The Interaction of TR1-N Terminus with Steroid Receptor Coactivator-1 (SRC-1) Serves a Full Transcriptional Activation Function of
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《内分泌学杂志》
Department of Integrative Physiology (T.I., W.M., N.K.), Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan
Core Research for Evolutional Science and Technology (CREST) (T.I., W.M., N.K.), Japan Science and Technology Corporation (J.S.T.), Kawaguchi, Saitama 332-0012, Japan
Brigham and Women’s Hospital and Harvard Medical School (T.I., A.T., W.W.C., N.K.), Boston, Massachusetts 02115
Division of Endocrinology and Metabolism (A.T.), Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo 105-8470, Japan
Abstract
Steroid receptor coactivator-1 (SRC-1) plays a crucial role in nuclear receptor-mediated transcription including thyroid hormone receptor (TR)-dependent gene expression. Interaction of the TR-ligand binding domain and SRC-1 through LXXLL motifs is required for this action. However, potential interactions between the TR1-N terminus (N) and SRC-1 have not been explored and thus are examined in this manuscript. Far-Western studies showed that protein construct containing TR1-N + DNA binding domain (DBD) bound to nuclear receptor binding domain (NBD)-1 (amino acid residue, aa 595–780) of SRC-1 without ligand. Mammalian two-hybrid studies showed that NBD-1, as well as SRC-1 (aa 595-1440), bound to TR1-N+DBD in the absence of ligand in CV-1 cells. However, NBD-2 (aa 1237–1440) did not bind to this protein. Glutathione-S-transferase pull-down studies showed that TR1-N (aa 1–105) bound to the broad region of SRC-1-C terminus. Expression vectors encoding a series of truncations and/or point mutations of TR1 were used in transient transfection-based reporter assays in CV-1 cells. N-terminal truncated TR1 (N-TR1) showed lower activity than that of wild-type in both artificial F2-thyroid hormone response element and native malic enzyme response element. These results suggest that there is the interaction between N terminus of TR1 and SRC-1, which may serve a full activation of SRC-1, together with activation function-2 on TR1-mediated transcription.
Introduction
THE NUCLEAR HORMONE receptors (NRs) are transcription factors that regulate target gene transcription in response to ligands such as steroid and thyroid hormones (THs) (1, 2). The DNA binding domain (DBD) of NR binds to the cognate hormone response element, located in the promoter regions of target genes. One of these, TH receptor (TR) belongs to type II nuclear hormone receptors that form heterodimers with retinoid X receptor (3, 4, 5).
At least three functional isoforms of TR (TR1, 1, and 2) have been reported to be expressed in mammalian cells at protein level (3, 4, 5). These TRs are highly similar within their DNA binding, hinge, and ligand binding domains (LBDs), but are divergent in their amino-terminal regions (3, 4, 5). These forms are expressed in distinct developmental- and tissue-specific manners, suggesting that they may possess unique functional properties (2).
In mammalian cells, TH binding to TR results in dissociation of corepressor complexes followed by recruitment of coactivator complexes to activate transcription. Functional analyses of NRs have shown that there are two major activation domains: activation function (AF)-1 and AF-2 (3, 4). The extreme C-terminal region of the LBD (AF-2) exhibits ligand-dependent transactivation. AF-2 interacts with coactivators, such as SRC-1 (6, 7), in a ligand-dependent manner (1, 8, 9). Members of SRC-1 family (1, 8, 10, 11, 12, 13), cAMP response element binding protein-binding protein (CBP) (1, 8, 14) and p300/CBP-associated factor (1, 8, 15), and participate in a protein complex on AF-2 possess intrinsic histone acetyltransferase activity. These cofactors may remodel chromatin structure, allowing the efficient access of basal transcriptional machinery to DNA (1). In addition to these coactivators, several other proteins, such as coactivator-associated arginine methyltransferase-1 (16) and TR binding protein (17)—this is also named as AIB3/ASC-2/RAP250/PRIP/NRC (18, 19, 20, 21, 22)—are involved in this complex and contribute to the precise transcriptional regulation.
On the other hand, the AF-1 region, located within the N-terminal region, contains a ligand-independent activation function (23, 24). The potency of the AF-1 transactivation domain varies widely, depending on the cell and promoter context (25, 26, 27, 28). The AF-1 domain of TR1 is represented by 19 amino acids located between residues 69 and 89 (29). Unlike the AF-2 domain, this AF-1 domain is not homologous to those of TR1, TR2, or the other steroid hormone receptors (27). This observation suggests that the AF-2 domain may evolutionarily represent the primary transactivation domain of TR, whereas the AF-1 domain, with differences in diverse sequences in the amino termini, may provide the molecular basis for differential activities of different TR isoforms to allow unique promoter- or cell-specific actions. Thus, in the present study, to examine the role of the TR1-N terminus on SRC-1-mediated transactivation, we investigated the binding between TR1-N terminus and SRC-1.
