The Adenovirus E1A Protein Is a Potent Coactivator for Thyroid Hormone Receptors

Gunilla M. Wahlström1, Björn Vennström and Maria Bondesson Bolin1

Department of Cell and Molecular Biology Medical Nobel Institute Karolinska Institutet S-171 77 Stockholm, Sweden


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The thyroid hormone receptors interact with several different cofactors when activating transciption. In this study, we show that the adenovirus E1A oncoprotein functions as a strong coactivator for the thyroid hormone receptor (TR), and that TR and E1A synergistically activate transcription via direct (DR4) or palindromic (IR0) hormone-responsive sites. Cotransfection experiments using different isoforms of the chicken TR and E1A show synergistic, ligand-enhanced transactivation. This transactivation is accomplished through a direct, ligand-independent interaction between TR and E1A. The interaction domains in TR are localized to the DNA-binding domain and to the carboxy-terminal part of the ligand-binding domain. In E1A, the regions of interactions are localized to the conserved regions 1 and 3. Both of these domains in E1A are required for a 40-fold enhancement of TR-mediated activation in transfection experiments. Taken together, we show that E1A strongly enhances transcriptional activation, which suggests that it serves as a bridging factor between the receptor and other components of the transcription machinery.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Thyroid hormones play a fundamental role in the regulation of normal cell function and differentiation by interacting with intracellular thyroid hormone receptors (TRs). These receptors belong to a family of nuclear hormone receptors that includes receptors for steroids as well as those for retinoic acid (RAR), 9-cis-retinoic acid (RXR), and vitamin D3. TRs interact with specific thyroid response elements (TREs) within the promoters of target genes and thereby activate or repress transcription. TREs consist of hexameric motifs (half-sites) arranged as inverted, everted, or direct repeats separated by a specific number of nucleotides (1, 2, 3). TR preferentially binds as a heterodimer with RXR to a direct repeat spaced by four nucleotides (DR4). In the same manner, heterodimers of RXR/RAR activate transcription via elements of the DR5 and DR2 type (4, 5, 6, 7). Corepressors, such as N-CoR and SMRT, have been shown to interact with TR in its unliganded state. These corepressors bind to the hinge region in between the DNA- and ligand-binding domain (DBD and LBD) in the absence of T3 (8, 9, 10, 11). Upon binding of T3, the corepressors are released and coactivators are recruited. The coactivators, such as SRC-1 and CBP/p300, have been suggested to preferentially bind to an activation domain (AF2) in the C-terminal part of TR (12).

The adenovirus E1A gene products have been shown to be multifunctional, playing central roles in the control of viral and cellular gene expression and transformation. Two structurally homologous proteins, of 243 and 289 amino acids (243R and 289R), are the major products encoded by E1A. E1A-289R contains three, among adenoviruses, highly conserved regions, designated conserved region (CR) 1, 2, and 3. These regions are important for the multiple activities ascribed to E1A. The major E1A transcription activation domain is contained within CR3 and is unique to E1A-289R. This domain participates in transcriptional activation by physically interacting with both basal and upstream binding transcription factors (13).

E1A has been shown to directly interact with retinoic acid receptor ß (RARß) and thereby function as a cofactor for activation of the RARß2 promoter (14). In cotransfection experiments, efficient activation of RARß2 by E1A and RARß also requires the addition of the TATA binding protein (TBP) (15). Furthermore, E1A has been shown to directly bind to TBP (16, 17, 18). These results suggest that E1A-289R mediates transcriptional activation by providing a physical bridge between TBP and RARß.

Retinoids play an important role in differentiation of embryonic carcinoma (EC) cells. Undifferentiated murine P19 EC cells differentiate into neurons, astrocytes, and fibroblast-like cells after addition of retinoic acid (RA) (19). An early and essential step in the differentiation process is the activation of the RARß2-promoter. However, activation of the RARß2-promoter in P19 cells does not require the viral E1A protein; instead, an endogenous E1A-like activity (E1A-LA) is used as a bridging factor between RARß and TBP (15). The existence of E1A-LA was first suggested when nonviable adenovirus E1A mutants were able to grow in undifferentiated EC cells (20).

