the transcriptional activity of ligand bound receptors (30-34). The molecular studies demonstrated that the helical motifs containing the LXXLL core consensus in co-activators play important roles for the interaction with nuclear receptors (35, 36). The nuclear receptor co-repressor (N-CoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT) also contain three repeated receptor interaction domains that include the conserved hydrophobic core motif (I/L)XXII, which can interact with unliganded nuclear receptors, such as retinoic acid receptor alpha , thyroid hormone receptor, and the orphan receptor, chicken ovalbumin upstream promoter-transcription factor I (37-43). Both N-CoR and SMRT proteins function as co-repressors with HDACs activity that can recruit a complex containing Sin3, HDACs, and several additional proteins (44-48). Lavinsky et al. (49) reported that both N-CoR and SMRT might interact with ER in the presence of the antagonist trans-hydroxytamoxifen, but forskolin or epidermal growth factor, which can change trans-hydroxytamoxifen function from antagonist to agonist, can decrease the ER/N-CoR interaction via phosphorylation of ER at amino acid Ser118. Their data suggested that multiple signal transduction pathways regulate the actions of both N-CoR and SMRT.

On the other hand, there are some co-repressors that can exert their suppression on nuclear receptors via interfering with the binding between nuclear receptors and their DNA response elements. For example, a TR uncoupling protein may inhibit TR and retinoic acid receptor-mediated transactivation via binding to the TR hinge region and the N-terminal portion of the ligand-binding domain (50). Another co-repressor, calreticulin, was suggested to be able to inhibit the transactivation of glucocorticoid receptor and AR via interruption of the binding between receptors and DNA response elements (51, 52). Cyclin D1, a cell cycle regulating protein that functions as an AR co-repressor, may rely on its cell cycle regulating function to suppress AR (53). There are also several proteins, such as a small ubiquitous nuclear co-repressor that may function as nuclear receptor co-repressor via forming complexes with N-CoR (54). Finally, Mathur et al. (55) found that polypyrimidine tract-binding protein-associated splicing factor could suppress TR and retinoid X receptor mediated transcription through a novel polypyrimidine tract-binding protein-associated splicing factor/Sin3-mediated pathway to recruit HDACs to the receptor DBD.

For the suppression of TR4-mediated transactivation, early reports suggested that AR could also function as repressor to interrupt the binding between TR4 and TR4 DNA response elements (16). Here we report the cloning and characterization of a novel TR4 repressor, TRA16, whose sequence was not previously reported. The amino acid sequence comparison also shows that TRA16 lacks the classic hydrophobic core motif (I/L)XXII of other repressors. The other interesting characteristic was the distribution of TRA16. We also detected the expression of TRA16 mRNA level in different cell lines with Northern blot assay (data not shown) with the same result as immunocytofluorescence assays. TRA16 expression in human lung cancer cell line H1299 cells was found to be higher than in normal human lung tissue, suggesting that TRA16 could play roles in some kinds of cancer tissues. TSA showed little effect on the TRA16-mediated suppression of TR4 transactivation, suggesting that HDAC activity may not play any roles for TRA16. Instead, we demonstrated that TRA16 may suppress TR4 transactivation via either interruption of the interaction between TR4 and its DNA response element or as competitor to block the interaction between TR4-DL and TR4-LBD. Together, our data suggest that TRA16 may function as a novel repressor to suppress TR4 function.

    ACKNOWLEDGEMENT

We thank Karen Wolf for preparation of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK56784 and a George Whipple Professorship Endowment.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. E-mail: chang@urmc.rochester.edu.

Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M207116200

2 E. Kim and C. Chang, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: TR4, testicular orphan receptor-4; TRA16, TR4 associated protein; AR, androgen receptor; TR, thyroid hormone receptor; TR4RE, TR4 response element; TR4-N, TR4-N terminus; TR4-DL, TR4 DNA binding domain (DBD) and ligand binding domain (LBD); DR, direct repeat; HDACs, histone deacetylases; TSA, Trichostatin; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; LUC, luciferase; -UL, minus uronolactone; RACE, rapid amplification of cDNA ends; IP, immunoprecipitation; N-CoR, nuclear receptor co-repressor; SMRT, silencing mediator for retinoid and thyroid hormone receptor; DAPI, 4'6-diamidino-2-phenylendole; MMTV, mouse mammary tumor virus.

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