Materials and Methods
Plasmids
cDNAs encoding glutathione-S-transferase (GST)-fusion proteins including the LBD of the rat TR1 (aa 174–461) has been described previously (7). GST-TR1-N terminus + DBD (aa 1–174), GST-TR1-N terminus (N) (aa 1–105), GST-TR1 (aa 1–54), GST-TR1 (aa 55–105), and GST-TR1-DBD (aa 106–174) were constructed by inserting the PCR-amplified fragments from TR1 into the EcoRI and HindIII sites of pGEX-4T-1 (Amersham Biosciences, Buckinghamshire, UK). GST-SRC-1-NR binding domain (NBD)-1 was constructed by inserting PCR-generated fragments of SRC-1 (7) (residues aa 595–780) into the GST plasmid (30), as described previously (31). Expression vectors TR1, TR1-AF2 (E457A) (glutamate at aa 457 was replaced to alanine, AF-2 mutant), N terminus (aa 1–105)-deleted TR1 (N-TR1) and N-TR1-AF2 (E457A) were constructed by inserting the PCR-amplified fragments from TR1 or TR1-AF2 (E457A) (31) into the EcoRI and HindIII sites of pBK/CMV (Stratagene, La Jolla, CA). Expression vectors encoding SRC-1 (aa 1154–1440) and SRC-1 (aa 595-1440) were constructed by inserting the PCR-generated fragments from human SRC-1 into EcoRI and HindIII sites into pBK/CMV. The luciferase (LUC) reporter constructs, the chick lysozyme thyroid hormone response element (TRE) (F2)-thymidine kinase (TK)-LUC, artificial direct repeat TRE, DR4-TRE-TK-LUC, and malic enzyme (ME) promoter fragment containing three TRE half-sites (GATCCATCCAGGACGTTGGGGTTAGGGGAGGACA GTGGA) in the PT109 vector (ME-LUC) were described previously (31, 32). 5x Upstream activation signal (UAS)-LUC has been described previously (31). Gal4-NBD-1 (aa 595–780), Gal4-NBD-2 (aa 1237–1440), Gal4-NBD- (1 + 2) (aa 595-1440) were constructed by insertion of PCR fragment into EcoRI site of pM (Invitrogen, Carlsbad, CA). VP16 transactivation domain fusion construct VP16-TR1-N+DBD (aa 1–174) was constructed by insertion of PCR fragment into EcoRI site of pVP16 (Invitrogen).
Far Western blotting
GST-SRC-1 protein was produced in Escherichia coli BL21 (DE3) and purified by gluthathione-Sepharose resin (Amersham Biosciences). Indicated amounts of GST-SRC-1 were spotted on nitrocellulose membrane. The production of the probe was described previously (7). Briefly, after affinity purification of the GST-fused TR construct proteins on glutathione-Sepharose resin, cleavage of the fusion protein by human thrombin (Sigma, St. Louis, MO) was performed. The supernatant containing TR protein was passed through a NICK column (Amersham Biosciences) preequilibrated with the kinase buffer [20 mM Tris (pH 7.5), 100 mM NaCl, 12 mM MgCl2, 5 mM NaF, 2 mM dithiothreitol, 100 μg/ml BSA]. Labeling of these proteins was performed in 100 μl of kinase buffer containing 100 U of the catalytic subunit of protein kinase A from bovine heart (Sigma) and 300 μCi of [-32P]-labeled-ATP for 1 h at room temperature. After blocking, [-32P]-ATP-labeled TR1-LBD or TR1-N+DBD was incubated in the absence or presence of 1 x 10–6 M of T3 for 1 h at room temperature. After extensive washing with cold buffer, the membrane was subjected to autoradiography.
In vitro-translated proteins and GST pull-down assays
The GST and GST-fusion proteins were produced in E. coli BL21 (DE3) and purified. Mutants of SRC-1 in pBK/CMV were transcribed and translated in rabbit reticulocyte lysates (Promega, Madison, WI) with [35S]-methionine according to the manufacturer’s instructions. GST pull-down assays were performed as described previously (31). Briefly, GST-fusion proteins containing wild-type and cognate mutant TR1 were bound to glutathione-Sepharose beads (Amersham Biosciences). Similar amounts of loading proteins bound to the beads were used, as determined by Coomassie Blue staining/SDS-PAGE analysis. The beads were resuspended in the binding buffer [20 mM HEPES (pH 7.7), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.05% Nonidet P-40, 2 mM dithiothreitol, 10% glycerol], and incubated with 5 μl of in vitro-translated, [35S]-labeled proteins in the presence or absence of 1 x 10–6 M of T3 for 1 h at room temperature. Beads were then washed with the binding buffer three times in the presence or absence of the ligands, followed by resuspension in 20 μl of sodium dodecyl sulfate sample buffer, and analysis by SDS-PAGE and autoradiography.
Cell culture and transient transfection assay
CV-1 cells were maintained in DMEM supplemented with 10% TH-free fetal bovine serum and penicillin/streptomycin at 37 C, 5% CO2. Cells were plated in six- or 24-well plates 2 d before transfection. Cells were transfected with the indicated expression and reporter vectors using the calcium phosphate method as described previously (31). Sixteen to 24 h after transfection, cells were refilled with fresh medium containing the indicated concentration of ligand. After 36–48 h, cells were harvested for luciferase activities as described (31). Total amounts of DNA for each well were normalized by adding vector pBK/CMV (STRATAGENE). Data shown represent means of triplicate transfections ± SEs. Statistical analysis was performed using ANOVA, followed by post hoc comparison with Duncan’s multiple range test.