Since RAR uses E1A/E1A-LA as a cofactor, we investigated whether TR and E1A/E1A-LA also cooperate in transcriptional activation. In this paper, we show that E1A and TR synergistically activate transcription of promoters containing TR recognition sites. This activation is seen for three different TR recognition elements [DR4>IR0>{alpha}-myosin heavy chain ({alpha}MHC)] and is mediated by all the isoforms of chicken TRs tested.

The synergistic activation is accomplished through a direct interaction between TR and E1A. We have mapped the interacting domains of E1A to CR1 and CR3, both of which are also required for efficient enhancement of TR-mediated transcription. E1A interacts with the LBD and DBD of TR. The results presented here indicate that a complex containing TR and E1A is formed on a promoter containing a DR4 type of cis-acting element and that this complex is of sufficient importance to gain efficient transcriptional activation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TR and E1A Synergistically Activate Transcription of Promoters Containing TREs
Since E1A interacts with several DNA-bound transcription factors that are localized to promoter regions, we tested whether E1A had the potential to activate TR-mediated transcription. For this, plasmids expressing either E1A or chicken TR{alpha}-p46 were cotransfected along with a chloramphenicol acetyltransferase (CAT)-reporter construct (pBLCAT-TRE) containing a TR-responsive element into human chorion carcinoma cells (JEG). These cells contain low endogenous TR (Fig. 1AGo, right) (21) and no endogenous E1A-like activity. We first studied the effects of E1A on thyroid-mediated transcription via a direct repeat separated by four nucleotides, since this is the the most commonly found TRE. Figure 1AGo shows that E1A increased the level of TR-mediated transcription at least 40-fold. As shown previously, TR alone activated transcription 3-fold via the single DR4 element in the presence of T3 (21). It is noteworthy that the basal activity in the absence of T3 was also several fold increased by E1A, although not to the same extent as when the ligand was added, 11- and 40-fold, respectively (Fig. 1Go). When E1A was transfected without exogenous TR, a small amount of T3-induced transcription was seen on all elements tested (Fig. 1Go). This is most likely explained by interaction of E1A with endogenous expressed TR in the JEG cells (21).



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Figure 1. E1A Potentiates TR-Mediated Transactivation

Three different response elements, cloned into the pBLCAT2 reporter vector, were tested for transcriptional activation in JEG cells by the TR and E1A proteins. Panel A, left, shows the relative TR-mediated activation on the thyroid DNA element DR4 in the presence or absence of RXR and E1A, as indicated. To the right, the endogenous levels of TR in JEG and HeLa cells are shown by Western blot anlysis. The numbers refer to the protein sizes. Panel B shows the relative TR-mediated activation on the natural response element rat {alpha}MHC and the palindromic element IR0 in the presence or absence of E1A, as indicated. pBLCAT2 represents an reporter vector containing no binding sites for TR. Addition of T3 to the samples is presented by striped bars. The organization of the half-sites in the response element are indicated at the top of the graphs.

 
To investigate the activation of TR and E1A on a DR4 type of element located in a natural gene, we analyzed the level of activation of a reporter construct that contained an element from the promoter region of the {alpha}MHC gene, which is normally expressed in heart muscle cells. This element consists of two half-sites oriented as direct (DR4) and palindromic (IR2) repeats separated by four and two nucleotides, respectively. E1A was shown to enhance the TR-mediated activation approximately 40-fold both via the heart-specific element and via a palindromic (IR0) TR-responsive element (Fig. 1BGo).

Neither TR nor E1A could activate transcription of the corresponding control reporter plasmid containing only the RSV (Rous sarcoma virus) TATA box in front of the CAT-gene (pBLCAT), thus verifying that the activation obtained on the various TREs was TR and DNA specific.

The ubiquitously expressed RXR is known to heterodimerize with TR and thereby increase transactivation. However, exogenous RXR had no additional effect on the E1A-enhanced TR-mediated activation in our cotransfection studies (Fig. 1AGo). Endogenous RXRs are present in low amounts in JEG cells as shown by Wahlström et al. (21).

During an adenovirus infection E1A efficiently activates transcription of the adenoviral E4 and E1A promoters. Therefore, the influence of TR on E1A- regulated transcription was assayed using these promoters as reporter constructs in cotransfections. Addition of TR had no effect on the activation by E1A (data not shown).

Different Isoforms of TR Cooperate with E1A in Transactivation
Several variants of the chicken thyroid hormone receptor that are expressed in different tissues and at different developmental times have been described (22, 23, 24, 25). The receptors differ in their N termini, regions that have been shown to contain putative phosphorylation sites and nuclear localization signals (26). We therefore investigated whether the synergism between E1A and cTR{alpha} was TR isoform specific. Cotransfection experiments were done in the presence of either TR{alpha}-p46, TR{alpha}-p40, TRß0, or TRß2 in the absence or presence of E1A and T3 hormone (Fig. 2Go). Figure 2BGo shows that the synergistic effect was obtained with all the chicken thyroid hormone receptors, tested on a DR4 type of DNA element. These different receptors have previously been shown to bind equally well to DR4 half-sites in vitro (Ref. 3 and data not shown). Interestingly, the N-terminal shorter variants (TR{alpha}-p40 and TRß0) gave a slightly lower level of activation.



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Figure 2. Synergistic Effect with Different Isoforms of Chicken TR

Panel A shows the schematic representation of the various chicken TRs tested in transactivation studies. TR{alpha}-p40 is an N-terminal shorter variant lacking the two phosphorylation sites present in the longer variant p-46. In addition to the {alpha}-receptors, two TRß receptors and the E1A early protein with three conserved regions are shown. Panel B shows the relative transcriptional activation obtained with the various TRs in the presence or absence of E1A and T3. Addition of T3 to the samples is indicated by stripes.

 
Efficient Activation Requires CR1 and CR3 in E1A
The conserved regions in E1A have been shown to exert different functions in transcription. CR1 is of importance in the activation of the proliferating cell nuclear antigen (PCNA) promoter, while CR3 activates for example the adenoviral E4 promoter (27). Both CR3 and CR1 separately activate transcription as Gal4 fusion proteins on promoters with Gal4-binding sites (28).

To investigate how the CR1, CR2, and CR3 domains of the E1A protein exert their effects on TR-mediated activation, we transfected plasmids encoding E1A deletion mutants along with TR into JEG cells. Figure 3Go shows that the {Delta}CR3 mutant activated transcription to 30% of the wild-type (wt) E1A protein, indicating that the E1A-enhanced activation is mainly dependent on this domain. A mutant lacking CR1 activated TR-regulated transcription to 50% of E1Awt, and the mutant lacking CR2 enhanced the transactivation to the same extent as E1Awt, whereas a double mutant of CR1 and CR3 gave, as expected, the lowest transactivation. Taken together, preferentially CR3, but also CR1, contributes to E1A enhancement of TR-mediated transactivation in JEG cells.



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Figure 3. Transcriptional Activation of E1A Deletion Mutants

The transcriptional activation by TR and the full-length E1A protein (E1Awt) was compared with various E1A deletion mutants in cotransfection experiments in JEG cells. The relative CAT activity obtained by constructs deleted in one of the three conserved regions is shown. Addition of T3 to the samples is as indicated.

 
TR and E1A Interact Directly; Domains of Interaction in E1A
E1A has been shown to directly interact with several upstream binding transcription factors, such as RARß and ATF-2 (14, 29, 30). To study a potential direct interaction between E1A and TR, 35S-labeled in vitro translated E1A proteins were added to column-bound glutathione-S-transferase (GST)-TR fusion proteins. The retained E1A proteins were analyzed using SDS-PAGE. Figure 4BGo shows that the wt E1A protein strongly bound to GST-TR, both in the presence and absence of T3. As expected, in vitro translated RXR was also bound by GST-TR. The E1A carboxy-terminal binding protein, CtBP (31), here used as a negative control, did not bind efficently to GST-TR compared with input levels. The binding of the E1A 289-amino acid protein to GST-TR was strong since E1A still bound efficiently in high-salt buffer (500 mM NaCl, data not shown).



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Figure 4. Direct Interaction between E1A and GST-TR

Panel A shows a schematic representation of the E1A-truncated or -deleted proteins tested in GST-TR binding experiments. Panel B shows retention of 35S-labeled E1Awt, RXR, RAR, and CtBP proteins on columns with GST-TR, GST, or Sepharose beads alone. In panels C and D, E1A proteins with truncations or internal deletions are retained by GST-TR in the presence or absence of T3, as indicated.