Results
TR1-N+DBD bound to SRC-1 in vitro
To investigate whether TR1-N+DBD binds to SRC-1, we first performed far-Western studies. Radiolabeled TR1-N+DBD (aa 1–174) bound to NBD-1 of SRC-1 (aa 595–780) without T3 (Fig. 1), whereas it did not bind to GST. In contrast, TR1-LBD (aa 174–461) did not bind to SRC-1 without T3, whereas it bound to SRC-1 with T3 as expected. These results suggest that SRC-1 binds to TR1-N or DBD in a ligand-independent manner.
TR1-N+DBD bound to SRC-1 in vivo
To confirm in vivo interaction of TR-N+DBD and SRC-1, we constructed a series of Gal4-SRC-1 and VP16-TR1-N+DBD constructs as shown in Fig. 2A. VP16 did not influence the transcription when Gal4-NBD-1, NBD-2, or NBD-(1 + 2) was cotransfected with 5xUAS-LUC. On the other hand, VP16-TR1-N+DBD activated the transcription when Gal4-NBD-1 or NBD- (1 + 2) was transfected (Fig. 2B). When Gal4-NBD-2 was transfected, the transcription was not significantly activated (Fig. 2B). These results suggest that TR1-N+DBD binds to SRC-1 in the absence of ligand in vivo.
TR1-N terminus binds to SRC-1 in vitro
Next, we constructed a series of truncation mutants of TR1 and SRC-1, shown in Fig. 3, A and B, and examined which region(s) of TR1 bind(s) to SRC-1, using GST pull-down assays. We also investigated which region(s) of SRC-1 bind(s) to TR1. TR1-LBD (aa 174–461) has been shown to interact with two nuclear receptor-binding domains (NBDs) in a ligand-dependent manner (31). As predicted by far-Western studies, the TR1-N (aa 1–105) bound to the SRC-1 at residue aa 595-1440. (Fig. 3B). On the other hand, TR1-DBD did not bind to SRC-1 (aa 595-1440), suggesting that the major binding surface to SRC-1 may be N terminus, not DBD, in the absence of T3. The deletion of either aa 1–54 or aa 55–105 region result in reduction of binding to SRC-1 (aa 595-1440), suggesting that relatively broad region of N terminus may contribute the binding to SRC-1. SRC-1 (aa 1154–1440), which is corresponding to NBD-2, did not bind to either TR1-N or DBD. These results indicate that SRC-1 (aa 1154–1440) is insufficient for binding, and that aa 595-1440 does bind to TR1-N (aa 1–105).
TR1-N terminus is required for the full activation function of SRC-1-mediated transcription on both artificial TREs and native ME response element
Next, we made a series of truncation and/or point mutants of TR1 (Fig. 4A) to investigate the contribution of the TR1-N terminus on SRC-1-mediated transcription on F2-TRE in CV-1 cells. To investigate the effect of the binding between N terminus of TR1 and SRC-1 on the native response element, we used ME promoter fragment containing three half-sites of TREs. This fragment was inserted into PT109 vector containing TK minimum promoter at N terminus of luciferase gene (ME-LUC). Without transfecting SRC-1, significant differences were observed in the magnitude of T3-activated transcription between wild-type and N-TR1 on both F2-TRE-LUC and ME-LUC (Fig. 4B). These results suggest that a little amount of intrinsic SRC-1 may contribute to the activation of transcription. When SRC-1 was cotransfected, N-TR1 showed lower activity than that of wild-type TR1 in the presence of T3; however, no significant difference was observed without T3 (Fig. 4C). The magnitude of SRC-1 activated transcription of constructs were wild-type> N (77.3%; ratio of wild-type and N-TR1 interacting with SRC-1 in the presence of T3) > AF2 (E457A) (37.2%) > N-AF2 (E457A) (30.4%). N-TR1-AF2 (E457A) showed the lowest activity of the mutants we tested (Fig. 4C). Using DR4-TRE-LUC, we obtained similar results (data not shown). In the presence of SRC-1, as on F2-TRE, N-TR1 showed lower activity than that of wild-type on ME fragment (Fig. 4C). The difference of activity is greater (47.7% of that of wild type) than that on F2-TRE (77.3%). The activities of the mutants were wild-type> N (47.7%; ratio of wild-type and N-TR1 interacting with SRC-1 in the presence of T3) > AF2 (E457A) (37.5%) > N-AF2 (E457A) (29.9%). No significant difference was observed between AF2 (E457A) and N-AF2 (E457A). Finally, cotransfection of the increasing amount of TR1 showed similar results (Fig. 4D) on F2-TRE, suggesting that this suppression is due to lack of the N terminus. These observations support the idea that the TR1-N terminus is required for the full activation of SRC-1-mediated transcription on TRE.
Discussion
In the present study, we show that the TR1-N terminus (aa 1–105) binds to a region of SRC-1 (aa 595-1440), and this binding may permit the full activity of SRC-1 on TR1-mediated transcription in the presence of ligand.
Several investigators have previously demonstrated the interaction between N termini of NRs and SRC-1. For instance, progesterone receptor-N terminus synergistically interacts with SRC-1 with progesterone receptor-LBD (33). Recent data also detail the enhancement of androgen receptor AF-1 activities by SRC-1 (34, 35). Thus, the activity of an NR cannot be viewed in the context of the AF-2 domain alone because the AF-1 domain may influence AF-2 function either through allosteric effects, or recruitment of other nuclear factors independently. In case of TRs, it has also been reported that TR2 binds to SRC-1 through its N terminus (36), and this binding promotes a greater activity than that seen with the other TRs in the absence of ligand (36). The present study shows that TR1-N terminus may also play an important role in SRC-1-mediated transactivation.
In the present study, we have demonstrated that the TR1-N+DBD bound to SRC-1-NBD-1 (aa 595–780) in the absence of T3 in far-Western studies (Fig. 1). In mammalian two-hybrid studies, we showed the binding of TR1-N+DBD to SRC-1-NBD-1 (aa 595–780) and NBD-(1 + 2) (aa 595-1440), but not to NBD-2 (aa 1237–1440) (Fig. 2). We further investigated the main binding site of the interaction between TR1-N terminus and SRC-1 using GST-pull-down studies (Fig. 3). The TR1-N terminus (aa 1–105) but not DBD mainly bound to the SRC-1 (aa 595-1440). On the other hand, TR1-N (aa 55–105) containing previously mapped AF-1 domain of TR1 (aa 69–89) (29), only very weakly bound to SRC-1 (aa 595-1440) (Fig. 3B). These results suggest that a broad range of N terminus of TR1 may contribute to the binding to SRC-1. On the other hand, SRC-1 (aa 1154–1440), which is corresponding to NBD-2, did not bind to either TR1-N terminus or -DBD, suggesting that SRC-1 (aa 1154–1440) is insufficient for binding, and that aa 595-1440 does bind to TR1-N terminus. This result is compatible to the mammalian two-hybrid studies. Therefore, we concluded that TR1-N terminus (aa 1–105) binds to relatively broad region of SRC-1.
Although our binding studies have shown the interaction between TR1-N terminus and SRC-1, SRC-1-mediated transcriptional activation in the presence of ligand was only modestly reduced with N-TR1. This indicates that binding between N terminus of TR1 and SRC-1 may not play dominant roles in activation of TR-mediated transcription in CV-1 cells. Using native ME promoter containing fragment, we observed more efficient reduction by deleting the N terminus of TR1 (Fig. 4C). On the other hand, no significant difference was observed between AF2 (E457A) and N-AF2 (E457A) on ME-LUC. Because ME promoter containing fragment contains three TRE half-sites, an additional effect may be involved. Increasing number of TRE half-sites may affect the transcription on different TREs. On the other hand, our results show that TR1 may be able to recruit both corepressors and coactivators (through the amino terminus) in the absence of ligand. Interaction of SRC-1 at TR1-N terminus alone may not be able to activate transcription. However, this binding may facilitate binding between AF-2 of TR1 and SRC-1 by apparently increasing the local concentration of SRC-1 relative to TR1. Thus, once ligand binds, transcription may be activated to a greater extent in the presence of the N terminus. There are several lines of evidence indicating that AF-2 is not the only region that mediates ligand-dependent activation. First, a number of ligand-dependent TR-interacting proteins do not contain LXXLL motifs (37), which are necessary for TR-AF-2 binding. Second, SRC-1 is able to enhance estrogen-stimulated transcriptional activity of an AF-2 mutant of ER (38). Third, it has been shown that SRC-1 functions as a general integrator like CBP/p300 (39). The apparent cooperativity of the two activation function (AFs) within a single receptor to achieve optimal transcriptional regulation, suggests that the proper assembly of the individual AFs of the TR is required to render the TR-DNA complex transcriptionally fully productive.
In conclusion, coactivators provide, in part, a mechanism by which the separate AFs of the TR collaborate within a transcriptional complex on target DNA elements to lead to full activation of gene expression in the presence of T3.
Acknowledgments
We thank Dr. M. Ikeda for kindly preparing the constructs. We thank M. Ohta for technical assistance throughout the study.
Footnotes
Present address for W.W.C.: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indianapolis 46285.
This work was supported by Grant-in-Aid for Scientific Research 14370020 (to N.K.) and 17510039 (to T.I.) from the Japanese Ministry of Education, Science, Sports and Culture, and a fund from CREST, Japan Science and Technology Corporation (JST) (to N.K.).
First Published Online December 15, 2005
Abbreviations: aa, Amino acid; AF-1, activation function-1; CBP, cAMP response element binding protein-binding protein; DBD, DNA binding domain; GST, gluthathione-S-transferase; LBD, ligand binding domain; LUC, luciferase; ME, malic enzyme; NBD, nuclear receptor binding domain; NR, nuclear receptor; SRC-1, steroid receptor coactivator-1; TH, thyroid hormone; TK, thymidine kinase; TR; thyroid hormone receptor; TRE, thyroid hormone response element; UAS, upstream activation signal.