 
To map the TR-interacting domains in E1A, the E1A protein was truncated at amino acids 221, 130, and 89. All three truncated proteins were retained by GST-TR, although the proteins truncated at amino acids 130 and 89 bound to a lesser extent than E1A 1–221 and E1A wt (Fig. 4CGo). As the E1A 1–130 and E1A 1–89 proteins both lack CR3, the result suggests that CR3 is important for efficient binding to TR.

An E1A mutant containing both CR1 and CR3, but lacking the carboxy-terminal amino acids 193–245, was retained by GST-TR at the same efficiency as the wt E1A protein (Fig. 4CGo). This deletion spans the AR1 region that is required for activation of the E4F transcription factor (32).

The E1A mutant with an internal deletion of CR1 bound to GST-TR, although to a lesser extent than the wt protein (Fig. 4DGo). The mutant with an internal deletion of CR3 also weakly bound to GST-TR (Fig. 4DGo). The double mutant, lacking both CR1 and CR3, was totally deficient in binding GST-TR. These results demonstrate that the CR1 and CR3 domains of E1A cooperate to form a stable complex with TR.

The bacterially expressed GST-TR bound to the DR4 recognition sequence, as determined by gel-shift analysis. The GST-TR protein was also recognized by an {alpha}-TR antibody in Western blots (data not shown). This indicates that expression of TR in bacteria does not destroy its conformation and function.

Domains of Interaction in TR
In a set of reciprocal experiments, different GST-E1A fusion proteins were used to assay binding of in vitro translated TR proteins. Both the TR{alpha} variants, p46 and p40, bound to GST-E1A containing either CR1 or CR3 (GST-E1A{Delta}CR3 and GST-E1A76–289) (Fig. 5BGo). None of the TR proteins bound to the GST-E1ACt mutant, which contains amino acids 200–243 of the carboxy-terminal domain of E1A (Fig. 5BGo). This is in agreement with the results presented above in Fig. 4Go. RAR{alpha} bound to GST-E1A proteins containing CR1 or CR3, whereas RXR did not associate with any of the GST-E1A proteins. CtBP, now used as a positive control, was retained by all E1A proteins, as they all have the carboxy-terminal domain present.



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Figure 5. Direct Interaction between TR and GST-E1A

Panel A shows a schematic illustration of GST-E1A proteins and TR isoforms and mutant proteins. Panel B shows binding of 35S-labeled in vitro translated p46 and p40 TR isoforms, as well as RAR, RXR, and CtBP to GST-E1A 76–289 (containing CR3), GSTE1A{Delta}CR3 (containing CR1), and GST-E1ACt (containing amino acids 200–243 of E1A). Panels C and D show retention of PCR-amplified, in vitro translated TR mutants on the various GST-E1A fusion protein columns.

 
To further map the domains of interaction in TR, a number of TR mutants were synthesized and in vitro translated using PCR primers (Fig. 5AGo). The TR mutant encoding amino acids 49–408 bound efficiently to GST-E1A 76–289 and GST-E1A{Delta}CR3. This TR mutant contains both the DBD and LBD. The p46 122–408 mutant, containing the LBD, also bound to GST-E1A 76–289 and GST-E1A{Delta}CR3, although not as strongly as p46 49–408 (Fig. 5CGo). The mutants p46 1–256, p46 49–256, and p46 49–118, all containing the DBD, bound weakly to GST-E1A 76–289, but not to GST-E1A{Delta}CR3 (Fig. 5Go, C and D). This suggests that there is a weak interaction between TR DBD and E1A CR3.

A clear interaction was found between p46 257–408 and GSTE1A 76–289 (Fig. 5DGo). This result indicates that the main interactions are made between the carboxy-terminal part of the LBD in TR and CR3 in E1A. The p46 1–118 and p46 122–256 mutants were not retained by any of the GST-E1A proteins.

The shorter amino terminus TR variant, p40, bound more efficiently to GST-E1A than the full-length variant, p46 (Fig. 5BGo). Similarly, the mutants p46 49–256 and p46 49–118 bound GST-E1A better than the corresponding amino termini containing mutants p46 1–256 and p46 1–118, respectively (Fig. 5Go, C and D). In all these cases, addition of the amino terminus to the TR mutant weakened its ability to bind to E1A. This indicates that the amino terminus has a negative influence on the interaction between TR and E1A.