Accepted for publication December 2, 2005.
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Core Research for Evolutional Science and Technology (CREST) (T.I., W.M., N.K.), Japan Science and Technology Corporation (J.S.T.), Kawaguchi, Saitama 332-0012, Japan
Brigham and Women’s Hospital and Harvard Medical School (T.I., A.T., W.W.C., N.K.), Boston, Massachusetts 02115
Division of Endocrinology and Metabolism (A.T.), Toranomon Hospital, Okinaka Memorial Institute for Medical Research, Tokyo 105-8470, Japan
Abstract
Steroid receptor coactivator-1 (SRC-1) plays a crucial role in nuclear receptor-mediated transcription including thyroid hormone receptor (TR)-dependent gene expression. Interaction of the TR-ligand binding domain and SRC-1 through LXXLL motifs is required for this action. However, potential interactions between the TR1-N terminus (N) and SRC-1 have not been explored and thus are examined in this manuscript. Far-Western studies showed that protein construct containing TR1-N + DNA binding domain (DBD) bound to nuclear receptor binding domain (NBD)-1 (amino acid residue, aa 595–780) of SRC-1 without ligand. Mammalian two-hybrid studies showed that NBD-1, as well as SRC-1 (aa 595-1440), bound to TR1-N+DBD in the absence of ligand in CV-1 cells. However, NBD-2 (aa 1237–1440) did not bind to this protein. Glutathione-S-transferase pull-down studies showed that TR1-N (aa 1–105) bound to the broad region of SRC-1-C terminus. Expression vectors encoding a series of truncations and/or point mutations of TR1 were used in transient transfection-based reporter assays in CV-1 cells. N-terminal truncated TR1 (N-TR1) showed lower activity than that of wild-type in both artificial F2-thyroid hormone response element and native malic enzyme response element. These results suggest that there is the interaction between N terminus of TR1 and SRC-1, which may serve a full activation of SRC-1, together with activation function-2 on TR1-mediated transcription.
Introduction
THE NUCLEAR HORMONE receptors (NRs) are transcription factors that regulate target gene transcription in response to ligands such as steroid and thyroid hormones (THs) (1, 2). The DNA binding domain (DBD) of NR binds to the cognate hormone response element, located in the promoter regions of target genes. One of these, TH receptor (TR) belongs to type II nuclear hormone receptors that form heterodimers with retinoid X receptor (3, 4, 5).
At least three functional isoforms of TR (TR1, 1, and 2) have been reported to be expressed in mammalian cells at protein level (3, 4, 5). These TRs are highly similar within their DNA binding, hinge, and ligand binding domains (LBDs), but are divergent in their amino-terminal regions (3, 4, 5). These forms are expressed in distinct developmental- and tissue-specific manners, suggesting that they may possess unique functional properties (2).
In mammalian cells, TH binding to TR results in dissociation of corepressor complexes followed by recruitment of coactivator complexes to activate transcription. Functional analyses of NRs have shown that there are two major activation domains: activation function (AF)-1 and AF-2 (3, 4). The extreme C-terminal region of the LBD (AF-2) exhibits ligand-dependent transactivation. AF-2 interacts with coactivators, such as SRC-1 (6, 7), in a ligand-dependent manner (1, 8, 9). Members of SRC-1 family (1, 8, 10, 11, 12, 13), cAMP response element binding protein-binding protein (CBP) (1, 8, 14) and p300/CBP-associated factor (1, 8, 15), and participate in a protein complex on AF-2 possess intrinsic histone acetyltransferase activity. These cofactors may remodel chromatin structure, allowing the efficient access of basal transcriptional machinery to DNA (1). In addition to these coactivators, several other proteins, such as coactivator-associated arginine methyltransferase-1 (16) and TR binding protein (17)—this is also named as AIB3/ASC-2/RAP250/PRIP/NRC (18, 19, 20, 21, 22)—are involved in this complex and contribute to the precise transcriptional regulation.
On the other hand, the AF-1 region, located within the N-terminal region, contains a ligand-independent activation function (23, 24). The potency of the AF-1 transactivation domain varies widely, depending on the cell and promoter context (25, 26, 27, 28). The AF-1 domain of TR1 is represented by 19 amino acids located between residues 69 and 89 (29). Unlike the AF-2 domain, this AF-1 domain is not homologous to those of TR1, TR2, or the other steroid hormone receptors (27). This observation suggests that the AF-2 domain may evolutionarily represent the primary transactivation domain of TR, whereas the AF-1 domain, with differences in diverse sequences in the amino termini, may provide the molecular basis for differential activities of different TR isoforms to allow unique promoter- or cell-specific actions. Thus, in the present study, to examine the role of the TR1-N terminus on SRC-1-mediated transactivation, we investigated the binding between TR1-N terminus and SRC-1.
Materials and Methods
Plasmids
cDNAs encoding glutathione-S-transferase (GST)-fusion proteins including the LBD of the rat TR1 (aa 174–461) has been described previously (7). GST-TR1-N terminus + DBD (aa 1–174), GST-TR1-N terminus (N) (aa 1–105), GST-TR1 (aa 1–54), GST-TR1 (aa 55–105), and GST-TR1-DBD (aa 106–174) were constructed by inserting the PCR-amplified fragments from TR1 into the EcoRI and HindIII sites of pGEX-4T-1 (Amersham Biosciences, Buckinghamshire, UK). GST-SRC-1-NR binding domain (NBD)-1 was constructed by inserting PCR-generated fragments of SRC-1 (7) (residues aa 595–780) into the GST plasmid (30), as described previously (31). Expression vectors TR1, TR1-AF2 (E457A) (glutamate at aa 457 was replaced to alanine, AF-2 mutant), N terminus (aa 1–105)-deleted TR1 (N-TR1) and N-TR1-AF2 (E457A) were constructed by inserting the PCR-amplified fragments from TR1 or TR1-AF2 (E457A) (31) into the EcoRI and HindIII sites of pBK/CMV (Stratagene, La Jolla, CA). Expression vectors encoding SRC-1 (aa 1154–1440) and SRC-1 (aa 595-1440) were constructed by inserting the PCR-generated fragments from human SRC-1 into EcoRI and HindIII sites into pBK/CMV. The luciferase (LUC) reporter constructs, the chick lysozyme thyroid hormone response element (TRE) (F2)-thymidine kinase (TK)-LUC, artificial direct repeat TRE, DR4-TRE-TK-LUC, and malic enzyme (ME) promoter fragment containing three TRE half-sites (GATCCATCCAGGACGTTGGGGTTAGGGGAGGACA GTGGA) in the PT109 vector (ME-LUC) were described previously (31, 32). 5x Upstream activation signal (UAS)-LUC has been described previously (31). Gal4-NBD-1 (aa 595–780), Gal4-NBD-2 (aa 1237–1440), Gal4-NBD- (1 + 2) (aa 595-1440) were constructed by insertion of PCR fragment into EcoRI site of pM (Invitrogen, Carlsbad, CA). VP16 transactivation domain fusion construct VP16-TR1-N+DBD (aa 1–174) was constructed by insertion of PCR fragment into EcoRI site of pVP16 (Invitrogen).
Far Western blotting
GST-SRC-1 protein was produced in Escherichia coli BL21 (DE3) and purified by gluthathione-Sepharose resin (Amersham Biosciences). Indicated amounts of GST-SRC-1 were spotted on nitrocellulose membrane. The production of the probe was described previously (7). Briefly, after affinity purification of the GST-fused TR construct proteins on glutathione-Sepharose resin, cleavage of the fusion protein by human thrombin (Sigma, St. Louis, MO) was performed. The supernatant containing TR protein was passed through a NICK column (Amersham Biosciences) preequilibrated with the kinase buffer [20 mM Tris (pH 7.5), 100 mM NaCl, 12 mM MgCl2, 5 mM NaF, 2 mM dithiothreitol, 100 μg/ml BSA]. Labeling of these proteins was performed in 100 μl of kinase buffer containing 100 U of the catalytic subunit of protein kinase A from bovine heart (Sigma) and 300 μCi of [-32P]-labeled-ATP for 1 h at room temperature. After blocking, [-32P]-ATP-labeled TR1-LBD or TR1-N+DBD was incubated in the absence or presence of 1 x 10–6 M of T3 for 1 h at room temperature. After extensive washing with cold buffer, the membrane was subjected to autoradiography.
In vitro-translated proteins and GST pull-down assays
The GST and GST-fusion proteins were produced in E. coli BL21 (DE3) and purified. Mutants of SRC-1 in pBK/CMV were transcribed and translated in rabbit reticulocyte lysates (Promega, Madison, WI) with [35S]-methionine according to the manufacturer’s instructions. GST pull-down assays were performed as described previously (31). Briefly, GST-fusion proteins containing wild-type and cognate mutant TR1 were bound to glutathione-Sepharose beads (Amersham Biosciences). Similar amounts of loading proteins bound to the beads were used, as determined by Coomassie Blue staining/SDS-PAGE analysis. The beads were resuspended in the binding buffer [20 mM HEPES (pH 7.7), 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.05% Nonidet P-40, 2 mM dithiothreitol, 10% glycerol], and incubated with 5 μl of in vitro-translated, [35S]-labeled proteins in the presence or absence of 1 x 10–6 M of T3 for 1 h at room temperature. Beads were then washed with the binding buffer three times in the presence or absence of the ligands, followed by resuspension in 20 μl of sodium dodecyl sulfate sample buffer, and analysis by SDS-PAGE and autoradiography.
Cell culture and transient transfection assay
CV-1 cells were maintained in DMEM supplemented with 10% TH-free fetal bovine serum and penicillin/streptomycin at 37 C, 5% CO2. Cells were plated in six- or 24-well plates 2 d before transfection. Cells were transfected with the indicated expression and reporter vectors using the calcium phosphate method as described previously (31). Sixteen to 24 h after transfection, cells were refilled with fresh medium containing the indicated concentration of ligand. After 36–48 h, cells were harvested for luciferase activities as described (31). Total amounts of DNA for each well were normalized by adding vector pBK/CMV (STRATAGENE). Data shown represent means of triplicate transfections ± SEs. Statistical analysis was performed using ANOVA, followed by post hoc comparison with Duncan’s multiple range test.