In summary, the CR3 in E1A interacts with both the carboxy terminus and the DBD of TR, while CR1 in E1A makes contact with TR somewhere in the LBD. The strongest binding is seen when both the DBD and LBD are present in TR. In contrast, the amino terminus of TR has a negative effect on binding to E1A.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
On the basis of transfection experiments and binding studies, we here show that E1A directly interacts with TR and that E1A enhances TR-dependent activation of promoters driven via TREs. E1A has previously been shown to directly bind to the basal transcription factor TBP (16, 17, 18). Based on these and our results, we propose that E1A may simultaneously interact with TR and TBP, thereby functioning as a supplementary bridge between TR and the basal transcription machinery.

E1A has been shown, in an analogous way, to interact with RARß, and thereby activate transcription of the RARß2 gene. The direct interaction between E1A and RARß was shown to depend on CR3 in E1A and the LBD including AF2 in RARß (14). In our study, two regions in E1A, CR1 and CR3, bound to TR, with stronger affinity for CR3. These two regions appear to cooperate to obtain an efficient binding. Our results from the binding experiments are in agreement with our cotransfection experiments, which demonstrated that a mutant containing CR3 gave 50% and a mutant containing CR1 gave 30% of the activation normally observed with E1Awt. In the study of Folkers and van der Saag (14), RARß-mediated transactivation was dependent on CR3. The observed differences between their study and the results presented in this paper may be explained by different requirements of E1A domains for TR- and RARß-induced transactivation and by the use of different cell lines.

The region in TR required for interaction with E1A is primarly the carboxy-terminal portion of the LBD, including AF-2. This is in agreement with the results of Folkers and van der Saag, as they have suggested that the region required for E1A binding in RARß is in the LBD. In addition, we also see a minor binding between E1A and the DBD of TR. Thus, we suggest that two separate regions in E1A interact with two separate regions in TR.

Several TR-interacting cofactors have been identified recently. Two of these cofactors, p300/CBP and SRC-1, have been found to possess intrinsic histone acetyltransferase activities; consequently, it was suggested that these cofactors may remodel transcriptionally repressed chromatin to make the promoter accessible for general transcription factors (33, 34, 35, 36). Other cofactors are thought to enhance and stabilize the assembly of the preinitiation complex. The latter is likely one function of E1A, since it interacts with both upstream and basal transcription factors. On the other hand, recent reports have suggested that E1A may inhibit p300/CBP histone acetyltransferase activity and also the transcription mediated by nuclear hormone receptors. (37, 38). However, the histone acetyltransferase activity of p300/CBP is necessary for transcriptional activation by the transcription factor Stat1{alpha}, but not for the nuclear retinoid receptor RAR, (39, 40). Instead, E1A inhibits RAR transactivation by preventing the association of CBP with nuclear receptor coactivators (40). Taken together, the recent reports suggest that E1A activates transcription of nuclear receptors when E1A interacts with the receptor and thereby localizes to the promoter. Not juxtaposed to the promoter, E1A would instead repress nuclear receptor-mediated transcription through disassembling receptor-coactivator complexes. The ability of E1A to function as a coactivator or a corepressor may be influenced by the promoter context and by the composition of nuclear receptor dimers as well as by cell type-specific cofactors.