Results
TR1-N+DBD bound to SRC-1 in vitro
To investigate whether TR1-N+DBD binds to SRC-1, we first performed far-Western studies. Radiolabeled TR1-N+DBD (aa 1–174) bound to NBD-1 of SRC-1 (aa 595–780) without T3 (Fig. 1), whereas it did not bind to GST. In contrast, TR1-LBD (aa 174–461) did not bind to SRC-1 without T3, whereas it bound to SRC-1 with T3 as expected. These results suggest that SRC-1 binds to TR1-N or DBD in a ligand-independent manner.
TR1-N+DBD bound to SRC-1 in vivo
To confirm in vivo interaction of TR-N+DBD and SRC-1, we constructed a series of Gal4-SRC-1 and VP16-TR1-N+DBD constructs as shown in Fig. 2A. VP16 did not influence the transcription when Gal4-NBD-1, NBD-2, or NBD-(1 + 2) was cotransfected with 5xUAS-LUC. On the other hand, VP16-TR1-N+DBD activated the transcription when Gal4-NBD-1 or NBD- (1 + 2) was transfected (Fig. 2B). When Gal4-NBD-2 was transfected, the transcription was not significantly activated (Fig. 2B). These results suggest that TR1-N+DBD binds to SRC-1 in the absence of ligand in vivo.
TR1-N terminus binds to SRC-1 in vitro
Next, we constructed a series of truncation mutants of TR1 and SRC-1, shown in Fig. 3, A and B, and examined which region(s) of TR1 bind(s) to SRC-1, using GST pull-down assays. We also investigated which region(s) of SRC-1 bind(s) to TR1. TR1-LBD (aa 174–461) has been shown to interact with two nuclear receptor-binding domains (NBDs) in a ligand-dependent manner (31). As predicted by far-Western studies, the TR1-N (aa 1–105) bound to the SRC-1 at residue aa 595-1440. (Fig. 3B). On the other hand, TR1-DBD did not bind to SRC-1 (aa 595-1440), suggesting that the major binding surface to SRC-1 may be N terminus, not DBD, in the absence of T3. The deletion of either aa 1–54 or aa 55–105 region result in reduction of binding to SRC-1 (aa 595-1440), suggesting that relatively broad region of N terminus may contribute the binding to SRC-1. SRC-1 (aa 1154–1440), which is corresponding to NBD-2, did not bind to either TR1-N or DBD. These results indicate that SRC-1 (aa 1154–1440) is insufficient for binding, and that aa 595-1440 does bind to TR1-N (aa 1–105).
TR1-N terminus is required for the full activation function of SRC-1-mediated transcription on both artificial TREs and native ME response element
Next, we made a series of truncation and/or point mutants of TR1 (Fig. 4A) to investigate the contribution of the TR1-N terminus on SRC-1-mediated transcription on F2-TRE in CV-1 cells. To investigate the effect of the binding between N terminus of TR1 and SRC-1 on the native response element, we used ME promoter fragment containing three half-sites of TREs. This fragment was inserted into PT109 vector containing TK minimum promoter at N terminus of luciferase gene (ME-LUC). Without transfecting SRC-1, significant differences were observed in the magnitude of T3-activated transcription between wild-type and N-TR1 on both F2-TRE-LUC and ME-LUC (Fig. 4B). These results suggest that a little amount of intrinsic SRC-1 may contribute to the activation of transcription. When SRC-1 was cotransfected, N-TR1 showed lower activity than that of wild-type TR1 in the presence of T3; however, no significant difference was observed without T3 (Fig. 4C). The magnitude of SRC-1 activated transcription of constructs were wild-type> N (77.3%; ratio of wild-type and N-TR1 interacting with SRC-1 in the presence of T3) > AF2 (E457A) (37.2%) > N-AF2 (E457A) (30.4%). N-TR1-AF2 (E457A) showed the lowest activity of the mutants we tested (Fig. 4C). Using DR4-TRE-LUC, we obtained similar results (data not shown). In the presence of SRC-1, as on F2-TRE, N-TR1 showed lower activity than that of wild-type on ME fragment (Fig. 4C). The difference of activity is greater (47.7% of that of wild type) than that on F2-TRE (77.3%). The activities of the mutants were wild-type> N (47.7%; ratio of wild-type and N-TR1 interacting with SRC-1 in the presence of T3) > AF2 (E457A) (37.5%) > N-AF2 (E457A) (29.9%). No significant difference was observed between AF2 (E457A) and N-AF2 (E457A). Finally, cotransfection of the increasing amount of TR1 showed similar results (Fig. 4D) on F2-TRE, suggesting that this suppression is due to lack of the N terminus. These observations support the idea that the TR1-N terminus is required for the full activation of SRC-1-mediated transcription on TRE.
Discussion
In the present study, we show that the TR1-N terminus (aa 1–105) binds to a region of SRC-1 (aa 595-1440), and this binding may permit the full activity of SRC-1 on TR1-mediated transcription in the presence of ligand.