What is the biological significance of an interaction between a nuclear receptor and the adenovirus E1A oncoprotein? As these two proteins can only coexist in adenovirus-infected cells, one function could be that the virus uses TR for modifying E1A-regulated gene transcription. However, since TR did not affect E1A-induced activation of the E1A- and E4-promoters, this is not likely to be the case. Instead, we hypothesize that the adenovirus E1A oncoprotein mimics the function of E1A-LA of undifferentiated EC cells, and that the interaction between TR and E1A-LA would be confined to early, pluripotent cells. E1A-LA would, in such a model, enhance the activating potential of TR on differentiation-specific genes. Our preliminary results suggest that activation of TR-driven transcription through DR4 sites is not further enhanced by the addition of viral E1A in undifferentiated P19 cells (data not shown). This indicates that E1A-LA could cooperate with TR to induce efficient transactivation in P19 cells. Furthermore, T3 treatment of P19 cells induces the cells to differentiate into cardiac myocytes (41). The authors suggest that this differentiation is accompanied by preferential binding of as yet undefined factors to DR4-sites (42). Whether or not these factors are TR and E1A-LA remains to be elucidated.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructs
One copy of the double-stranded DR4 oligonucleotide with the four- nucleotide spacer sequence CTTC was inserted into the HindIII site of the pBLCAT2 vector. The MHC and IR0 elements were cloned in the same way (21). The chicken TR{alpha}1 (p40 and p46) and TRß0 and ß2 receptors were cloned into the pSG5 expression vector (3, 43). The plasmid pML005, used for expressing E1Awt in transfections, contains nucleotides 1–1773 of genomic adenovirus type 2. The CR1, CR2, and CR3 mutants of pML005 has been described previously (44). GSTE1A 76–289 and GSTE1A{Delta}CR3 (GSTE1A12S) have been described previously (45). GST-TR was cloned by inserting the EcoRI fragment of pSG65-p46 into the EcoRI site of the pGEX1lambdaT vector (Pharmacia Biotech, Piscataway, NJ). Gst-E1ACt (aa 200–243) and pcDNA3-CtBP have been described previously (31, 46). pML00512S, pML00513S (47), and pSG5p46 were used as templates for making in vitro translated E1A and TR proteins, respectively.

Transfections
Human chorion carcinoma cells (JEG) were plated at a density of 2 x 105 per 3-cm dish in DMEM (Biological Industries) supplemented with 8% FCS. One day after plating, the medium was replaced with DMEM containing 8% calf serum depleted of T3 and T4 by ion exchange resin (48). Approximately 2 h later the cells were cotransfected with expression vectors encoding 100–200 ng of different chicken TRs, E1A, or E1A mutants plus 500 ng of reporter constructs containing a TRE. The cells were maintained in the presence or absence of 12–30 nM T3, harvested 24 h after hormone treatment, and assayed for chloramphenicol acetyltransferase activity. Quantifications were done with a Molecular Dynamics, Inc. (Sunnyvale, CA) phosphorimager (49). All transfections were repeated at least three times with similar results. Duplicate sample points were used in each experiment and varied by less than 15%.

Protein Binding Analysis
In vitro GST-fusion proteins were produced in Escherichia coli and purified with gluthathione agarose beads (Current Protocols in Molecular Biology). Protein concentrations were estimated on a Coomassie-stained SDS-polyacrylamide gel. Approximately equal amounts of GST fusion proteins were mixed with 5–10 µl of [35S]methionine-labeled in vitro synthesized proteins (TNT-coupled reticulocyte lysate systems, Promega Corp., Madison, WI). The proteins were incubated rotating at 4 C for 3 h in binding buffer (250–500 mM NaCl, 50 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.1% NP40, 1 mM dithiothreitol, and 0.2 mM phenylmethylsulfonylfluoride). Beads were washed four times in binding buffer, and bound proteins were separated on a polyacrylamide gel and visualized by autoradiography. In Figs. 4DGo and 5Go, C and D, the 35S-labeled proteins were synthesized using PCR-amplified DNA templates. The 5'-PCR primers contained the sequence for a T7 RNA polymerase start site.

Western Blot
Equal amounts of crude extracts made from human chorion carcinoma (JEG) and human cervical carcinoma (HeLa) cells were run on a 10% denatured gel. Separated proteins were transferred overnight to enhanced chemiluminescence (ECL) nitrocellular membranes, which were then hybridized to a primary rabbit polyclonal antibody directed against TR diluted 1:1000 (FL-408, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After incubation with a secondary peroxidase-conjugated goat antirabbit antibody diluted 1:1000 (DAKO Corp., Carpinteria, CA), the membranes were washed and the migrated proteins were detected with the ECL system (Amersham, Arlington Heights, IL).


    ACKNOWLEDGMENTS
 
We are grateful to Dr. K. Sollerbrandt for the gift of plasmids.


    FOOTNOTES
 
Address requests for reprints to: Maria Bondesson Bolin, Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, S-171 77 Stockholm, Sweden.

This project was funded by grants from Cancerfonden, the Lars Hierta Foundation, the Magnus Bergvalls Foundation, Human Fronties, Karolinska Institutet, and the Swedish Society for Medical Research. M.B-B. was also supported by the Swedish Natural Science Research Council.

1 These authors have contributed equally to the work. Back

Received for publication December 17, 1998. Revision received April 12, 1999. Accepted for publication April 16, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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