Several investigators have previously demonstrated the interaction between N termini of NRs and SRC-1. For instance, progesterone receptor-N terminus synergistically interacts with SRC-1 with progesterone receptor-LBD (33). Recent data also detail the enhancement of androgen receptor AF-1 activities by SRC-1 (34, 35). Thus, the activity of an NR cannot be viewed in the context of the AF-2 domain alone because the AF-1 domain may influence AF-2 function either through allosteric effects, or recruitment of other nuclear factors independently. In case of TRs, it has also been reported that TR2 binds to SRC-1 through its N terminus (36), and this binding promotes a greater activity than that seen with the other TRs in the absence of ligand (36). The present study shows that TR1-N terminus may also play an important role in SRC-1-mediated transactivation.
In the present study, we have demonstrated that the TR1-N+DBD bound to SRC-1-NBD-1 (aa 595–780) in the absence of T3 in far-Western studies (Fig. 1). In mammalian two-hybrid studies, we showed the binding of TR1-N+DBD to SRC-1-NBD-1 (aa 595–780) and NBD-(1 + 2) (aa 595-1440), but not to NBD-2 (aa 1237–1440) (Fig. 2). We further investigated the main binding site of the interaction between TR1-N terminus and SRC-1 using GST-pull-down studies (Fig. 3). The TR1-N terminus (aa 1–105) but not DBD mainly bound to the SRC-1 (aa 595-1440). On the other hand, TR1-N (aa 55–105) containing previously mapped AF-1 domain of TR1 (aa 69–89) (29), only very weakly bound to SRC-1 (aa 595-1440) (Fig. 3B). These results suggest that a broad range of N terminus of TR1 may contribute to the binding to SRC-1. On the other hand, SRC-1 (aa 1154–1440), which is corresponding to NBD-2, did not bind to either TR1-N terminus or -DBD, suggesting that SRC-1 (aa 1154–1440) is insufficient for binding, and that aa 595-1440 does bind to TR1-N terminus. This result is compatible to the mammalian two-hybrid studies. Therefore, we concluded that TR1-N terminus (aa 1–105) binds to relatively broad region of SRC-1.
Although our binding studies have shown the interaction between TR1-N terminus and SRC-1, SRC-1-mediated transcriptional activation in the presence of ligand was only modestly reduced with N-TR1. This indicates that binding between N terminus of TR1 and SRC-1 may not play dominant roles in activation of TR-mediated transcription in CV-1 cells. Using native ME promoter containing fragment, we observed more efficient reduction by deleting the N terminus of TR1 (Fig. 4C). On the other hand, no significant difference was observed between AF2 (E457A) and N-AF2 (E457A) on ME-LUC. Because ME promoter containing fragment contains three TRE half-sites, an additional effect may be involved. Increasing number of TRE half-sites may affect the transcription on different TREs. On the other hand, our results show that TR1 may be able to recruit both corepressors and coactivators (through the amino terminus) in the absence of ligand. Interaction of SRC-1 at TR1-N terminus alone may not be able to activate transcription. However, this binding may facilitate binding between AF-2 of TR1 and SRC-1 by apparently increasing the local concentration of SRC-1 relative to TR1. Thus, once ligand binds, transcription may be activated to a greater extent in the presence of the N terminus. There are several lines of evidence indicating that AF-2 is not the only region that mediates ligand-dependent activation. First, a number of ligand-dependent TR-interacting proteins do not contain LXXLL motifs (37), which are necessary for TR-AF-2 binding. Second, SRC-1 is able to enhance estrogen-stimulated transcriptional activity of an AF-2 mutant of ER (38). Third, it has been shown that SRC-1 functions as a general integrator like CBP/p300 (39). The apparent cooperativity of the two activation function (AFs) within a single receptor to achieve optimal transcriptional regulation, suggests that the proper assembly of the individual AFs of the TR is required to render the TR-DNA complex transcriptionally fully productive.
In conclusion, coactivators provide, in part, a mechanism by which the separate AFs of the TR collaborate within a transcriptional complex on target DNA elements to lead to full activation of gene expression in the presence of T3.
Acknowledgments
We thank Dr. M. Ikeda for kindly preparing the constructs. We thank M. Ohta for technical assistance throughout the study.
Footnotes
Present address for W.W.C.: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indianapolis 46285.
This work was supported by Grant-in-Aid for Scientific Research 14370020 (to N.K.) and 17510039 (to T.I.) from the Japanese Ministry of Education, Science, Sports and Culture, and a fund from CREST, Japan Science and Technology Corporation (JST) (to N.K.).
First Published Online December 15, 2005
Abbreviations: aa, Amino acid; AF-1, activation function-1; CBP, cAMP response element binding protein-binding protein; DBD, DNA binding domain; GST, gluthathione-S-transferase; LBD, ligand binding domain; LUC, luciferase; ME, malic enzyme; NBD, nuclear receptor binding domain; NR, nuclear receptor; SRC-1, steroid receptor coactivator-1; TH, thyroid hormone; TK, thymidine kinase; TR; thyroid hormone receptor; TRE, thyroid hormone response element; UAS, upstream activation signal.
Accepted for publication December 2, 2005.
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